Ira R. Horowitz

Phase II trial of trastuzumab in women with advanced or recurrent, HER2-positive endometrial carcinoma: A Gynecologic Oncology Group study

PURPOSE This study evaluated efficacy of single-agent trastuzumab against advanced or recurrent HER2-positive endometrial carcinoma (EC), and explored predictors for HER2 amplification. PATIENTS AND METHODS Eligible patients had measurable stage III, IV, or recurrent EC. There was no limit on prior therapy although total prior doxorubicin dose was limited to 320 mg/m(2). Tumors were required to have HER2 overexpression (2+ or 3+ immunohistochemical staining) or HER2 amplification (FISH HER2/CEP 17 ratio >2.0). Trastuzumab was administered intravenously at a dose of 4 mg/kg in week 1, then 2 mg/kg weekly until disease progression. The primary endpoint was tumor response. RESULTS Of the 286 tumors centrally screened by LabCorp, 33 (11.5%) were HER2-amplified. Three of 8 clear (38%) cell carcinomas and 7 of 25 serous carcinomas (28%) screened exhibited HER2 amplification compared with 7% (2/29) of endometrioid adenocarcinomas. HER2 overexpression was correlated with HER2 amplification (r=0.459; p<0.0001). Thirty-four women were enrolled; 1 was excluded (refused treatment); and 18 had tumors with known HER2 amplification. No major tumor responses were observed. Twelve women experienced stable disease, 18 had increasing disease, and 3 were indeterminate for tumor response. Neither HER2 overexpression nor HER2 amplification appeared to be associated with progression-free survival or overall survival. CONCLUSION Trastuzumab as a single agent did not demonstrate activity against endometrial carcinomas with HER2 overexpression or HER2 amplification, although full planned accrual of women with HER2 amplified tumors was not achieved due to slow recruitment. Serous and clear cell endometrial carcinomas appear to be more likely to demonstrate HER2 amplification.

Angiogenic role for glycodelin in tumorigenesis

Angiogenesis plays an important role in neovascularization in tumors. Glycodelin, a hormone-responsive protein, has been detected in tumors of reproductive organs and is found in high levels in the plasma of subjects with gynecological malignancies. Glycodelin is also found in the endothelial cells of the umbilical cord and in the blood vessels of tumors. In this study, we tested whether glycodelin-rich amniotic fluid and a synthetic peptide derived from the sequence of glycodelin peptide (Gp) might promote angiogenic response by examining the migration and tube formation in human umbilical cord vein endothelial cells (HUVECs). Increased migration and tube formation of HUVECs were found in the presence of amniotic fluid and Gp, and this increase was blocked by antibody to Gp and by an anti-vascular endothelial growth factor (VEGF) antibody, suggesting that the angiogenic effects of glycodelin might be mediated by VEGF. The results also showed that Gp significantly increased the release of VEGF protein and mRNA expression in HUVECs, RL-95 (human endometrial carcinoma cells), OVCAR-3 (human ovarian adenocarcinoma cells), EM42 (human endometrial epithelial cells), THP-1 (human monocyte), and MCF-7 and MDA-MB-231 (human breast adenocarcinoma cells) cell lines. VEGF receptor Fit-1 mRNA expression in HUVECs was also increased in the presence of Gp. These findings, together with the suggestion from the literature that glycodelin may have immunosuppressive properties, suggest that glycodelin might play an important role in neovascularization during embryogenesis and tumor development.

Epidemiologic and viral factors associated with cervical neoplasia in HPV-16-positive women

While infection with high‐risk HPV is the most important risk factor for cervical cancer, HPV alone is insufficient. Our purpose was to identify viral and epidemiologic factors associated with cervical disease in HPV‐16 DNA‐positive women referred to colposcopy. We used a standardized interview to collect epidemiologic data from consenting women. Total nucleic acids from exfoliated cervical cells were used for all viral assays (HPV detection and typing using L1 consensus PCR with line probe hybridization, variant classification by sequencing, viral load and transcript copy determination by quantitative PCR and transcript pattern by nested RT‐PCR). Cervical disease was based on colposcopic biopsy. Logistic regression was used to calculate ORs with 95% CIs. There were 115 HPV‐16 positive women among 839 enrollees. By univariate analyses, age >25 years (OR = 3.05, 95% CI 1.20–7.76), smoking (OR = 3.0, 95% CI 1.19–7.56), high viral load (OR = 5.27, 95% CI 2.05–13.60), detection of both E6 and E6*I transcripts (OR = 10.0, 95% CI 2.1–47.58) and high transcript copies (OR = 5.56, 95% CI 2.05–13.60) were significant risk factors for CIN III with reference to No CIN/CIN I. Less than a third of the women (31.5%) had prototype HPV‐16 detected, and variants showed no association with disease, viral load or transcription. Viral DNA and transcript copies were highly correlated, and the ratio of transcript copies to DNA copies was not changed with disease status. While viral load, transcript copies and transcript pattern were statistically associated with CIN III, none of these measures effectively discriminated between HPV‐16 women with disease requiring treatment and those who could be followed. Cellular proliferation and differentiation pathways affected by HPV should be investigated as biomarkers for cervical cancer screening.

Phase I Trial of Escalating Doses of Paclitaxel Combined With Fixed Doses of Cisplatin and Doxorubicin in Advanced Endometrial Cancer and Other Gynecologic Malignancies: A Gynecologic Oncology Group Study

PURPOSE The primary objective of this phase I trial was to determine the feasibility of administering a combination of paclitaxel, cisplatin, and doxorubicin with or without granulocyte colony-stimulating factor (G-CSF) in patients with advanced endometrial and other gynecologic cancers. PATIENTS AND METHODS Patients were chemotherapy-naive. Doxorubicin was administered as a brief infusion, paclitaxel for 3 hours, and cisplatin for 60 minutes. Treatments were repeated every 3 weeks. For most dose levels, the cisplatin and doxorubicin were fixed at 60 mg/m(2) and 45 mg/m(2), whereas the paclitaxel was escalated in successive cohorts from 90 to 250 mg/m(2). Patients who had received previous radiotherapy to the whole pelvis were escalated separately from those who had not. RESULTS Eighty patients received 320 cycles of therapy. When G-CSF was not used, myelosuppression prevented escalation beyond the starting dose for patients with or without previous pelvic radiotherapy. When G-CSF was added, neurotoxicity became dose-limiting for both groups. Ten patients were removed from the study for asymptomatic declines in ejection fraction, but no symptomatic congestive heart failure was observed. Major antitumor responses occurred in 46% of patients (six of 13) with measurable endometrial carcinoma and 50% of patients (eight of 16) with measurable cervical carcinoma. CONCLUSION The combination of paclitaxel, doxorubicin, and cisplatin at relevant single-agent doses is active and feasible with the addition of G-CSF. A regimen of cisplatin 60 mg/m(2), doxorubicin 45 mg/m(2), and paclitaxel 160 mg/m(2) with G-CSF support is recommended for further testing.

Enhanced Protein Profiling Arrays with ELISA-Based Amplification for High-Throughput Molecular Changes of Tumor Patients’ Plasma

Purpose: The purpose of this study is to develop a high-throughput approach to detect protein expression from hundreds and thousands of samples and to apply this technology to profile circulating angiogenic factor protein levels in patients with gynecological tumors. Experimental Design: Analytes containing a mixture of protein are immobilized onto antibody-coated surface of support in array format. The presence of protein in analytes is detected with biotin-labeled antibody coupled with an enhanced chemiluminescence or fluorescence detection system. The exact amount of protein can be quantitatively measured. The expression levels of five angiogenic factors (angiogenin, interleukin 8, vascular endothelial growth factor, platelet-derived growth factor, and epidermal growth factor) from 157 samples were quantitatively measured using this novel protein array technology and were statistically analyzed. The expression patterns of angiogenic factors were analyzed using two-way hierarchical cluster analysis approach. Results: A novel protein array technology, which can simultaneously and quantitatively measure few protein levels from hundreds and thousands of samples was developed. Only minute amounts of sample are required for the assay. This approach also features high sensitivity and specificity. Using this novel protein array approach, we analyzed the plasma expression levels of five angiogenic factors in 137 patients diagnosed with a tumor and 20 controls. Statistical analysis reveals different expression levels of angiogenic factors between patients and controls. Cluster analysis suggests a possible classification of normal subjects from patients. Conclusions: Enhanced protein profiling arrays provide a high-throughput and sensitive system to detect one or few protein from hundreds and thousands of samples. Such an approach should have broad application in biomedical discovery.

Large prospective study of ovarian cancer screening in high-risk women: CA125 cut-point defined by menopausal status

Previous screening trials for early detection of ovarian cancer in postmenopausal women have used the standard CA125 cut-point of 35 U/mL, the 98th percentile in this population yielding a 2% false positive rate, whereas the same cut-point in trials of premenopausal women results in substantially higher false positive rates. We investigated demographic and clinical factors predicting CA125 distributions, including 98th percentiles, in a large population of high-risk women participating in two ovarian cancer screening studies with common eligibility criteria and screening protocols. Baseline CA125 values and clinical and demographic data from 3,692 women participating in screening studies conducted by the National Cancer Institute–sponsored Cancer Genetics Network and Gynecologic Oncology Group were combined for this preplanned analysis. Because of the large effect of menopausal status on CA125 levels, statistical analyses were conducted separately in pre- and postmenopausal subjects to determine the impact of other baseline factors on predicted CA125 cut-points on the basis of 98th percentile. The primary clinical factor affecting CA125 cut-points was menopausal status, with premenopausal women having a significantly higher cut-point of 50 U/mL, while in postmenopausal subjects the standard cut-point of 35 U/mL was recapitulated. In premenopausal women, current oral contraceptive (OC) users had a cut-point of 40 U/mL. To achieve a 2% false positive rate in ovarian cancer screening trials and in high-risk women choosing to be screened, the cut-point for initial CA125 testing should be personalized primarily for menopausal status (50 for premenopausal women, 40 for premenopausal on OC, and 35 for postmenopausal women). Cancer Prev Res; 4(9); 1401–8. ©2011 AACR.

Granular cell tumors of the vulva

Granular cell tumor (GCT), although nearly ubiquitous, is seen infrequently in the vulva. A review of the surgical pathology files from Grady Memorial Hospital, Atlanta, Georgia, from 1983 through 1987 identified eight cases of vulvar GCT. Five of the eight patients had more than one skin and soft tissue lesion. Two of the five had biopsy-proven multicentric GCT with a unique clinical course. One of the patients was a 32-year-old woman with multiple vulvar, lingual, laryngeal, bronchial and pulmonary GCT, necessitating multiple excisions and ultimately pneumonectomy. The second patient had multiple GCTs in the vulva and inguinal area and finally in both lungs, resulting in her death at age 39. No dependable microscopic features could be identified to distinguish benign GCT from its more aggressive variant. However, Feulgen DNA histomorphometry demonstrated aneuploidy in the patient with apparent lung metastases, whereas the tumors from patients with a benign course as well as from the patient with multiorgan involvement were diploid. In three of five patients who could be interviewed there was a history of soft tissue tumors in members of the family. The multifocal nature and possible familial component of GCT need to be explored further.

Proteomics-based approach to elucidate the mechanism of antitumor effect of curcumin in cervical cancer.

Cervical cancer is the second leading cause of cancer death for women in the world. A potential target for preventing and treating cervical cancer is cyclooxygenase-2 (cox-2). Curcumin is an anti-inflammatory agent that is known to have anti-cox-2 activity. In this study we examined the expression of cox-2 in cervical cancer and its precursors by immunohistochemistry. The effect of curcumin in inhibiting cervical cancer cells was determined via 2-dimensional gel electrophoresis, data analysis, and ingenuity pathway analysis. No significant differences in the expression of cox-2 in squamous cell carcinoma, and carcinoma in situ were observed. However, there was a statistically significant difference in the expression of cox-2 in adenocarcinoma in comparison to normal (p value=0.01) and squamous cell carcinoma (p value=0.02) tissues. Proteins associated with cancer and cell cycle were significantly altered in cultured cells. Curcumin may have antitumor effect in cervical cancer.

The promise of cytokine antibody arrays in the drug discovery process

The introduction of cytokine antibody arrays has added a new approach for investigators to simultaneously measure multiple cytokine levels in biological samples. Several different platforms have been developed. The ability to measure hundreds of cytokine levels with high specificity and sensitivity within a very limited amount of samples is a powerful tool. Many investigators worldwide have applied this novel technology in their biomedical research, particularly in drug discovery. Undoubtedly, the technology will continue to be improved and the application increased in the next several years.

Multiple Anticancer Activities of EF24, a Novel Curcumin Analog, on Human Ovarian Carcinoma Cells

Curcumin, a component of turmeric, has been reported to exhibit potential antitumor activities. This study assessed the effects of a novel synthetic curcumin analog, EF24, on proliferation, apoptosis, and vascular endothelial growth factor (VEGF) regulation in platinum-sensitive (IGROV1) and platinum-resistant (SK-OV-3) human ovarian cancer cells. EF24 time- and dose-dependently suppressed the growth of both cell lines and synergized with cisplatin to induce apoptosis. Although treatment with EF24 had no significant effect on VEGF messenger RNA (mRNA) expression,VEGF protein secretion into conditioned media was dose-dependently reduced with EF24 demonstrating ∼8-fold greater potency than curcumin (P < .05). EF24 significantly inhibited hydrogen peroxide (H2O2)-induced VEGF expression, as did the phenolic antioxidant tert-butylhydroquinone (t-BHQ). EF24 upregulated cellular antioxidant responses as observed by the suppression of reactive oxygen species (ROS) generation and activation of antioxidant response element (ARE)-dependent gene transcription. Given its high potency, EF24 is an excellent lead candidate for further development as an adjuvant therapeutic agent in preclinical models of ovarian cancer.