FFPEcap-seq: a method for sequencing capped RNAs in formalin-fixed paraffin-embedded samples.

The majority of clinical cancer specimens are preserved as formalin-fixed paraffin-embedded (FFPE) samples. For clinical molecular tests to have wide-reaching impact, they must be applicable to FFPE material. Accurate quantitative measurements of RNA derived from FFPE specimens is challenging because of low yields and high amounts of degradation. Here, we present FFPEcap-seq, a method specifically designed for sequencing capped 5' ends of RNA derived from FFPE samples. FFPEcap-seq combines enzymatic enrichment of 5' capped RNAs with template switching to create sequencing libraries. We find that FFPEcap-seq can faithfully capture mRNA expression levels in FFPE specimens while also detecting enhancer RNAs that arise from distal regulatory regions. FFPEcap-seq is a fast and straightforward method for making high-quality 5' end RNA-seq libraries from FFPE-derived RNA.

Recurrent early stage endometrial cancer: Patterns of recurrence and results of salvage therapy.

OBJECTIVE: To analyze our institutional experience and oncologic outcomes for salvage treatment for the recurrence of early-stage endometrial cancer patients. METHODS: We included women of all ages diagnosed with FIGO stage I-II, any grade endometrial cancer from 2000 to 2016 at our institutions who were treated with at least a hysterectomy. Recurrences in the pelvis and/or vagina were considered locoregional recurrences (LRR). Overall survival (OS) was assessed using Kaplan-Meier survival analysis. Univariate (UV) and multivariate (MV) Cox proportional hazards modeling was also used. RESULTS: A total of 2691 women were analyzed. The majority had endometrioid histology (91%), stage IA disease (61%), and were grade 1 (57%). With a median follow-up of 6.1 years, the overall rate of recurrence was 7.2%, and the rate of LRR was 3.7%. Women with vaginal-only recurrences had a longer median OS after recurrence (14.0 years) compared to both pelvic (1.2 years) and distant (1.0 year) failures. For women with vaginal-only recurrences, salvage radiotherapy (RT) was the only factor associated with improved OS on MVA (HR 0.1, p = .04). For women with pelvic recurrences, salvage surgery (HR 0.3, p = .01), salvage RT (HR 0.3, p < .01), and salvage chemotherapy (HR 0.4, p = .03) were associated with improved OS. CONCLUSIONS: Failure rates for women with early-stage endometrial cancer are low. Women with vaginal-only recurrences have improved OS compared to pelvic or distant recurrences. Salvage RT appears to be an important factor for treatment of women with vaginal-only recurrences. Aggressive multimodality treatment may be beneficial for women with pelvic recurrences.

Hyperglycosylated hCG and Placenta Accreta Spectrum.

OBJECTIVE: We aimed to evaluate the relationship between hyperglycosylated human chorionic gonadotropin (hCG-H) and placenta accreta spectrum (PAS) in the second and third trimesters of pregnancy. STUDY DESIGN: This was a case-control study of PAS and controls. hCG-H was measured in the second and third trimesters of pregnancy in women with pathologically confirmed cases of PAS and in gestational age-matched controls without PAS. We compared serum hCG-H levels in cases and controls, calculated summary statistics for diagnostic accuracy, and used receiver operating characteristic (ROC) curves to define an optimal cut-point for diagnosis of PAS using hCG-H. RESULTS: Thirty case samples and 30 control samples were evaluated for hCG-H. Mean hCG-H was lower in the case compared with control group (7.8 ± 5.9 µg/L vs. 11.8 ± 8.8 µg/L, p = 0.03). At an optimal cut-point for hCG-H of ≤7.6 µg/L, the sensitivity, specificity, positive likelihood ratios, negative likelihood ratios, and area under the ROC curve were 66.7%, 69.7%, 2.20%, 0.48%, and 0.68%, respectively. CONCLUSION: Hyperglycosylated hCG levels in the second and third trimesters of pregnancy were lower in patients with PAS than in controls, but hCG-H showed only modest capability as a diagnostic test for PAS.

Familial clustering of endometrial cancer in a well-defined population.

OBJECTIVE: Using a genealogical database, we examined risk of endometrial cancer among family members of individuals with endometrial cancer. METHODS: We identified endometrial cancer cases in the Utah Population Database (UPDB), a computerized archive of genealogy data linked to the Utah Cancer Registry. We tested for excess relatedness and estimated relative risks (RR) among first-, second-, and third-degree relatives of endometrial cancer cases and stratified analyses by tumor histology and body mass index (BMI). RESULTS: We identified 3911 cases; 3546 were Type I cancers and 365 Type II cancers. The RR for all endometrial cancer cases and for cases with type I histology was significantly increased for first-, second-, and third-degree relatives. An almost three-fold risk was observed among first-degree relatives of individuals with Type I cancers and a 2.24-fold risk among second-degree relatives of Type I morbidly obese cases. The magnitude of endometrial cancer risk among relatives appeared to increase with case BMI. CONCLUSIONS: The elevated risks for endometrial cancer among first-, second-, and third-degree relatives support a genetic contribution to predisposition to endometrial cancer. The increased risk appears to be limited to Type I endometrial cancer. We observed increased risks for endometrial cancer among relatives of obese and morbidly obese Type I cases, which may be indicative of a synergistic relationship between underlying genetic propensity and shared environment. This study quantifies risk of developing cancer among relatives of patients with disease and provides the basis for further analysis of high risk pedigrees and gene identification for genetic etiologies of endometrial cancer.

Do vaginal recurrence rates differ among adjuvant vaginal brachytherapy regimens in early-stage endometrial cancer?

PURPOSE: We sought to retrospectively examine clinical outcomes for three adjuvant vaginal high-dose-rate (HDR) brachytherapy regimens after hysterectomy for early-stage endometrial cancer. METHODS: Included were women of all ages from two independent hospital systems diagnosed with Stage I-II endometrial cancer of any grade between 2000 and 2016 who underwent hysterectomy followed by adjuvant vaginal cylinder HDR brachytherapy with either 7.0 Gy × 3 fractions prescribed to 0.5 cm vaginal depth, 6.5 Gy × 3 fractions prescribed to 0.5 cm vaginal depth, or 6.0 Gy × 5 fractions prescribed to the vaginal surface. Outcomes included vaginal recurrence (VR), pelvic recurrence, distant recurrence, locoregional recurrence, recurrence-free survival, and overall survival. RESULTS: Of the 348 women, 45 (13%) received 7.0 Gy × 3 fractions, 259 (74%) received 6.5 Gy × 3 fractions, and 44 (13%) received 6.0 Gy × 5 fractions. Women receiving 5-fraction brachytherapy were more likely to be younger with a higher performance status. At a median follow-up of 4.5 years, VR rates were 2.2%, 0.8%, and 4.5%, respectively. Multivariate analysis revealed no significant differences in the risks for VR among brachytherapy regimens. Risks for VR, pelvic recurrence, distant recurrence, locoregional recurrence, recurrence-free survival, and overall survival did not differ between propensity score-matched five- and 3-fraction brachytherapy cohorts. CONCLUSIONS: VR rates after hysterectomy and adjuvant vaginal brachytherapy for early-stage endometrial cancer were low and not significantly different by HDR dose fractionation.

Radiosensitization of cervical cancer cells via double-strand DNA break repair inhibition.

PURPOSE: LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, has been found to radiosensitize various human cancer cells. However, its potential to act as an effective therapeutic agent is diminished by its toxicity levels. The purposes of this study were to determine the mechanism by which LY294002 radiosensitizes. MATERIALS AND METHODS: Cell growth curves and clonogenic assays were performed with increasing LY294002 exposure times proximate to the radiation dose. Protein levels of downstream PI3K effectors were analyzed. Detection of phosphorylated histone H2AX (gammaH2AX) was used to identify DNA double-strand breaks at various time points post-radiation. RESULTS: LY294002 significantly radiosensitized HeLa cervical cancer cells when administered for just 12 h following radiation. Cell growth curves also decreased with brief LY294002 application. DNA double-strand breaks are typically repaired within 2-6 h following radiation. Interestingly, at 48, 72, and 96 h post-irradiation, gammaH2AX was still significantly elevated in cells radiated in combination with LY294002. Protein expressions of ATM and ATR downstream effectors showed no differences among the treated groups, however, DNA-PK activity was significantly inhibited by LY294002. CONCLUSIONS: These results lead us to conclude that the central mechanism by which LY294002 radiosensitizes is via DNA-PK inhibition which induces DNA double-strand break repair inhibition. We are currently investigating radiosensitization induced by DNA-PK-specific inhibition in efforts to find a less toxic, yet equally effective, chemotherapeutic agent than LY294002.

Self-test device for cytology and HPV testing in rural Appalachian women: an evaluation.

OBJECTIVE: To determine the efficacy of a self-test device for obtaining vaginal/cervical samples for HPV DNA and cytologic testing. STUDY DESIGN: We recruited 878 Appalachian women; 775 were able to submit a self-test sample and a provider-collected sample. Each specimen was assessed for cytologic abnormalities and for high-risk HPV infection. RESULTS: Specimen adequacy: 869 of 878 (99%) women had sufficient material for cytologic analysis in provider-collected samples compared to 771 of 775 (99%) self-collected specimens (p = NS). There was sufficient cellular material (DNA) for HPV testing in 724 of 834 (87%) provider-collected samples compared to 690 of 736 (94%) self-collected specimens (p <0.04). In the provider-collected samples, 654 of 869 (75%) women had endocervical cells compared to 103 of 771 (13%) self-collected specimens (p <0.001). HPV assessment: 142 (19%) had high-risk HPV DNA detected. Of those women, 28 (20%) had HPV infection detected on the provider-collected specimen only, 51 (36%) on the self-collected specimen only, and 63 (44%) had HPV DNA on both specimens. CONCLUSION: Our device is sufficient to obtain an adequate number of epithelial cells for cytologic and HPV testing.

Phosphatidylinositol 3-kinase inhibition by LY294002 radiosensitizes human cervical cancer cell lines.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K) catalytic subunit is amplified in cervical cancers, implicating PI3K in cervical carcinogenesis. We evaluated the radiosensitizing effect of PI3K inhibition by LY294002 on clonogenic survival, growth characteristics, and gene expression in cervical cancer cell lines (HeLa and CaSki). EXPERIMENTAL DESIGN: Cervical cancer cells were treated separately and concurrently with the PI3K inhibitor LY294002 (10 micromol/L) and radiation (2 Gy) with serial analysis of cell count, apoptosis, and flow cytometry. PI3K inhibition was assessed by protein analysis of phosphorylated Akt. Clonogenic assays were done with varying doses of radiation and LY294002 and varied time points of administration of LY294002 proximate to the radiation dose. Surviving fractions and dose modification factors (DMF) were calculated. Each experiment was done in triplicate and analyzed using ANOVA regression analysis and Dunnett's t Test. Microarray gene expression analysis was done on the HeLa cell line. RESULTS: PI3K inhibition with LY294002 alone did not decrease cell survival. However, treatment with LY294002 significantly radiosensitized HeLa and CaSki cell lines with DMFs (1 log cell kill) of 1.95 and 1.37, respectively. Compared with post-irradiation, pretreatment produced more radiosensitization (P < 0.0001). DMFs were 2.2, 2.0, 2.0, and 1.2 for LY294002 added at 6, 2, and 0.5 hours before irradiation and 6 hours after irradiation, respectively. LY294002 pretreatment in irradiated HeLa cells led to altered gene expression. CONCLUSIONS: Although LY294002 alone did not produce cytotoxic effects, PI3K inhibition with LY294002 produced significant radiosensitization, showed significant time-dependent effects, increased apoptosis, and altered gene expression. These findings support future investigation of PI3K inhibitors in combination with radiation therapy for carcinoma of the cervix.

Successful treatment of low-grade endometrial cancer in premenopausal women with an aromatase inhibitor after failure with oral or intrauterine progesterone.

INTRODUCTION: Young women with endometrial intraepithelial hyperplasia or low-grade endometrial carcinoma are potential candidates for conservative fertility sparing therapy utilizing progesterone rather than hysterectomy. High-dose progesterone treatment is associated with 55-80% initial response but high relapse rates. Using aromatase inhibitors in conjunction with high-dose progesterone has largely been unstudied. CASE DESCRIPTIONS: Three obese premenopausal women with endometrial cancer failed to respond to oral or intrauterine progesterone as first line therapy. Due to their desire to continue to pursue fertility sparing treatment options, an aromatase inhibitor was added to their treatment regimen. This resulted in resolution of their malignancy in each case. DISCUSSION: In obese premenopausal women, the mechanism of malignant transformation in endometrial carcinoma is considered to be an association with relatively high levels of serum estrogen from peripheral conversion of androgens to estrone in adipose tissue with a deficiency in progesterone exposure due to chronic anovulation. Using aromatase inhibitors seems reasonable as an adjunct to progesterone given the high likelihood that this population has a significant proportion of their estrogen production coming from peripheral conversion in adipose tissue. This case series is unique in that each woman initially failed to respond to progesterone but had resolution when an aromatase inhibitor was added to their treatment regimen. This would suggest that obese women with low grade malignancy or hyperplasia who have no radiographic evidence of deep myometrial invasion, ovarian or retroperitoneal metastases and who wish to retain their fertility may be treated with intrauterine progesterone and an aromatase inhibitor.

Survival Analysis of Cancer Patients With FIGO Stage IIIA Endometrial Cancer.

OBJECTIVE: Endometrial cancer patients with positive serosa and/or adnexae (FIGO stage IIIA) have a variable prognosis and are at a significant risk for recurrence. We investigated how tumor characteristics and adjuvant treatments influence the overall survival (OS) and recurrence patterns in these patients and patients with positive cytology alone (previously classified as stage IIIA before 2009). MATERIALS AND METHODS: This multi-institution retrospective study reviewed 55 patients with positive serosa and/or adnexae and 18 patients with positive cytology only, surgically staged from 1990 to 2010. The study cohort was evaluated using the Kaplan-Meier estimates of OS and Cox proportional hazards modeling. RESULTS: The 5-year OS for all IIIA patients was 55%. Administration of adjuvant therapy was associated with improved OS when compared with surgery alone (P=0.0018). The 5-year OS was 20% for patients treated with surgery alone (n=10), 55% with surgery and radiation therapy (n=26), 75% with surgery and chemotherapy (n=7), and 79% with surgery followed by both radiation therapy and chemotherapy (n=12; P=0.005). The tumor characteristics showed that nonendometrioid histology (P=0.0143) and lymph vascular space invasion (P=0.0483) had a poorer OS. Recurrence occurred in 29% of IIIA patients, with 9% locoregional failures and 20% distant failures. Patients with positive cytology only had a similar OS to patients with positive serosa and/or adnexae (76% vs. 55%; P=0.104) and recurrence rate (22% vs. 29%; P=0.4101). CONCLUSIONS: This retrospective study suggests benefit from the use of adjuvant radiotherapy and chemotherapy for stage IIIA patients. We recommend further investigation of adjuvant therapies for IIIA patients in prospective studies and randomized clinical trials.

Superiority of electrocautery over the suture method for achieving cervical cone bed hemostasis.

OBJECTIVE: To compare the efficacy of electrocautery with that of the suture method for achieving hemostasis of the cervical cone bed. METHODS: We performed a retrospective chart review of all patients who underwent cold-knife conization of the cervix over a 5-year period. Patients were categorized into two groups: the cautery group, in which the cone bed was electrocauterized with a hand-held electrocoagulation device; and the suture group, in which hemostasis was achieved by a continuous locking suture placed circumferentially around the cone bed. Outcome measures evaluated include estimated blood loss, operative time, and incidence of complications, including secondary hemorrhage, cervicitis, and cervical stenosis. Data were analyzed by Student t test, chi(2) test, linear regression, and multiple logistic regression where appropriate. RESULTS: There were 156 women in the cautery group and 35 in the suture group. The cautery group had significantly lower estimated blood loss (27 mL versus 101 mL; P <.01) and shorter operative time (34 versus 43 minutes; P <.01) than the suture group. The procedure-related complication rate was 6.4% in the cautery group, compared with 14.3% in the suture group (P = nonsignificant). A higher use of lateral sutures, vasopressors, and thrombotic agents was seen in the cautery group. However, even after adjusting for these variables, mean estimated blood loss (33 mL, P <.01) and mean operative time (34 minutes, P <.01) were significantly less in the cautery group than in the suture group. CONCLUSIONS: Cauterization of the cone bed is superior to suture as a method of achieving hemostasis, with significantly less blood loss and shorter operative time.

Cephalic vein cutdown for inserting indwelling subclavian vein catheters in gynecologic oncology patients.

To decide on an alternate procedure for placement of central vein catheters other than percutaneous access of the subclavian vein, we conducted a study of 46 women who underwent placement of a central venous catheter utilizing the cephalic vein procedure at Ruby Memorial Hospital at West Virginia University in Morgantown. Of these patients, 96% had a successful placement of a catheter into the subclavian vein following dissection of the cephalic vein within the deltopectoral groove. There were no cases of pneumothorax or injury to the great vessels during the surgical procedure, which is a significant advantage for women who often have significant pleural effusions.

Survival analysis of endometrial cancer patients with cervical stromal involvement.

OBJECTIVE: Stage II endometrial cancer is relatively uncommon. There is no consensus for appropriate adjuvant therapy in endometrial cancer patients with cervical stromal involvement (International Federation of Gynecology and Obstetrics [FIGO] stage II). This study investigates how adjuvant treatments and tumor characteristics influence overall survival (OS) and disease-free survival (DFS) in stage II patients in order to establish better treatment guidelines. METHODS: This multi-institution, Institutional Review Board approved, study is a retrospective review of 40 endometrial cancer patients with cervical stromal involvement treated from 1993 to 2009. Kaplan-Meier estimates were used to evaluate OS and DFS. RESULTS: OS was 85% at three years and 67% at five years. There were no significant differences in age, histology, depth of invasion, comorbid conditions, surgical staging or recurrence between patients who received radiation therapy (RT) and those who did not. However, patients with FIGO grade 1 cancers were less likely to receive RT (p=0.007). Patients treated with RT had a similar 5 year OS (n=33, 69%) to those treated with surgery only (n=7, 60%, p=0.746). There were no OS differences when evaluating by grade, histology, or depth of invasion between patients who did and did not receive RT. Four patients recurred: three were locoregional failures only, and one failed locally and distant. CONCLUSION: Patients receiving RT had higher grade tumors. Despite this, OS was comparable between the RT and the no RT cohorts. Local failure was the predominant pattern of failure. Endometrial cancer patients with cervical stromal involvement likely receive better locoregional control with the addition of adjuvant RT and we continue to advocate for RT in most cases.

Mucoadhesive hybrid gel improves intraperitoneal platinum delivery.

A leading cause of death and suffering in patients with abdominal or pelvic malignancies is progression of peritoneal surface disease. Changes in the use of chemotherapy have shown significant survival benefits for intraperitoneal or combined intraperitoneal and intravenous treatment following optimal surgical cytoreduction. However, broader clinical use of intraperitoneal therapy has not reached its full potential due to limited efficacy, accessibility and nonspecific toxicity. To overcome these problems, we developed a mucoadhesive hybrid gel (HG) for a local, intraperitoneal drug delivery. In vivo studies confirmed reliable adherence and residence of the gel to the peritoneal sidewall for at least 72 h exhibiting no signs of tissue toxicity. Functionally active CDDP was released from HG within 2h and was equal to free CDDP in vitro. Moreover, intraperitoneal application of HG-CDDP significantly enhanced CDDP accumulation in the genomic DNA of peritoneal tissues compared to the same CDDP dose administered intravenously. These findings indicate the potential application of this hybrid gel as a mucoadhesive drug carrier amendable to use for intraperitoneal drug delivery and possible expansion for use on other mucosal surfaces of the female reproductive tract.

Characterization and evaluation of pre-clinical suitability of a syngeneic orthotopic mouse ovarian cancer model.

BACKGROUND/AIM: To develop and characterize the pre-clinical suitability of a syngeneic mouse epithelial ovarian cancer model in immunocompetent hosts. MATERIALS AND METHODS: ID8 mouse ovarian surface epithelium cells were implanted into the left ovarian bursa of C57BL/6 mice. Using conventional as well as ultrasound-based techniques and histopathological analysis, the tumor weights, volumes, metastases, ascites and vascularity were observed over a period of 16 weeks. RESULTS: Ovarian weights and volume increased 12- and 7-fold, respectively. Ultrasound measurements of ovarian ID8 tumors correlated with the actual size obtained following surgical excision. Ascites and metastasis were first observed at 12 weeks post-orthotopic implantation. Histopathological analysis indicated similarities between orthotopically-generated ovarian tumors and human ovarian tumors. However, there was less evidence of angiogenesis in this animal model. CONCLUSION: The development of this mouse model closely replicates characteristics seen in human ovarian cancer with feasibility of using ultrasound to assess tumor formation, progression and vascularization.

Fertility-sparing management of endometrial adenocarcinoma.

UNLABELLED: Approximately 15% of patients with endometrial cancer are premenopausal. Previous studies largely support the conservative treatment of endometrial cancer in women desiring future fertility. From these studies, 75% to 80% of patients demonstrate a complete response to progestin therapy and the average recurrence rate is 30% to 35%. Conservative therapy should be reserved for women with International Federation of Gynecology and Obstetrics grade I tumors. Before conservative management, patients should be informed of the elevated risk (11%-29%) of concurrent ovarian cancer in cases of premenopausal endometrial cancer, and screening and ongoing surveillance during the treatment period is mandatory. A suggestion of myometrial invasion or metastatic disease is a contraindication to conservative management. Individuals meeting criteria for Lynch syndrome testing should be referred to genetic counseling. Fertility treatment should be individualized, and close surveillance is required during treatment. Staging hysterectomy is recommended after the completion of the childbearing period. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians Learning Objectives: After participating in this activity, physicians should be better able to select appropriate candidates with endometrial cancer for fertility-sparing treatment. Educate patients with endometrial cancer regarding the risks and benefits of standard of care therapy and conservative therapy and screen appropriate patients for Lynch syndrome.

Maternal morbidity in cases of placenta accreta managed by a multidisciplinary care team compared with standard obstetric care.

OBJECTIVE: To compare maternal morbidity in cases of placenta accreta managed by a multidisciplinary care team with similar cases managed by standard obstetric care. METHODS: This was a retrospective cohort study of all cases of placenta accreta identified in the State of Utah from 1996 to 2008. Cases of placenta accreta were identified using International Classification of Diseases (ICD-9) codes for placenta accreta, placenta previa, and cesarean hysterectomy. Maternal morbidity was compared for cases managed by a multidisciplinary care team in two tertiary care centers and similar cases managed at 26 other hospitals using multivariable logistic regression analysis. RESULTS: One-hundred forty-one cases of placenta accreta were identified including 79 managed by a multidisciplinary care team and 62 cases managed by standard obstetric care. Women managed by a multidisciplinary care team were less likely to require large-volume blood transfusion (4 or more units of packed red blood cells) (43% compared with 61%, P=.031) and reoperation within 7 days of delivery for bleeding complications (3% compared with 36%, P<.001) compared with women managed by standard obstetric care. Women with suspected placenta accreta managed by a multidisciplinary team were less likely to experience composite early morbidity (prolonged maternal admission to the intensive care unit, large-volume blood transfusion, coagulopathy, ureteral injury, or early reoperation) than women managed by standard obstetric care (47% compared with 74%, P=.026). The odds ratio of composite early morbidity in women managed by a multidisciplinary team was 0.22, (95% confidence interval, 0.07- 0.70) in the multivariable model. CONCLUSION: Maternal morbidity is reduced in women with placenta accreta who deliver in a tertiary care hospital with a multidisciplinary care team. LEVEL OF EVIDENCE: II

Endometrial cancer--current state of the art therapies and unmet clinical needs: the role of surgery and preoperative radiographic assessment.

Endometrial carcinoma is the fourth most common cancer among women in the United States. Surgical pathologic staging has been the standard of care since 1988, which consists of analysis of collected peritoneal fluid, hysterectomy/oophorectomy, and pelvic and para-aortic lymphadenectomy. In 2005, it was further recommended that essentially all women with endometrial cancer who choose to undergo surgery have pelvic and para-aortic lymph node analysis. Despite this recommendation, there still remains controversy as to whether all patients with endometrial cancer should undergo full lymph node dissection. In this review, we assess the evidence surrounding this controversy and conclude that women with endometrial cancer should undergo complete lymphadenectomy at the time of surgery. Furthermore, we evaluate the evidence regarding laparoscopic surgical staging as a safe and effective alternative to the more invasive traditional laparotomy. Finally, for those patients who a gynecologic oncologist is not readily available to perform a complete lymph node dissection, we evaluate the various imaging studies and their utility as preoperative triage modalities.

C/EBPbeta activity and HPV-16 E6/E7 mRNA expression are not altered by imiquimod (ALDARA) in human cervical cancer cells in vitro.

OBJECTIVE: The purpose of this study was to determine the potential relationship between imiquimod and C/EBPbeta by investigating the extent to which imiquimod could alter C/EBPbeta binding activity to known sequences of the HPV-16 NCR, which could lead to the repression of HPV-16 E6/E7 oncogene expression, possibly impacting a major mechanism by which HPV causes cellular transformation. METHODS: The effect of imiquimod treatment on C/EBPbeta binding activity to its consensus sequence as well as to two specific regions of the HPV-16 NCR was determined by electromobility shift assay (EMSA) in CaSki cervical cancer cells. HPV-16 E6/E7 gene expression was evaluated by RNase protection assay (RPA) in CaSki and in W12-E cells treated with imiquimod. In addition, C/EBPbeta mRNA expression and protein production in response to imiquimod were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively, in the cervical cancer cell lines, CaSki, HeLa, and C33A. RESULTS: C/EBPbeta binding activity, mRNA expression, and protein production remained unchanged with imiquimod treatment. Initially, HPV-16 E6/E7 expression appeared to be increased with imiquimod treatment in CaSki cells, but this effect was not reproducible. HPV-16 E6/E7 expression was not reproducibly altered with imiquimod treatment in W12-E cells. CONCLUSION: While these results indicate that imiquimod does not alter C/EBPbeta binding activity, nor does it appear to decrease HPV-16 E6/E7 oncogene expression in vitro, it remains possible that imiquimod may have utility in treating cervical dysplasia or cervical cancer via another mechanism.