DNA damage-induced ephrin-B2 reverse signaling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53.

Mutation in the TP53 gene positively correlates with increased incidence of chemoresistance in different cancers. In this study, we investigated the mechanism of chemoresistance and epithelial-to-mesenchymal transition (EMT) in colorectal cancer involving the gain-of-function (GOF) mutant p53/ephrin-B2 signaling axis. Bioinformatic analysis of the NCI-60 data set and subsequent hub prediction identified EFNB2 as a possible GOF mutant p53 target gene, responsible for chemoresistance. We show that the mutant p53-NF-Y complex transcriptionally upregulates EFNB2 expression in response to DNA damage. Moreover, the acetylated form of mutant p53 protein is recruited on the EFNB2 promoter and positively regulates its expression in conjunction with coactivator p300. In vitro cell line and in vivo nude mice data show that EFNB2 silencing restores chemosensitivity in mutant p53-harboring tumors. In addition, we observed high expression of EFNB2 in patients having neoadjuvant non-responder colorectal carcinoma compared with those having responder version of the disease. In the course of deciphering the drug resistance mechanism, we also show that ephrin-B2 reverse signaling induces ABCG2 expression after drug treatment that involves JNK-c-Jun signaling in mutant p53 cells. Moreover, 5-fluorouracil-induced ephrin-B2 reverse signaling promotes tumorigenesis through the Src-ERK pathway, and drives EMT via the Src-FAK pathway. We thus conclude that targeting ephrin-B2 might enhance the therapeutic potential of DNA-damaging chemotherapeutic agents in mutant p53-bearing human tumors.

HDAC8 and STAT3 repress BMF gene activity in colon cancer cells.

Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

Referral and ascertainment bias in patients with synchronous and metachronous endometrial malignancy.

The purpose of this study was to evaluate the frequency in patients with endometrial cancer of other malignancies and the influence of referral and ascertainment biases on these associations. Analysis of 1,028 local and referred patients who had a hysterectomy for endometrial cancer was based on residence at the time of diagnosis. Altogether, 208 patients had a history of another malignancy, most frequently breast, colon, and ovary. At the time of surgery for endometrial cancer, the prevalence of lymphoma and breast and ovarian cancers was greater than expected although the higher prevalence of lymphoma was limited to referred patients. During follow-up after hysterectomy, the incidence of lung cancer was lower than expected, whereas the incidence of lymphoma was higher. Breast, colorectal, and bladder cancers were more common than expected although this finding was limited to local patients. We concluded that results of epidemiologic studies from tertiary care centers may be misleading if they do not account for referral and ascertainment biases.

STK11 status and intussusception risk in Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is caused by germline STK11 mutations and characterised by gastrointestinal polyposis. Although small bowel intussusception is a recognised complication of PJS, risk varies between patients. OBJECTIVE: To analyse the time to onset of intussusception in a large series of PJS probands. METHODS: STK11 mutation status was evaluated in 225 PJS probands and medical histories of the patients reviewed. RESULTS: 135 (60%) of the probands possessed a germline STK11 mutation; 109 (48%) probands had a history of intussusception at a median age of 15.0 years but with wide variability (range 3.7 to 45.4 years). Median time to onset of intussusception was not significantly different between those with identified mutations and those with no mutation detected, at 14.7 years and 16.4 years, respectively (log-rank test of difference, chi(2) = 0.58, with 1df; p = 0.45). Similarly no differences were observed between patient groups on the basis of the type or site of STK11 mutation. CONCLUSIONS: The risk of intussusception in PJS is not influenced by STK11 mutation status.

A comparison of endoscopic ultrasound, magnetic resonance imaging, and exam under anesthesia for evaluation of Crohn's perianal fistulas.

BACKGROUND & AIMS: To determine accuracy of endoscopic ultrasound (EUS) and magnetic resonance imaging (MRI) for evaluation of Crohn's disease perianal fistulas. METHODS: Thirty-four patients with suspected Crohn's disease perianal fistulas were prospectively enrolled in a blinded study comparing EUS, MRI, and examination under anesthesia (EUA). Fistulas were classified according to Parks' criteria, and a consensus gold standard was determined for each patient. Acceptable accuracy was defined as agreement with the consensus gold standard for > or =85% of patients. RESULTS: Three patients did not undergo MRI; 1 did not undergo EUS or EUA; and consensus could not be reached for 1. Thirty-two patients had 39 fistulas (20 trans-sphincteric, 5 extra-sphincteric, 6 recto-vaginal, 8 others) and 13 abscesses. The accuracy of all 3 modalities was > or =85%: EUS 29 of 32 (91%, confidence interval [CI] 75%-98%), MRI 26 of 30 (87%, CI 69%-96%), and EUA 29 of 32 (91%, CI 75%-98%). Accuracy was 100% when any 2 tests were combined. CONCLUSIONS: EUS, MRI, and EUA are accurate tests for determining fistula anatomy in patients with perianal Crohn's disease. The optimal approach may be combining any 2 of the 3 methods.

A search for germline APC mutations in early onset colorectal cancer or familial colorectal cancer with normal DNA mismatch repair.

Twenty percent of colorectal cancers (CRCs) arise in people who have a family history of CRC in at least one other relative. Although a fraction of these CRCs are explained by two well-described autosomal dominant syndromes-5% by hereditary nonpolyposis colorectal cancer (HNPCC) and 1% by familial adenomatous polyposis (FAP)-the cause of the remaining 14% of familial aggregates of CRC is unknown. Many cases of HNPCC are due to germline mutations in DNA mismatch repair genes, leading to the tumor phenotype of microsatellite instability (MSI), and most cases of FAP are caused by germline APC mutations. To date, non-FAP familial CRC aggregates have not been evaluated for germline APC mutations. In this study, we examined the involvement of germline APC mutations in 79 individuals with CRC who had early-age onset of their cancer (age < 50 years) and/or a family history of CRC. Cases with FAP or HNPCC due to defective mismatch repair were excluded from the study. Using conformation-sensitive gel electrophoresis and the protein truncation test as the screening methods, no functionally significant germline mutations were detected for any of the cases. An apparently silent polymorphism resulting in a 1-bp alteration of A --> G (proline --> proline) in exon 4 was observed. Additionally, four intervening sequence (IVS) alterations were detected: IVS2-53t-->c in 3 cases; IVS4-17ins T in 3 cases; IVS5+32t-->c in 16 cases; and IVS5+33g-->a in 1 case. All appeared to be polymorphisms present in similar proportions in an average-risk population. We conclude that germline APC mutations do not account for familial MSS (stable microsatellite) CRC associated with few synchronous polyps.

Association of Peutz-Jeghers-like mucocutaneous pigmentation with breast and gynecologic carcinomas in women.

Most reports describe an increased risk of malignancy in Peutz-Jeghers syndrome (PJS). We identified individuals with PJS-like pigmentation but no polyposis, designated as isolated mucocutaneous melanotic pigmentation (IMMP), and 1) characterized their clinical features, 2) assessed them for cancer events, and 3) screened a sample of these subjects for mutations in LKB1, a gene responsible for a portion of PJS cases. Review of Mayo Clinic records from 1945 to 1996 identified 26 patients with IMMP. All were then interviewed or their medical records reviewed to determine if cancer had developed. Conformation-sensitive gel electrophoresis (CSGE) screening for LKB1 mutations was followed by direct sequencing. Ten of these 26 individuals (38%) developed 12 malignancies that arose in the cervix (n = 3), endometrium (n = 3), breast (n = 1), kidney (n = 1), lung (n = 2), colon (n = 1), and lymphatic tissue (n = 1). In females with IMMP, the relative risk for cancer was 3.2 (95% CI, 1.2-6.9), while that for males was not increased. The relative risk for breast and gynecologic cancers was 7.8 (95% CI, 2.5-18.1) in affected females. Of 9 individuals tested, no LKB1 mutations were detected. Classical PJS is associated with an increased cancer risk. Our results indicate that IMMP is another lentiginosis with cancer predisposition. In particular, the relative risk for cancer in females with IMMP was significantly increased, as is true in females with PJS. However, LKB1 mutations did not contribute to the development of IMMP in the patients tested.

Genetic heterogeneity in Peutz-Jeghers syndrome.

LKB1, the human gene encoding a serine threonine kinase, was recently identified as a susceptibility gene for Peutz-Jeghers syndrome (PJS), a disease characterized by the constellation of intestinal hamartomata, oral mucocutaneous hyperpigmentation, and an increased risk for gastrointestinal as well as extraintestinal malignancies. To date, the majority of individuals with PJS have been found to have genetic alterations in LKB1, most of which result in protein truncation. Additionally, linkage analyses have suggested a modicum of genetic heterogeneity, with the majority of PJS families showing linkage to the LKB1 locus. In this study, we evaluated five kindreds with greater than two affected family members, five PJS probands with only one other affected family member, as well as 23 individuals with sporadic PJS for mutations within the LKB1 gene. Conformation sensitive gel electrophoresis was utilized for the initial screen, followed by direct sequence analysis for characterization. Long-range PCR was used for the detection of larger genetic insertions or deletions. Mutation analysis revealed genetic alterations in LKB1 in two probands who had a family history of PJS. LKB1 mutations were detected in only four of the remaining 23 cases of sporadic PJS. These data suggest the presence of significant genetic heterogeneity for PJS and the involvement of other loci in this syndrome.

Positive cone biopsy specimen margins in women infected with the human immunodeficiency virus.

OBJECTIVES: The purpose of this study was to compare the positive margin rate associated with cervical conization among women who are seropositive for human immunodeficiency virus with that among women who are seronegative. STUDY DESIGN: This was a cross-sectional study of 245 women who underwent cervical conization for the following indications: biopsy-proven cervical intraepithelial neoplasia grade 2 or 3, abnormal endocervical curettage specimen, cytologic-histologic examination discrepancy, persistent cervical intraepithelial neoplasia grade 1, or abnormal cytologic characteristics with inadequate colposcopic examination. RESULTS: Twenty-two (47.8%) of 46 women who were seropositive for human immunodeficiency virus and 65 (32.7%) of 199 women who were seronegative had positive cone biopsy specimen margins. In a multivariable logistic regression the human immunodeficiency virus-seropositive women had a 2-fold increased risk of having a positive cone biopsy margin (odds ratio, 2.25; 95% confidence interval, 1.07-4.76). CONCLUSION: If the presence of positive cone biopsy specimen margins represents the potential for disease progression, then our findings of a positive margin rate of nearly 50% in a human immunodeficiency virus-positive population may argue against the kind of conservative management of colposcopic follow-up that has been proposed for immunocompetent women.

Representative cDNA libraries and their utility in gene expression profiling.

An increasing interest in gene expression profiles in human diseases has led to the use of microdissected tumors and biopsies in gene discovery approaches. Since many of these clinical samples yield extremely small amounts of RNA, reproducible methods are needed to amplify this RNA while maintaining the original message profile. Using the SMART cDNA Synthesis Method, we show that high-, medium- and low-abundance transcripts can be amplified in a representative fashion and that the resulting cDNA can also be used as a complex probe to confirm gene expression differences identified by other techniques.

Erdheim-Chester disease: clinical, radiologic, and histopathologic findings in five patients with interstitial lung disease.

Erdheim-Chester disease is a clinicopathologic entity defined by a characteristic pattern of symmetric osteosclerosis caused by an infiltrate of mononuclear cells that include prominent numbers of foamy histiocytes. About half of patients have extraskeletal manifestations, including involvement of the hypothalamus/posterior pituitary, orbit, retroperitoneum, skin, lung, and heart. Pulmonary involvement is an uncommon but important manifestation of Erdheim-Chester disease because it causes significant morbidity and mortality. A review of the Mayo Clinic files produced four patients with confirmed Erdheim-Chester disease in whom lung biopsy had been performed. One additional patient was included from the University of Pittsburgh. Four patients were women. The mean age was 53.6 years (range 25-70 years). All patients had bilateral and symmetric sclerotic bone lesions characteristic of Erdheim-Chester disease, although in three the skeletal abnormalities were discovered only after lung biopsy. Four patients had dyspnea, and one also had a dry cough. One patient died 17 months after diagnosis. Chest radiographs showed diffuse interstitial infiltrates in all patients, with an upper zone predominance in three. Thoracic computed tomography (CT) scans showed thickening of the visceral pleura and interlobular septa with patchy associated fine reticular and centrilobular opacities and ground glass attenuation. Lung biopsy specimens showed an infiltrate of foamy histiocytes, lymphocytes, and scattered Touton giant cells with associated fibrosis in a striking lymphatic distribution. The infiltrate involved visceral pleura, interlobular septa, and bronchovascular bundles. Immunohistochemical stains were positive for CD68 in all cases and S-100 protein in four cases. Stains for CD1a were consistently negative. Ultrastructural studies in one case showed no Birbeck granules. Although in bone the histologic features of Erdheim-Chester disease may overlap with Langerhans' cell histiocytosis, its expression in the lung is distinct. Lung involvement in Erdheim-Chester disease has emerged as a unique radiographic and histologic entity.

Increased risk for cancer in patients with the Peutz-Jeghers syndrome.

BACKGROUND: Some reports describe an increased risk for cancer in patients with the Peutz-Jeghers syndrome. OBJECTIVE: To characterize occurrences of cancer in a large cohort of patients with the Peutz-Jeghers syndrome. DESIGN: Retrospective cohort study. SETTING: Tertiary care center. PATIENTS: 34 patients with the Peutz-Jeghers syndrome identified from Mayo Clinic records from 1945 to 1996. MEASUREMENTS: Cases of cancer documented by chart review and telephone follow-up. RESULTS: 26 cases of noncutaneous cancer developed in 18 of the 34 patients: 10 cases of gastrointestinal cancer and 16 cases of extraintestinal cancer. With the use of SEER (Surveillance, Epidemiology, and End Results) data for comparison, the relative risk for cancer was 18.5 (95% CI, 8.5 to 35.2) in women with the Peutz-Jeghers syndrome and 6.2 (CI, 2.5 to 12.8) in men with the syndrome (P = 0.001). In women, the relative risk for breast and gynecologic cancer was 20.3 (CI, 7.4 to 44.2). CONCLUSIONS: The Peutz-Jeghers syndrome is associated with an increased risk for cancer. The relative risk for breast and gynecologic cancers is particularly high.

Research design and methods of quantitative synthesis of medical evidence.

OBJECTIVE: To review the scientific principles, strengths, and limitations of research designs and methods of quantitative synthesis of medical evidence. DATA SOURCES: We used MEDLINE to perform a systematic search for literature using the keywords research design, epidemiology, and biometry. Journals searched included six major journals in obstetrics and gynecology and three in general medicine. These sources were supplemented with texts and reviews from the general medical literature. METHODS OF STUDY SELECTION: We reviewed the publications identified by our search and evaluated critically the relevant reports. We summarized objectives and scientific guidelines for the common research methodologies and outlined their advantages and disadvantages. TABULATION, INTEGRATION, AND RESULTS: The standard of clinical research design is the randomized controlled trial (RCT), which, if performed with sufficient methodologic rigor, is least likely to have serious biases. Cohort, case-control, and cross-sectional studies are common observational studies used in reproductive health; such observational studies are more susceptible to biases that can distort the researcher's results and conclusions. Descriptive studies such as case series and case reports are often interesting as clinical vignettes but have limited scientific merit. Methods for quantitative synthesis of medical evidence, including meta-analysis, decision analysis, and cost-effectiveness analysis are being used with increased frequency in the reproductive health literature to summarize medical evidence. CONCLUSION: Various research methods have their own inherent advantages and disadvantages. An understanding of the scientific principles of these methods will enable the clinician to evaluate medical evidence critically.

Endovaginal sonography for the diagnosis of upper genital tract infection.

OBJECTIVE: To determine the clinical utility of transvaginal sonography for the diagnosis of upper genital tract infection. METHODS: Fifty-five women who either met the Centers for Disease Control and Prevention's minimal criteria for acute pelvic inflammatory disease or were being seen for non-classic signs of upper genital tract infection were evaluated. During abdominal and endovaginal ultrasound testing, fluid in the cul-de-sac, discrete tubes with or without tubal fluid, multicystic ovaries, and adnexal masses were noted. Upper genital tract infection was confirmed by laparoscopic visualization or histologic or microbiologic evidence of salpingitis of endometritis. RESULTS: The specificity of identifying fallopian tubes with or without intraluminal fluid on ultrasound was 97% (35 of 36); the sensitivity, however, was only 32% (six of 19). Calculated using Bayes theorem and based on a prevalence rate of 50%, the positive predictive value of visualizing fallopian tubes was 91%. The sensitivities associated with the visualization of a multicystic ovary or tubo-ovarian abscess were 42% (eight of 19) and 32% (six of 19), with specificities of 86% (31 of 36) and 97%, (35 of 36), and positive predictive values of 75% and 91%, respectively. Cul-de-sac fluid was associated with low sensitivity (37%; seven of 19), low specificity (58%; 21 of 36), and the lowest positive predictive value (47%). CONCLUSION: Endovaginal sonography has limited clinical utility in the diagnosis of upper genital tract infection due to its low sensitivity.

Performance of clinical and laparoscopic criteria for the diagnosis of upper genital tract infection.

OBJECTIVE: The purpose of this study was to validate the standard minimal clinical criteria and the laparoscopic triad of tubal edema, erythema, and purulent exudate used to diagnose acute upper genital tract infection. METHODS: Subjects included women who either met the Centers for Disease Control and Prevention's (CDC) minimal criteria for acute pelvic inflammatory disease or had other signs of upper genital tract infection (i.e., atypical pelvic pain, abnormal uterine bleeding, or cervicitis). The subjects were evaluated with a baseline interview comprehensive laboratory testing, and either an endometrial biopsy or laparoscopy with endometrial and fimbrial biopsies for definitive diagnosis of upper genital tract infection. Patients were considered positive for upper genital tract infection if they had any of the following findings: 1) histologic evidence of endometritis or salpingitis; 2) laparoscopic visualization of purulent exudate in the pelvis without another source; or 3) positive testing for Neisseria gonorrhoeae or Chlamydia trachomatis from the endometrium, fallopian tubes, or pelvis. RESULTS: One hundred twenty-nine women with adequate endometrial samples were evaluated between August 1993 and September 1997, and 62 had complete laparoscopic evaluations. The sensitivities of the CDC's minimal clinical criteria for pelvic inflammatory disease and the laparoscopic triad of edema, erythema, and purulent exudate were 65% and 60%, respectively. CONCLUSIONS: Commonly used minimal clinical criteria for pelvic inflammatory disease and the laparoscopic triad of tubal edema, erythema, and purulent exudate have limited sensitivity with correspondingly high false negative rates.