Michael A. Steller

Generation of tumor-specific cytolytic T lymphocytes from peripheral blood of cervical cancer patients by in vitro stimulation with a synthetic human papillomavirus type 16 E7 epitope.

OBJECTIVE Approximately 90% of squamous carcinomas of the cervix harbor the human papillomavirus and type 16 has been detected in nearly 50% of cases. Recent studies in mice have shown that the human papillomavirus type 16 E7 oncoprotein contains peptide epitopes that are processed and presented in association with a major histocompatibility antigen for recognition by cytolytic T lymphocytes. We investigated whether an epitope from human papillomavirus type 16 E7 could be used to generate specific human cytolytic T lymphocytes in patients with cervical carcinoma. STUDY DESIGN After radiation therapy, three patients with antigen HLA-A2 and with locally advanced cervical cancer underwent leukapheresis. Epitope-specific cytolytic T lymphocytes were generated from the peripheral blood mononuclear cells by in vitro stimulation with autologous peripheral blood mononuclear cells pulsed with a human papillomavirus type 16 E7, HLA-A2-restricted, synthetic peptide, E7(11-20) (YMLDLQPETT). RESULTS In two patients cytolytic T lymphocytes were capable of E7(11-20)-specific, HLA-A2-restricted cytolysis of the peptide-pulsed, HLA-matched, T2 target cell line. Cytolytic T lymphocytes from one of these patients also demonstrated specific cytolysis against the HLA-A2+, HPV-16+ CaSki cervical cancer cell line but did not lyse either HLA-A2+, HPV-16- MS-751 cells or HLA-A2-, HPV-16- HT-3 cells. CONCLUSIONS These experiments demonstrate that novel cytolytic T lymphocytes that recognize a human papillomavirus type 16 E7 epitope can be generated by using the peripheral blood mononuclear cells from irradiated patients with cervical cancer. In addition, because CaSki cells were specifically lysed by the cytolytic T lymphocytes, these data indicate that the peptide E7(11-20) is endogenously processed and presented on the cell surface of the CaSki cells. The demonstration of epitope-specific lysis of cytolytic T lymphocytes of HPV-16+ cervical cancer cells supports further efforts to develop human papillomavirus peptide-based vaccines or antigen-specific adoptive immunotherapy for the prevention and treatment of cervical carcinoma.

Sentinel node identification and the ability to detect metastatic tumor to inguinal lymph nodes in squamous cell cancer of the vulva

OBJECTIVES The goal of this study was to identify one or more inguinal sentinel nodes in patients with primary squamous cell carcinoma of the vulva and to determine the ability of the sentinel node to predict metastasis to the inguinal lymphatic basin. METHODS Techniques employing technetium-99m (Tc-99m) sulfur colloid and isosulfan blue dye were utilized to identify sentinel nodes in the inguinal lymphatic beds. Technetium-99m sulfur colloid was injected intradermally at the tumor margins 90-180 min preoperatively followed by a similar injection of isosulfan blue dye 5-10 min before the groin dissection. A handheld collimated gamma counter was employed to identify Tc-99m-labeled sentinel nodes. Lymphatic tracts that had taken up blue dye and their corresponding sentinel node were also identified and retrieved. A completion inguinal dissection was then performed. Each sentinel node was labeled as hot and blue, hot and nonblue, or cold and blue. The sentinel nodes were subjected to pathologic examination with step sections and nonsentinel nodes were evaluated in the standard fashion. RESULTS Twenty-one patients with a median age of 79 were entered onto protocol and a total of 31 inguinal node dissections were performed. A sentinel node was identified in 31/31 (100%) groin dissections with the use of Tc-99m. Isosulfan blue dye identified a sentinel node in 19/31 (61%) groin dissections. Surgical staging revealed 7 patients with stage I disease, 5 with stage II disease, 5 with stage III disease, and 4 with stage IV disease. Lymph nodes in 9 groin dissections were found to have metastatic disease, and in 4 of these dissections, the sentinel node was the only positive node. Lymph nodes in 22 groin dissections had no evidence of metastasis. No false-negative sentinel lymph nodes were obtained (sentinel node negative and a nonsentinel node positive). CONCLUSION Tc-99m sulfur colloid is superior to isosulfan blue dye in the detection of sentinel nodes in inguinal dissections of patients with vulvar cancer. A sentinel node dissection utilizing Tc-99m alone can identify a sentinel node in all inguinal dissections. Pathologic examination with step sections has shown the sentinel node to be an accurate predictor of metastatic disease to the inguinal nodal chain.

A pilot phase I trial of continuous hyperthermic peritoneal perfusion with high-dose carboplatin as primary treatment of patients with small-volume residual ovarian cancer.

Purpose: Because intraperitoneal (i.p.) therapy may provide a therapeutic advantage and because hyperthermia enhances carboplatin (CBDCA) cytotoxicity, we evaluated the feasibility, toxicity, and pharmacokinetics of CBDCA given via continuous hyperthermic peritoneal perfusion (CHPP) in patients with small-volume residual ovarian cancer. Patients and Methods: Six patients underwent optimal cytoreductive procedures (residual disease ≤5 mm) as initial treatment of stages II and III epithelial ovarian adenocarcinoma. All patients received a 90-min CHPP at a CBDCA dose of 800–1200 mg/m2, with the perfusate being recirculated rapidly from a reservoir through a heat exchanger, resulting in i.p. temperatures of 41–43 °C. Plasma, perfusate, and urine samples were collected and platinum was quantified by flameless atomic absorption spectrophotometry. Results: At no time did any patients core temperature exceed 40 °C. Peak perfusate platinum concentrations were 8- to 15-fold higher than peak ultrafilterable plasma concentrations. The permeability-area product was extremely high and variable (14–90 ml/min), resulting in a regional advantage of 1.9–5.3. The percentage of the dose absorbed ranged widely from 27% to 77%. Dose-limiting hematologic toxicity was observed at a dose of 1200 mg/m2 and this was associated with a CBDCA AUC in plasma of 11 mg min ml−1. Conclusions: CHPP with CBDCA was safely given to three patients at a dose of 800 mg/m2, and dose-limiting hematologic toxicities observed at 1200 mg/m2, correlated with the plasma CBDCA exposure established when lower doses of CBDCA are given systemically. The pharmacokinetic data are consistent with the expected effect of vigorous mixing on the exposed peritoneal surface area. Variable drug absorption and clearance make the prediction of systemic exposure highly uncertain. These findings may have important implications for novel therapies given i.p.

The role of cytoreductive surgery in the management of stage IV epithelial ovarian carcinoma

Patients with Stage IV epithelial ovarian carcinoma are generally treated in the same manner as are patients with disease confined to the abdomen--cytoreductive surgery followed by combination chemotherapy. Between 1980 and 1990, 35 women with histologically or cytologically documented Stage IV ovarian carcinoma were treated in this fashion. Sixteen women (45%) underwent optimal initial cytoreductive surgery, defined as less than 2 cm maximum residual disease. Eleven of the 19 women undergoing suboptimal initial procedures underwent interval cytoreduction after two to four cycles of chemotherapy, with 7 achieving an optimal status after the interval procedure. Overall, 23 of 35 patients (66%) were successfully cytoreduced to less than 2 cm either initially or at an interval procedure. Thirty-one of the 35 patients received combination regimens containing platinum as part of their initial therapy. Kaplan-Meier survival curves demonstrated no significant difference in survival between those groups of women cytoreduced intervally or initially, or between those groups of women optimally cytoreduced at some point during their initial therapy and those who were not. The 5-year survival for the entire group was less than 5%, with no significantly prolonged survival seen in those patients undergoing successful cytoreduction.

Human papillomavirus vaccines for cervical cancer.

Cervical cancer is one of the most common causes of cancer-related death in women. As a result of several recent advances in molecular biology, the association between human papillomavirus (HPV) infection and cervical cancer has been firmly established, and the oncogenic potential of certain HPV types has been clearly demonstrated. Several lines of evidence suggest the importance of the hosts immune response, especially cellular immune response, in the pathogenesis of HPV-associated cervical lesions. These observations form a compelling rationale for the development of vaccine therapy to combat HPV infection. Both prophylactic and therapeutic HPV vaccine strategies are being developed. Prophylactic strategies currently under investigation focus on the induction of effective humoral immune responses against subsequent HPV infection. In this respect, impressive immunoprophylactic effects have been demonstrated in animals using papillomavirus-like particles (VLPs). VLPs are antigenic and protective, but are devoid of any viral DNA that may be carcinogenic to the host. For treatment of existing HPV infection, techniques to improve cellular immunity by enhancing viral antigen recognition are being studied. For this purpose, the oncogenic proteins E6 and E7 of HPV-16 and -18 are the focus of current clinical trials for cervical cancer patients. The development of successful HPV-specific vaccines may offer an attractive alternative to existing screening and treatment programs for cervical cancer.

Cervical Cancer Vaccines: Progress and Prospects

Cervical cancer remains a leading cause of cancer-related mortality in women, particularly in developing countries. The causal association between genital human papillomavirus (HPV) infection and cervical cancer has been firmly established and the oncogenic potential of certain HPV types has been clearly demonstrated. In recognition of the causal association of cervical cancer with this sexually transmitted viral infection, substantial interest has arisen to develop effective prophylactic and therapeutic vaccines. Prophylactic strategies currently under investigation focus on the induction of effective humoral and cellular immune responses that are potentially protective against subsequent HPV infection. Papillomavirus-like particles have been synthesized to induce neutralizing antibody responses, and impressive immunoprophylactic effects have been demonstrated in both animals and humans. For the treatment of existing HPV infection, techniques to augment cellular immunity by enhancing viral antigen recognition are under investigation, Vaccines targeting the oncogenic proteins E6 and E7 of HPV-16 and -18 are the focus of current clinical trials for cervical cancer patients. It is hoped that the development of successful HPV-specific vaccines will diminish the costs of existing cervical cancer screening programs and reduce the morbidity and mortality associated with the treatment of cervical neoplasias.

Insulin-like growth factor-II participates in the biphasic effect of a gonadotropin-releasing hormone agonist on ovarian cancer cell growth

OBJECTIVE To examine the involvement of insulin-like growth factors (IGFs) in growth regulation of an ovarian cancer cell line and to investigate whether the GnRH agonist tryptorelin might influence a potential autocrine or paracrine loop involving IGFs. DESIGN In vitro, prospective, randomized controlled study. SETTING In vitro experiments at the Section of Gynecologic Oncology, Surgery Branch, National Cancer Institute. PATIENT(S) None. Human ovarian adenocarcinoma cell line IGROV-1. INTERVENTION(S) The proliferative effect of tryptorelin on IGROV-1 cells was analyzed by using the MTT (93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) colorimetric assay. The ribonuclease protection assay was used to investigate whether an autocrine pathway involving IGF-I or IGF-II might participate in the growth of these cells. The expression of GnRH receptor was assessed by the 125I-GnRH binding assay. MAIN OUTCOME MEASURE(S) Changes in cell growth and expression of IGF-I and IGF-II messenger RNA (mRNA). RESULT(S) Tryptorelin exhibited a bimodal, dose- and time-dependent effect on IGROV-1 cells when compared with untreated control cells: Cellular proliferation was enhanced during the first 24 hours of exposure, but longer incubations resulted in growth inhibition. The mitogenic effect of tryptorelin was inhibited when cells were co-incubated with either IGF binding protein-5 (IGFBP-5) or anti-IGF-II antibody, which can both bind to IGF-II and neutralize it. Insulin-like growth factor-I mRNA was not detected in IGROV-1 cells. However, IGF-II transcripts were detected after incubation with tryptorelin for 12 hours, but thereafter, no mRNA was observed, even after prolonged exposure. Binding analysis revealed a specific, high-affinity GnRH binding site. CONCLUSION(S) These data suggest that tryptorelin exerts a bimodal growth effect on ovarian cancer cells by a mechanism involving the autocrine production of IGF-II. The effect of tryptorelin on IGF-II gene transcription in these ovarian cancer cells appears to mirror the desensitizing effects of prolonged GnRH exposure on pituitary gonadotropin production.

Human Papillomavirus (HPV) Detection Using In Situ Hybridization in Histologic Samples Correlations With Cytologic Changes and Polymerase Chain Reaction HPV Detection

Although in situ hybridization (ISH) and polymerase chain reaction (PCR) have extensively been used on cytology specimens, there have been limited reports of the usefulness of these techniques in relation to confirmed histologic findings. In this study, we used PCR and ISH to detect human papillomavirus (HPV) in cytologic and histologic specimens, respectively. By using positive and negative likelihood ratios, we attempted to identify any predictive role of ISH testing alone or in combination with PCR for the development of high-grade histologic lesions (cervical intraepithelial neoplasia [CIN] 2+). In our study, ISH was a useful method for detection of HPV, even in a large fraction of samples with normal cytologic or biopsy findings. We suggest that when used together and evaluated in conjunction with histologic sections, ISH is a useful tool for ancillary molecular testing of HPV infection in cervical lesions, especially in CIN 2+ histological lesions where its analytic sensitivities and specificities were as good as those of PCR testing.

Videoconferencing for gynaecological cancer care: an international tumour board.

Sharing expertise between health-care staff is particularly important in the care of cancer patients, for whom treatment, even at its best, is not always effective, readily obvious or available. All relevant therapeutic options should be considered before deciding on cancer management. Self-evident though this may be, it is not always easy to do in practice, as it requires substantial collaboration1–4. Using videoconferencing, large, geographically dispersed groups can be convened and can function in realtime5–10. Case-specific discussions can occur with colleagues over great distances. In October 1998, we created an international tumour board (ITB) to improve the care of gynaecological cancer patients and to exchange medical knowledge between multidisciplinary groups in Providence, Rhode Island, USA, and in Safed, Israel. The ITB consists of doctors trained in cancer surgery, chemotherapy, radiation oncology, diagnostic radiology and tumour pathology, as well as nurses, nutritionists and oncology social workers. The prospective, focused, multidisciplinary process brings special insight and balance to the formulation of treatment recommendations. The establishment of such boards is difficult even in the largest medical institutions. ITB equipment

Interferon-γ receptors on human gestational choriocarcinoma cell lines: Quantitative and functional studies†

Objectives: This study was performed to further define the effects of interferon-γ on choriocarcinoma cell lines and to determine whether variations in response among cell lines are attributable to quantitative differences in interferon-γ receptors. Study design: Interferon-γ receptors were quantified on BeWo, JEG-3 and Jar choriocarcinoma cell lines by a radiolabeled interferon-γ ligand binding assay. The response of these cell lines to interferon-γ was measured in two functional assays: a cell proliferation assay and a cell lysis assay after exposure to interferon-γ with and without actinomycin-D. Results: The number of interferon-γ receptors on BeWo, Jar, and JEG-3 cells did not differ significantly (650, 560, and 420 interferon-γ receptors per cell, respectively). Proliferation of all three choriocarcinoma cell lines was significantly inhibited to a similar extent by interferon-γ. After treatment with interferon-γ actinomycin-D, each choriocarcinoma cell line exhibited dose-dependent cell lysis; lysis of Jar was significantly less than that of either BeWo or JEG-3. Conclusion: These data further document variations in the response of choriocarcinoma cell lines to interferon-γ and indicate that these differences are not the result of interferon-γ receptor number but of postreceptor mechanisms.