Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of Clostridium perfringens enterotoxin.

BACKGROUND: Emerging evidence has suggested that the capability to sustain tumor formation, growth, and chemotherapy resistance in ovarian as well as other human malignancies exclusively resides in a small proportion of tumor cells termed cancer stem cells. During the characterization of CD44(+) ovarian cancer stem cells, we found a high expression of the genes encoding for claudin-4. Because this tight junction protein is the natural high-affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian cancer stem cells to CPE treatment in vitro and in vivo. METHODS: Real-time polymerase chain reaction and flow cytometry were used to evaluate claudin-3/-4 expression in ovarian cancer stem cells. Small interfering RNA knockdown experiments and MTS assays were used to evaluate CPE-induced cytotoxicity against ovarian cancer stem cell lines in vitro. C.B-17/SCID mice harboring ovarian cancer stem cell xenografts were used to evaluate CPE therapeutic activity in vivo. RESULTS: CD44(+) ovarian cancer stem cells expressed claudin-4 gene at significantly higher levels than matched autologous CD44(-) ovarian cancer cells, and regardless of their higher resistance to chemotherapeutic agents died within 1 hour after exposure to 1.0 µg/mL of CPE in vitro. Conversely, small-interfering RNA-mediated knockdown of claudin-3/-4 expression in CD44(+) cancer stem cells significantly protected cancer stem cells from CPE-induced cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy-resistant CD44(+) ovarian cancer stem cells had a significant inhibitory effect on tumor progression leading to the cure and/or long-term survival of all treated animals (ie, 100% reduction in tumor burden in 50% of treated mice; P < .0001). CONCLUSIONS: CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy-resistant cancer stem cells.

Clostridium perfringens enterotoxin carboxy-terminal fragment is a novel tumor-homing peptide for human ovarian cancer.

BACKGROUND: Development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4. METHODS: Because claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, in this study we evaluated the in vitro and in vivo bioactivity of the carboxy-terminal fragment of CPE (i.e., CPE290-319 binding peptide) as a carrier for tumor imaging agents and intracellular delivery of therapeutic drugs. Claudin-3 and -4 expression was examined with rt-PCR and flow cytometry in multiple primary ovarian carcinoma cell lines. Cell binding assays were used to assess the accuracy and specificity of the CPE peptide in vitro against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy and biodistribution assays were performed to evaluate the localization and uptake of the FITC-conjugated CPE peptide in established tumor tissue. RESULTS: Using a FITC-conjugated CPE peptide we show specific in vitro and in vivo binding to multiple primary chemotherapy resistant ovarian cancer cell lines. Bio-distribution studies in SCID mice harboring clinically relevant animal models of chemotherapy resistant ovarian carcinoma showed higher uptake of the peptide in tumor cells than in normal organs. Imunofluorescence was detectable within discrete accumulations (i.e., tumor spheroids) or even single chemotherapy resistant ovarian cancer cells floating in the ascites of xenografted animals while a time-dependent internalization of the FITC-conjugated CPE peptide was consistently noted in chemotherapy-resistant ovarian tumor cells by confocal microscopy. CONCLUSIONS: Based on the high levels of claudin-3 and -4 expression in chemotherapy-resistant ovarian cancer and other highly aggressive human epithelial tumors including breast, prostate and pancreatic cancers, CPE peptide holds promise as a lead peptide for the development of new diagnostic tracers or alternative anticancer agents.

Higher sensitivity to patupilone versus paclitaxel chemotherapy in primary uterine serous papillary carcinoma cell lines with high versus low HER-2/neu expression in vitro.

OBJECTIVE: To compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine serous papillary carcinoma (USPC) with high versus low HER-2/neu expression. METHODS: Six primary USPC cell lines, half of which overexpress HER-2/neu at a 3+ level, were evaluated for growth rate and tested for their in vitro sensitivity/resistance to patupilone versus paclitaxel by MTS assays. Quantitative RT-PCR was used to identify potential mechanisms underlying the differential sensitivity/resistance to patupilone versus paclitaxel in primary USPC cell lines. RESULTS: Cell lines overexpressing HER-2/neu showed higher proliferation when compared to low HER-2/neu-expressing cell lines. Compared to low-expressing cell lines, high HER-2/neu expressors were significantly more sensitive to patupilone than to paclitaxel (P<0.0002). In contrast, there was no appreciable difference in sensitivity to patupilone versus paclitaxel in primary USPC cell lines with low HER-2/neu expression. Higher levels of ß-tubulin III (TUBB3) and P-glycoprotein (ABCB1) were detected in USPC cell lines with high versus low HER-2/neu expression (P<0.05). CONCLUSIONS: USPC overexpressing HER-2/neu display greater in vitro sensitivity to patupilone and higher levels of the patupilone molecular target TUBB3 when compared to low HER-2/neu expressors. Due to the adverse prognosis associated with HER-2/neu overexpression in USPC patients, patupilone may represent a promising novel drug to combine to platinum compounds in this subset of aggressive endometrial tumors.

High-grade, chemotherapy-resistant ovarian carcinomas overexpress epithelial cell adhesion molecule (EpCAM) and are highly sensitive to immunotherapy with MT201, a fully human monoclonal anti-EpCAM antibody.

OBJECTIVE: We evaluated the expression of epithelial cell adhesion molecule (EpCAM) and the potential of MT201 (adecatumumab), a human-monoclonal-antibody that targets EpCAM against chemotherapy-resistant ovarian disease. STUDY DESIGN: EpCAM expression was evaluated by real-time polymerase chain reaction and flow cytometry. Sensitivity to MT201 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in 4-hour chromium-release assays. The effect of interleukin-2 on MT201 ADCC was also studied. RESULTS: High messenger RNA expression by real-time polymerase chain reaction and high EpCAM surface expression by flow cytometry was detected in 71% of ovarian cancers (5 of 7 cell lines). Although these cell lines were highly resistant to complement-dependent cytotoxicity and natural killer-dependent cytotoxicity in vitro (range of killing, 0-7%), EpCAM-positive cell lines showed high sensitivity to MT201 ADCC (range of killing, 27-66%). Incubation with interleukin-2 further increased the cytotoxic activity against EpCAM-positive ovarian cancer cell lines. CONCLUSION: MT201 may represent a novel, potentially highly effective treatment option for patients with ovarian carcinoma whose body is harboring disease refractory to chemotherapy.

Differential sensitivity to platinum-based chemotherapy in primary uterine serous papillary carcinoma cell lines with high vs low HER-2/neu expression in vitro.

OBJECTIVE: We sought to identify effective chemotherapy regimens against uterine serous papillary adenocarcinoma (USPC). STUDY DESIGN: Six USPC, half of which overexpress HER-2/neu at 3+ level, were evaluated for growth rate and in vitro sensitivity to 14 single-agent chemotherapies and 5 combinations by ChemoFx (Precision Therapeutics Inc, Pittsburgh, PA). RESULTS: Cell lines overexpressing HER-2/neu showed higher proliferation when compared to low HER-2/neu-expressing cell lines and a lower half maximum inhibitory concentration (IC(50)) when exposed to the majority of single-agent chemotherapies. High HER-2/neu expressors were more sensitive to platinum compounds, manifesting a 5.22-fold decrease in carboplatin-IC(50) (P = .005) and a 5.37-fold decrease in cisplatin-IC(50) (P = .02). When all cell lines were analyzed as a group, chemotherapy agents tested demonstrated lower IC(50) when used in combination than as individual agents. CONCLUSION: USPC overexpressing HER-2/neu display greater in vitro sensitivity to platinum compounds when compared to low HER-2/neu expressors. Higher proliferative capability rather than increased drug resistance may be responsible for the adverse prognosis associated with HER-2/neu overexpression in USPC.

Primary cervical carcinoma cell lines overexpress epithelial cell adhesion molecule (EpCAM) and are highly sensitive to immunotherapy with MT201, a fully human monoclonal anti-EpCAM antibody.

INTRODUCTION: Epithelial cell adhesion molecule (EpCAM) is a surface glycoprotein highly differentially expressed in many epithelial malignancies. The goal of this study was to evaluate the expression of EpCAM and the potential of MT201 (adecatumumab), a human monoclonal antibody targeting EpCAM, against multiple primary cervical carcinoma cell lines. METHODS: Epithelial cell adhesion molecule expression was evaluated by real-time polymerase chain reaction and flow cytometry in a total of 8 primary cervical cancer cell lines. Sensitivity to MT201-mediated cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in standard 4-hour 51Cr release assays. To investigate the effect of interleukin-2 (IL-2) on MT201-mediated ADCC, 4-hour 51Cr release assays were also conducted in the presence of low doses of IL-2. RESULTS: High messenger RNA expression by real-time polymerase chain reaction and high EpCAM surface expression by flow cytometry were detected in 4 (50%) of 8 primary cervical carcinoma cell lines. With no exception, the primary cell lines derived from clinically aggressive tumors showed EpCAM overexpression. Whereas these cell lines were highly resistant to complement-dependent cytotoxicity and natural killer (NK)-dependent cytotoxicity in vitro (range of killing, 4%-19%), EpCAM-positive cell lines showed high sensitivity to MT201-mediated ADCC (range of killing, 23%-59%). Incubation with IL-2 in addition to MT201 significantly increased the cytotoxic activity against EpCAM-positive cervical cancer cell lines (P = 0.007). Addition of human serum also further increased the MT201-mediated killing of EpCAM-positive cell lines (P = 0.03). CONCLUSIONS: Epithelial cell adhesion molecule is highly expressed in primary cervical carcinoma cell lines, and these biologically aggressive tumors are highly sensitive to MT201-mediated cytotoxicity in vitro. MT201 may represent a novel, potentially highly effective treatment option for patients with cervical carcinoma, especially for those with advanced, recurrent, or metastatic disease refractory to standard salvage therapy.

Ovarian preservation and staging in reproductive-age endometrial cancer patients.

OBJECTIVES: The goal of this case review was to evaluate the influence of complete surgical versus clinical staging, including the impact of ovarian preservation, on the outcome of young patients with endometrial carcinoma. METHODS: A retrospective chart review was performed on all patients with endometrial cancer diagnosed at age 45 or younger from 1960 until 2006 who were treated at Yale-New Haven Hospital (YNHH). Clinical, epidemiological and histological data were extracted. Histological slides were reviewed by the gynecologic pathologist. Statistical analysis was performed, and p<0.05 was considered statistically significant. RESULTS: More than half of the patients underwent surgical staging. A bilateral salpingo-oophorectomy (BSO) was part of the surgery in most cases. Less than 5% of patients were diagnosed with recurrence. In patients with grade 1 disease, surgically staged patients had a significantly longer overall survival (p=0.003). Patients who underwent a BSO had a trend towards longer disease-free survival. Stage I disease patients who underwent BSO had significantly longer disease-free survival (p=0.013). CONCLUSIONS: BSO seems to lead to better disease-free survival in young endometrial cancer patients, especially with stage I disease, but not to improved overall survival. In the absence of risk factors, a more conservative approach to surgical staging may be possible in young women with early stage low grade endometrial cancer but BSO should be strongly considered as part of the surgical treatment.

Eclampsia complicated by abdominal compartment syndrome.

A primigravida with eclampsia and hemolytic anemia, elevated liver enzymes, and low platelet count (HELLP syndrome) developed intra-abdominal compartment syndrome requiring a decompressive laparotomy, underlining the importance of including abdominal compartment syndrome in the differential diagnosis in pregnant women.

Lack of apoptotic protease activating factor-1 expression and resistance to hypoxia-induced apoptosis in cervical cancer.

PURPOSE: Clinical observations suggest that intratumoral hypoxia increases the aggressiveness of tumors through clonal selection of cancer cells that have lost their apoptotic potential. The aim of this study, therefore, was to investigate the expression of the proapoptotic protein apoptotic protease activating factor-1 (Apaf-1) in cervical cancers and to analyze its relation to intratumoral hypoxia and apoptosis. Furthermore, the effect of hypoxia and apoptosis on survival was examined. EXPERIMENTAL DESIGN: In 56 patients, intratumoral oxygenation measurements and subsequent needle biopsies were done. The obtained tissue was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays and by immunohistochemistry with an Apaf-1 antibody. RESULTS: Apaf-1 was expressed in 86% of cancers. The median apoptosis rate was 1.0%. There was no correlation between Apaf-1 expression and intratumoral hypoxia. However, Apaf-1 expression was negative in 37.5% of hypoxic cervical cancers (pO(2)

Occult tumor cells in pelvic lymph nodes and parametrial tissue of small-sized FIGO IB1 squamous cell carcinomas of the uterine cervix--results of a pilot study.

Micrometastases (MM) and occult tumor cell deposits (OTCD) in pelvic tissue may cause recurrences, and immunohistochemistry may improve their detection. We used cytokeratine-immunohistochemistry to investigate 263 pelvic lymph nodes and parametrial tissue for MM and OTCD obtained from eight squamous cell carcinomas (maximum tumor size: 2.5 cm). These patients were treated with radical abdominal hysterectomy (Piver type III) with complete tumor resection without receiving any adjuvant therapy. The mean count of resected pelvic lymph nodes was 32.9 (range 24-47). All lymph nodes were completely embedded, and three step sections were performed for routine histopathologic evaluation. Three patients developed pelvic side wall and five central tumor recurrences within a median time of 25.9 (range 8-55) months. On immunohistochemistry, only one case (12.5%) showed OTCD in a venule in the parametrial tissue. In patients with small cervical carcinomas (< 2.5 cm in largest dimension), OTCD can only rarely be detected by immunohistochemistry after careful handling of resected lymph nodes using step sectioning for routine histologic examination and complete processing of parametrial pelvic tissue. Therefore, tumor recurrence in those patients appears to be due to occult residual tumor cells that were not resected during the classical Wertheim-Meigs-procedure or that were disseminated during the surgical procedure and persisted in situ.

HER-2/NEU overexpression in vulvar Paget disease: the Yale experience.

AIMS: To determine the level of HER-2/neu overexpression in vulvar Paget disease (PD) in order to assess the possibility of using HER-2/neu as a target for the treatment of Paget disease. METHODS: A medical record search identified 39 patients with a histologically confirmed diagnosis of vulvar PD between 1986 and 2009. A tissue microarray containing all 39 tumour samples was created, and corresponding sections were stained immunohistochemically with an anti-HER-2/neu-antibody (HercepTest). Staining results were reported on a scale from 0 to 3+. 2+ and 3+ were considered as HER-2/neu overexpression. The HER-2/neu expression was correlated with clinical, pathological and outcome data. RESULTS: Negative staining for HER-2/neu was noticed in four patients (12%), 1+ in 10 patients (30%), 2+ in 11 patients (33%) and 3+ in eight patients (25%), resulting in 19 patients with HER-2/neu overexpression (2+ and 3+, 58%) and 14 patients without HER-2/neu overexpression (0 and 1+, 42%). The proportion of HER-2/neu overexpression was higher in the patients with invasive than with non-invasive PD (71%, n=5/7 invasive PD vs 54%, n=14/26 non-invasive PD). There was no significant correlation between HER-2/neu staining results and clinical, pathological or outcome data. CONCLUSIONS: Surgical treatment of vulvar Paget disease is associated with a high recurrence rate and extensive reconstructive procedures. In this study, over 50% of the patients with vulvar Paget disease overexpress HER-2/neu. Anti-HER-2/neu-antibodies like trastuzumab may therefore be an interesting treatment option for HER-2/neu-positive vulvar Paget disease. Clinical trials are needed to evaluate the efficacy of trastuzumab in this disease.

p16, p14, p53, cyclin D1, and steroid hormone receptor expression and human papillomaviruses analysis in primary squamous cell carcinoma of the endometrium.

Pathogenetically, endometrioid adenocarcinomas of the endometrium are associated with hyperestrogenism and serous papillary carcinomas with alterations of p53. The etiology of primary endometrial squamous cell carcinoma (ESCC), however, is speculative. The purpose of this study was to evaluate the role of p14, p16, p53, cyclin D1, steroid hormone receptors, and human papillomaviruses (HPV) infection in the pathogenesis of primary endometrial squamous cell carcinoma. The expression of p16, p14, p53, cyclin D1, and steroid hormone receptors (estrogen, progesterone, and androgen) was examined immunohistochemically in 8 primary ESCCs. HPV analysis was performed using general primers and HPV typing. The median age of the patients was 62.1 years. Four cases showed positive nuclear and cytoplasmic p16 staining in an insular pattern, and 1 case nuclear positivity for p53 and estrogen receptors, respectively. Four of 8 cases were positive for progesterone receptor analysis and cyclin D1. All cases were negative for p14 and androgen receptor staining. All but one case were negative for HPV analysis. Five patients were alive with and without evidence of disease after a mean follow-up of 6.1 years. The results of this study suggest that alterations of the p16 pathway may play an etiologic role in at least a proportion of the ESCC, but without any association to HPV infection. Factors known to play a pathogenetic role in types 1 and 2 of endometrial carcinomas are not associated with primary ESCC. However, prognostically, ESCCs are more related to type 1 cancers.

Mixed small cell carcinomas of the uterine cervix: prognostic impact of focal neuroendocrine differentiation but not of Ki-67 labeling index.

Small cell neuroendocrine carcinomas sometimes represent a non-small cell component. Because of infection with the high-risk human papillomavirus of small cell carcinomas (SmCCs), several host cell regulatory proteins are altered, thus causing altered proliferative activity. Knowledge regarding the prognostic impact of focal neuroendocrine differentiation in mixed SmCCs and the value of proliferative activity in these tumors is very limited. Small cell carcinomas were selected for immunohistochemical staining with neuroendocrine markers and Ki-67. In cases with mixed tumors, the percentage of the SmCC component was calculated and correlated with survival. Of 677 tumors, 9 (1.3%) were classified as SmCCs after Grimelius staining (8/9 positive tumors) and immunohistochemical reaction against neuron-specific enolase, chromogranin A, synaptophysin (7/9 positive tumors), and CD56 (8/9 positive tumors); all specimens were positive for at least 2 of these. CD99 staining was completely negative. Two thirds of the SmCCs showed non-small cell differentiation. Four patients died of the tumor after a median time of 36.7 months (range, 15-56 months). Even an SmCC component of 17% was associated with a fatal course. Small cell carcinoma represented a significantly lower proliferation (Ki-67 labeling index) than did the non-small cell component in the same tumor (12.8% vs 70.8%; P < .001). Even a small SmCC component in mixed carcinomas of the uterine cervix was associated with adverse outcome. Proliferative activity, determined by Ki-67 labeling index, is of no prognostic value.

Juxtatumoral desmoplastic stromal reaction is associated with high tumor cell dissociation in squamous cell carcinomas of the uterine cervix.

There are different types of tumoral growth patterns invading host tissue. During tumor infiltration, cancer cells not only destroy the pre-existing extracellular matrix, but usually induce new matrix formation by activating the peritumoral stromal cells; that is, desmoplastic stromal reaction (DSR) at the front of invasion (juxtatumoral stroma). This study evaluates the association between different types of invasion and DSR. Eighty-eight squamous cell carcinomas (Fédération Internationale de Gynécologie et d'Obstétrique [FIGO] stage IB to IV) were evaluated histologically for different patterns of invasion (PI) using a 3-level scoring system (pushing, finger-like, and spray-like). Desmoplastic stromal reaction was scored from none to weak, moderate, or strong. The pattern of invasion and DSR were compared with patients' age, FIGO stage, clinical tumor size, tumor grade, and the presence of lymphovascular space involvement. Finger-like PI was the most common (72.7%), followed by the spray-like PI (27.3%), whereas pushing PI was not seen. Of the tumors, 23.9% showed no DSR; 51.1%, weak; 14.8%, moderate; and 10.2%, strong DSR. Tumors with spray-like PI showed a significantly stronger desmoplastic reaction compared with the finger-like PI (P < .0001) and were significantly associated with poor tumor cell differentiation (P = .018). Moderate or strong DSR was associated with G2 and G3 carcinomas (P = .027). No correlation was seen neither for PI and DSR to lymphovascular space involvement, FIGO stage, and tumor size. The intensity of DSR, as understood in the context of a remodeling of the juxtatumoral stroma to the infiltrative tumor growth, might be indicative of a highly dissociative tumor growth and is correlated to poorly differentiated tumors.