2005 Annual Meeting
ABSTRACT 1
An Autoregulatory Element Maintains HOXA10 Expression in Endometrial Epithelial CellsBy Invitation
Hugh Taylor, M.D.
Michael Kelly, M.D.
Gaurang Daftary, M.D.
OBJECTIVE: HOXA10 is necessary for endometrial receptivity and regulated by sex steroids. Secretory phase HOXA10 expression increases in endometrial epithelial cells despite the loss of PR. Stromal-epithelial molecular communication likely transmits progesterone signaling from PR containing stromal cells to epithelium. Here we investigated an alternative hypothesis, persistent HOXA10 expression by auto-regulation.
STUDY DESIGN: Nested segments of the HOXA10 5' regulatory region were cloned into a pGL3-Luciferase reporter construct and tested for HOXA10 induced reporter activity. Direct binding was assayed by electrophoretic mobility shift assay.
RESULTS: One 370 base pair element drove reporter gene expression specifically in response to HOXA10 in Ishikawa cells but not in BT-20 cells. This element contained a site which bound HOXA10 protein.
CONCLUSION: HOXA10 expression is driven by an auto-regulatory element in the 5' regulatory region of the gene. Autoregulation is a novel alternative molecular mechanism by which steroid induced gene expression can be maintained in the absence of steroid receptors.
DISCUSSANT: Bruce Carr, M.D.
ABSTRACT 2
The Effects of the Inhibition of Inducible Nitric Oxide Synthase on Angiogenesis of Epithelial Ovarian CancerBy Invitation
John Malone, M.D.
Ghassan M. Saed, M.D.
Michael P. Diamond, M.D.
Robert J. Sokol, M.D.
Adnan R. Munkarah, M.D.
OBJECTIVE: The release of nitric oxide by tumor cells, through the stimulation of inducible nitric oxide synthase (iNOS) expression, may play a critical role in ovarian cancer progression. In this study we have sought to determine the effects of inhibiting iNOS on angiogenesis induced by two ovarian cancer cell lines, SKOV and MDAH2774.
STUDY DESIGN: Real-time PCR and ELISA techniques were used to determine the expression levels of iNOS and VEGF in the ovarian cancer cell lines in response to treatments with L-NAME, an inhibitor of NOS, and SNAP, an NO donor. Ovarian cancer induced angiogenesis was assessed in vitro using an established assay based on the ability of human umbilical vein endothelial cells to form a tubular network in response to angiogenic agents.
RESULTS: SKOV and MDAH2774 cell lines exhibited over-expression of iNOS and have high baseline NO levels. This was associated with high levels of VEGF production and angiogenesis induction. Treatment of the ovarian cancer cell lines with L-NAME significantly reduced VEGF levels production and completely inhibited angiogenesis. In contrast, treatment with SNAP significantly increased VEGF levels and increased angiogenesis in both cell lines.
CONCLUSION: Our data suggest that the inhibition of iNOS may form a basis for a novel therapeutic treatment option for ovarian cancer therapy.
DISCUSSANT: Yusoff Dawood, M.D.
ABSTRACT 3
Biological Targets for Therapeutic Intervention in Endometrioid Endometrial Adenocarcinoma and Malignant Mixed Mullerian TumorsBy Invitation
Laurel Rice, M.D.
Rebecca L. Stone, M.D.
Miaohou Xu, M.D., Ph.D.
Mary Galgano, M.D.
Mark H. Stoler, M.D.
Elise N. Everett, M.D.
Amir A. Jazaeri, M.D.
OBJECTIVE: To investigate the AKT signaling cascade in endometrial cancers and assess its therapeutic potential.
STUDY DESIGN: Western blotting and immunohistochemistry were utilized to investigate expression of estrogen receptor, progesterone receptor, HER2, AKT, and 4EBP1 proteins in 27 atrophic endometria, 31 grade 1 and 24 grade 3 endometrioid endometrial cancers (EEC), and 19 malignant mixed mullerian tumors (MMMT). Inhibition of the AKT signaling cascade was investigated in cell-lines.
RESULTS: MMMT and grade 3 EEC demonstrated higher levels of AKT and 4EBP1 activation and hormone receptor loss compared to grade 1 EEC and atrophic samples. HER2 over-expression was most often identified in grade 3 tumors without gene amplification. In endometrial cancer cell-lines, AKT cascade inhibitors decreased cell proliferation by apoptosis and cell cycle arrest.
CONCLUSION: AKT cascade activation in grade 3 EEC and MMMT is a novel finding. Apoptosis and growth arrest resulting from AKT inhibition expose opportunities for therapeutic intervention.
DISCUSSANT: David Mutch, M.D.
ABSTRACT 4
Immunohistochemical Evidence for Presence and Location of Neurotrophin-Trk Receptor Family in Adult Human Preovulatory Ovarian FolliclesBy Invitation
David Seifer, M.D.
Bo Feng, M.D.
Robert M. Shelden, M.D.
OBJECTIVE: To evaluate the presence or absence of neurotrophins and their respective receptors within adult human preovulatory follicles.
STUDY DESIGN: Setting: University-based fertility program. Patient(s): Tissue (mural and cumulus granulosa, unfertilized oocytes) isolated from women undergoing follicular aspiration for in vitro fertilization/intracytoplasmic sperm injection. Intervention(s): Immunohistochemical visualization of neurotrophins and receptor proteins. Main outcome measures(s): Positive or negative immunohistochemical staining.
RESULTS: Both mural and cumulus granulosa cells were positive for BDNF, NT-4/5, NT-3 and NGF as well as for Trk B, Trk C and Trk A receptors. Unfertilized oocytes were positive for Trk B, Trk C and Trk A receptors.
CONCLUSION: Neurotrophins and their respective receptor proteins are present within the mural and cumulus granulosa cells of adult human preovulatory follicles. Neurotrophin receptors are present in human unfertilized oocytes. The location of the neurotrophins and their receptors suggest both an autocrine and paracrine function within the adult human ovarian follicle.
DISCUSSANT: Christos Coutifaris, M.D., Ph.D.
ABSTRACT 5
The Use of Needle Biopsy for Assessment of Placental Gene ExpressionBy Invitation
Yoel Sadovsky, M.D.
Solange M. Wyatt, M.D.
Lynne Collins, B.Sc., M.A.
Uriel Elchalal, M.D.
Fredrick T. Kraus, M.D.
D. Michael Nelson, M.D., Ph.D.
OBJECTIVE: To test the hypothesis that placental samples, obtained using needle aspiration ex vivo, are useful for determination of villus gene expression.
STUDY DESIGN: Placental biopsy was performed using a spinal needle after uncomplicated deliveries. Villi were inspected microscopically and RNA was extracted and analyzed using capillary electrophoresis. Gene expression was determined using quantitative PCR.
RESULTS: We obtained more placental villi per aspiration using a 20-gauge needle (5.2±1.8) than with a 22-gauge needle (3.3±1.6), p<0.01). RNA quality was adequate based on the 28S and 18S rRNA bands, with a mean 260/280 ratio of 1.88. The amount of extracted RNA correlated with the number of villi per aspirate. Importantly, the expression of NDRG1 and hPL, both markedly altered in hypoxia, was consistent between villi obtained by either needle or standard biopsy.
CONCLUSION: Placental samples, obtained using ex vivo needle aspiration, are useful for extracting RNA and for determination of villus gene expression.
DISCUSSANT: Karin Blakemore, M.D.
ABSTRACT 6
Maternal-Fetal Surgery for Myelomeningocele: Neurodevelopmental OutcomesBy Invitation
Mark Johnson, M.D.
Marsha Gerdes, Ph.D.
Natalie Rintoul, M.D.
Patrick Pasquariello, M.D.
Jeanne Melchionni, R.N.
Leslie N. Sutton, M.D.
N. Scott Adzick , M.D.
OBJECTIVE: To examine short-term neurodevelopmental outcomes in children with myelomeningocele (MMC) that underwent in utero neurosurgical closure.
STUDY DESIGN: Between 1998 and 2002, 51 fetuses underwent in utero MMC closure at our Center. Thirty of these children have returned for neurodevelopmental testing at two years of age using the Bayley Scales of Infant Development and Preschool Language Scales.
RESULTS: Overall shunt rate was 43% in this group. Neurodevelopmental testing found 67% with cognitive language and personal-social skills in the normal range, 20% with mild delays, and 13% with significant delays. Children with shunted hydrocephalus scored lower than those with unshunted ventriculomegaly.
CONCLUSION: Children who have undergone fetal MMC closure have characteristic neurodevelopmental deficits that do not appear worsened by fetal surgery, and developmental outcomes may be improved by decreasing the need for ventriculoperitoneal shunting.
DISCUSSANT: Mark Evans, M.D.
ABSTRACT 7
Learning Enhancements with NeuropeptidesBy Invitation
Catherine Spong, M.D.
Laura Toso, M.D.
Melanie Endres
Joy Vink, B.A.
Daniel T. Abebe, B.S.
Douglas E. Brenneman, Ph.D.
OBJECTIVE: Previous work has demonstrated that two synthetic peptides can prevent prenatal alcohol-induced damage as assessed by prevention of learning abnormalities in adult offspring as well as improve outcome from traumatic brain damage. The present studies were undertaken to evaluate whether these peptides could enhance performance in a learning and memory paradigm when administered either prenatally or to aged mice.
STUDY DESIGN: For prenatal treatment, C57Bl6/J mice were treated on gestational day 8 with one oral administration of D-NAP or D-SAL alone or D-NAP+D-SAL or a double dose of D-SAL. Control groups were same-regimen treated with vehicle alone. Learning was assessed in adult male offspring (35-50d) using the Morris watermaze. To evaluate aged animals, 12-month old mice were treated with D-NAP and D-SAL or vehicle alone daily and tested on the Morris watermaze.
RESULTS: Offspring exposed prenatally to D-NAP+D-SAL learned significantly faster than controls, with an earlier onset of learning and an overall decreased latency to find the hidden platform (P<0.05). Animals exposed prenatally to either D-NAP or D-SAL alone learned similar to control, with a trend toward faster latencies. Aged animals who received D-NAP+D-SAL learned significantly faster than age-matched controls, with an earlier onset of learning (P<0.05).
CONCLUSION: Combined D-NAP+D-SAL enhanced learning in healthy young mice and aged mice. These findings suggest potential therapeutic interventions not only during a critical developmental period, but also in aged animals.
DISCUSSANT: Robert Jaffe, M.D.
ABSTRACT 8
A Multifaceted Approach to Resolving the Professional Liability Crisis and Improving Patient SafetyBy Invitation
Louis Weinstein, M.D.
The current professional liability crisis is the third in the last 30 years. Similarities of the 3 crises are the rising cost of professional liability insurance, the decrease in the number of sources available to purchase coverage, and the decrease in available limits. Proposed tort reform with caps on non-economic damages and attorney contingency fees is a back end approach and will do little to solve this crisis or prevent future ones. The current situation can only be improved by placing an increased emphasis on improving patient safety and elimination of preventable medical errors. The present system of risk management has changed little over time and needs to be revamped. The protective devices of privileged communication and peer review need to be changed. Full and prompt disclosure of any medical error or injury needs to be made to the patient and family. Physicians need to be taught appropriate communication skills and providers with frequent patient or nursing complaints need to be critically reviewed. Claims management needs to be proactive in assisting the patient with early compensation when appropriate and oversee a vigorous defense when medical care is adequate. Major medical centers need to establish a panel of medical experts who when requested will render fair, unbiased reviews of patient care with all the findings being disclosed. All health care systems must identify practitioners likely to cause adverse outcomes, strive to eliminate preventable medical errors and improve patient safety.
DISCUSSANT: Stanley Zinberg, M.D., M.S.
ABSTRACT 9
Risk Factors for Previable Premature Rupture of Membranes or Advanced Cervical Dilation: A Case Control StudyBy Invitation
Sarah Kilpatrick, M.D., Ph.D.
Rajita Patil, M.D.
Jeness Connell, M.D.
Jessica Nichols, M.P.H.
Laura Studee, M.P.H.
OBJECTIVE: To identify risk factors for second trimester premature preterm rupture of membranes (PPROM) or advanced cervical dilation (ACD) in a high risk population.
STUDY DESIGN: A retrospective case control study was performed comparing women with PPROM or ACD to term controls. The cases included all singleton pregnancies between 14 and 24 weeks' gestation presenting with PPROM or ACD between 1996 and 2000. The next two term deliveries were chosen as controls. The variables compared between cases and controls included pregnancy history, infectious and medical histories, cervical/uterine procedures, and habits. This study had IRB approval.
RESULTS: There were 102 women with PPROM, 56 women with ACD and 316 controls. The mean gestational ages at presentation for PPROM or ACD were 20 ± 2.6 and 19.9 ± 2.6 weeks. Tobacco use, history of or current cervical incompetence, prior second trimester delivery, prior termination < 20 weeks, and prior PPROM were significantly associated with PPROM or ACD compared to term controls. When controlled for parity, age, marital status, and race these variables remained significant. BV in current pregnancy was significantly associated with only ACD but not PPROM compared to controls. History of Chlamydia was most common in the term controls (19.6%).
CONCLUSION: In a high risk population of inner city women only pregnancy history and tobacco use distinguished women with second trimester PPROM or ACD from term controls. Women with PPROM or ACD had similar risk factors suggesting that these may be presentations of the same disease process. We found little evidence that lower genital tract infection was a risk factor for PPROM or ACD. The only modifiable risk identified was tobacco use.
DISCUSSANT: Daniel Landers, M.D.
ABSTRACT 10
Clinical, Biologic, and Genetic Characteristics of Recurrent Spontaneous Preterm BirthBy Invitation
Brian Mercer, M.D.
Cora MacPherson, Ph.D.
Alice R. Goepfert, M.D.
Sylvie Haugel-de Mouzon, Ph.D.
Michael W. Varner, M.D.
Jay D, Iams, M.D.
Paul J. Meis, M.D.
Atef H. Moawad, M.D.
Menachem Miodovnik, M.D.
Steve N Caritis, M.D.
J. Peter Van Dorsten, M.D.
Mitchell Dombrowski, M.D.
Gary R. Thurnau, M.D.
Catherine Y. Spong, M.D.
OBJECTIVE: To determine if women with recurrent spontaneous preterm births (rSPBs) have clinical characteristics, systemic markers of inflammation and stress, or inflammatory polymorphisms that are different from those with isolated preterm (iSPBs) or recurrent term births (rTBs), when assessed remote from delivery.
STUDY DESIGN: We compared demographic/clinical characteristics and findings (including cervical ultrasound, bacterial vaginosis, fetal fibronectin), as well as systemic plasma markers obtained at 22-24 weeks gestation (inflammatory cytokines, cortisol, corticotrophin releasing hormone [CRH]), and genetic polymorphism allele frequencies/genotypes (IL1β-511, IL1β Exon 5 +3954, IL6-174, IL8-845, IL10-1082, TNFα-238, TNFα-308: and toll-like receptors TLR4 299, TLR4 399), between women having pregnancies resulting in rSPBs (2 or 3 consecutive SPBs with no term births), iSPBs (1 SPB with 1 or 2 term births), and rTBs (2 or 3 consecutive term births with no SPBs).
RESULTS: 1,257 women met our inclusion criteria; 47 rSPBs, 241 iSPBs (80 current and 161 prior SPB), and 969 rTBs. Women with rSPBs had lower pregravid weights (p<0.0001) and BMIs (p<0.001), and were more likely <100 pounds (p=0.008) or <19.8 kg/m2 BMI (p=0.001) before conception. At 22-24 weeks those with rSPBs remained leaner and had worse findings on digital cervical exam, including Bishop score, than iSPBs and rTBs. Transvaginal ultrasound demonstrated progressive decrease in cervical length for those with prior iSPBs, current iSPBs, and rSPBs as compared with rTBs, and a progressive increase in the frequency of a short cervix (p<0.001) with worsening history. Cervical length was shorter for women of lower pregravid weight and BMI, but not with shorter height. Fetal fibronectin levels were higher for those with an SPB in the current gestation regardless of prior outcome. At 22-24 weeks, women with rSPBs had more common uterine contractions and tocolytic Rx, but not more infections or antibiotic Rx. In the 22-24 week maternal plasma samples, cortisol and CRH levels were higher in women with iSPBs and rSPBs than in rTB controls, (p=0.001 and 0.0027). This was most apparent with SPB in the current pregnancy. However, maternal plasma cytokines were not increased among women with either iSPBs or rSPBs. Only the non-wild type TNFα–308 allele (A), heterozygous (G*A) and homozygous (AA) genotype were more common with iSPBs and rSPBs.
CONCLUSION: Women with rSPBs are constitutionally different (leaner, shorter cervical length, increased Bishop score) than women with either iSPBs or rTBs.
DISCUSSANT: George Macones, M.D., M.S.C.E.
ABSTRACT 11
Utility of Preoperative Lymphoscintigraphy in Patients Undergoing Radical Hysterectomy and Pelvic Lymphadenectomy for Cervical Cancer
By Invitation
Charles Levenback, M.D.
Michael Frumovitz, M.D., M.P.H.
Robert L. Coleman, M.D.
Isis W. Gayed, M.D.
Pedro T. Ramirez, M.D.
Judith K. Wolf, M.D.
David M. Gershenson, M.D.
OBJECTIVE: To assess the value of preoperative lymphoscintigraphy prior to intraoperative lymphatic mapping (IOLM) for sentinel lymph node (SLN) identification during radical hysterectomy.
STUDY DESIGN: Fifty patients underwent IOLM on our IRB-approved protocol. The location of SLN's found on lymphoscintigraphy and IOLM were compared.
RESULTS: Fifteen (71%) of 21 patients with solitary SLN's on lymphoscintigraphy had multiple SLN basins found intraoperatively. Thirteen (52%) of 25 patients with unilateral SLN basins on lymphoscintigraphy had bilateral SLN's at IOLM. Of 15 patients with 2 SLN locations identified on lymphoscintigraphy, 12 (80%) had 3 or more intraoperatively. Of the SLN's located on the external iliac basin (n=47) on lymphoscintigraphy, only 20 (43%) were found at that location intraoperatively. Concordance between the 2 methods was poor. Seven of the nine patients with lymph node metastases had disease limited to the SLN's.
CONCLUSION: Preoperative lymphoscintigraphy adds little value over IOLM for SLN identification during radical hysterectomy.
DISCUSSANT: Jeff Fowler, M.D.