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Dana M Roque,
Stefania Bellone,
Diana P English,
Natalia Buza,
Emiliano Cocco,
Sara Gasparrini,
Ileana Bortolomai,
Elena Ratner,
Dan-Arin Silasi,
Masoud Azodi, Thomas J Rutherford,
Peter E Schwartz,
Alessandro D Santin
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ABSTRACT: BACKGROUND: Uterine serous carcinoma (USC) is a subtype of endometrial cancer associated with chemoresistance and poor outcome. Overexpression of tubulin-β-III and p-glycoprotein has been linked to paclitaxel resistance in many cancers but has been undercharacterized among USCs. Epothilones have demonstrated activity in certain paclitaxel-resistant malignancies. In this study, relationships are clarified, in USCs relative to ovarian serous carcinomas (OSCs), between tubulin-β-III and p-glycoprotein expression, clinical outcome, and in vitro chemoresponsiveness to epothilone B, ixabepilone, and paclitaxel. METHODS: Tubulin-β-III and p-glycoprotein were quantified by real-time polymerase chain reaction in 48 fresh-frozen tissue samples and 13 cell lines. Copy number was correlated with immunohistochemistry and overall survival. Median inhibitory concentration (IC50 ) was determined using viability and metabolic assays. Impact of tubulin-β-III knockdown on IC50 was assessed with small interfering RNAs. RESULTS: USC overexpressed tubulin-β-III but not p-glycoprotein relative to OSC in both fresh-frozen tissues (552.9 ± 106.7 versus 202.0 ± 43.99, P = .01) and cell lines (1701.0 ± 376.4 versus 645.1 ± 157.9, P = .02). Tubulin-β-III immunohistochemistry reflected quantitative real-time polymerase chain reaction copy number and overexpression stratified patients by overall survival (copy number ≤ 400: 615 days; copy number > 400: 165 days, P = .049); p-glycoprotein did not predict clinical outcome. USCs remained exquisitely sensitive to patupilone in vitro despite tubulin-β-III overexpression (IC50,USC 0.245 ± 0.11 nM versus IC50,OSC 1.01 ± 0.13 nM, P = .006). CONCLUSIONS: Tubulin-β-III overexpression in USCs discriminates poor prognosis, serves as a marker for sensitivity to epothilones, and may contribute to paclitaxel resistance. Immunohistochemistry reliably identifies tumors with overexpression of tubulin-β-III, and a subset of individuals likely to respond to patupilone and ixabepilone. Epothilones warrant clinical investigation for treatment of USCs. Cancer 2013;000:000-000. © 2013 American Cancer Society.
Cancer 04/2013; · 4.77 Impact Factor
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Dana M Roque,
Stefania Bellone,
Natalia Buza,
Chiara Romani,
Emiliano Cocco,
Eliana Bignotti,
Antonella Ravaggi, Thomas J Rutherford,
Peter E Schwartz,
Sergio Pecorelli,
Alessandro D Santin
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ABSTRACT: OBJECTIVES: Clear cell carcinoma of the ovary (OCCC) is a distinct subtype of epithelial cancer associated with chemoresistance and poor outcome compared to serous papillary carcinomas (OSPC). Resistance to paclitaxel has been linked to overexpression of class III β-tubulin in several human cancers but inadequately characterized among OCCC. Chemoresistance has also been variably linked to the drug efflux pump p-glycoprotein. Epothilones are microtubule-stabilizing agents with putative activity in paclitaxel-resistant malignancies. In this study, we clarify the relationship between class III β-tubulin and p-glycoprotein expression in OCCC, clinical outcome, and in vitro responsiveness to patupilone and paclitaxel. STUDY DESIGN: Class III β-tubulin and p-glycoprotein were quantified by real time polymerase chain reaction (qRT-PCR) in 61 fresh-frozen tissue samples and 11 cell lines. Expression by PCR was correlated with immunohistochemistry and overall survival. IC50 was determined using viability/metabolic assays. Impact of class III β-tubulin downregulation on IC50 was assessed with siRNAs. RESULTS: OCCC overexpressed class III β-tubulin and p-glycoprotein relative to OSPC in fresh-frozen tissues and cell lines. Class III β-tubulin immunohistochemistry reflected qRT-PCR results and overexpression stratified patients by overall survival. P-glycoprotein correlated with in vitro paclitaxel resistance, but not clinical outcome. OCCC were exquisitely sensitive to patupilone in a manner that correlated with class III β-tubulin expression. CONCLUSIONS: Class III β-tubulin overexpression in OCCC discriminates poor prognosis, serves as a marker for sensitivity to patupilone, and may contribute to paclitaxel resistance. Immunohistochemistry reliably identifies tumors with overexpression of class III β-tubulin, and accordingly a subset of individuals likely to respond to patupilone.
American journal of obstetrics and gynecology 04/2013; · 3.28 Impact Factor
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Siming Zhao,
Murim Choi,
John D Overton,
Stefania Bellone,
Dana M Roque,
Emiliano Cocco,
Federica Guzzo,
Diana P English,
Joyce Varughese,
Sara Gasparrini, [......],
Elena Ratner,
Masoud Azodi,
Peter E Schwartz, Thomas J Rutherford,
Amy L Stiegler,
Shrikant Mane,
Titus J Boggon,
Joseph Schlessinger,
Richard P Lifton,
Alessandro D Santin
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ABSTRACT: Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. We analyzed the mutational landscape of USC by whole-exome sequencing of 57 cancers, most of which were matched to normal DNA from the same patients. The distribution of the number of protein-altering somatic mutations revealed that 52 USC tumors had fewer than 100 (median 36), whereas 5 had more than 3,000 somatic mutations. The mutations in these latter tumors showed hallmarks of defects in DNA mismatch repair. Among the remainder, we found a significantly increased burden of mutation in 14 genes. In addition to well-known cancer genes (i.e., TP53, PIK3CA, PPP2R1A, KRAS, FBXW7), there were frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling complex, and TAF1, an element of the core TFIID transcriptional machinery. Additionally, somatic copy-number variation was found to play an important role in USC, with 13 copy-number gains and 12 copy-number losses that occurred more often than expected by chance. In addition to loss of TP53, we found frequent deletion of a small segment of chromosome 19 containing MBD3, also a member of the NuRD-chromatin-modification complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (an upstream activator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiquitination). These findings identify frequent mutation of DNA damage, chromatin remodeling, cell cycle, and cell proliferation pathways in USC and suggest potential targets for treatment of this lethal variant of endometrial cancer.
Proceedings of the National Academy of Sciences 01/2013; · 9.68 Impact Factor
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ABSTRACT: BACKGROUND: To compare outcomes in women ≥ age 70 who receive neoadjuvant chemotherapy (NACT) for advanced epithelial ovarian cancer (EOC) followed by cytoreductive surgery with those undergoing upfront cytoreductive surgery followed by the same chemotherapy. METHODS: A retrospective cohort study was performed for women ≥ age 70 with Stage IIIC or Stage IV EOC from 1996 to 2009. RESULTS: Sixty-two patients who underwent upfront cytoreductive surgery and 42 patients who received NACT were eligible for analysis. Patients receiving NACT were significantly more likely to have Stage IV disease (P = 0.004). Cytoreduction to no macroscopic disease was achieved in 71.4% of women who received NACT and 28.1% of women undergoing upfront surgery (P < 0.001). NACT patients had significantly less blood loss at surgery (P = 0.01), required fewer small bowel resections (P = 0.009), had shorter ICU stays (P = 0.02) and fewer hospital days (P = 0.04). NACT patients experienced a trend toward an improved progression-free survival (P = 0.078); however, no statistically significant differences were found in either the progression-free or overall survival analyses. CONCLUSION: NACT is associated with reduced perioperative morbidity in elderly patients with advanced stage ovarian cancer. J. Surg. Oncol © 2012 Wiley Periodicals, Inc.
Journal of Surgical Oncology 05/2012; · 2.10 Impact Factor
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Federica Guzzo,
Stefania Bellone,
Natalia Buza,
Pei Hui,
Luisa Carrara,
Joyce Varughese,
Emiliano Cocco,
Marta Betti,
Paola Todeschini,
Sara Gasparrini,
Peter E Schwartz, Thomas J Rutherford,
Roberto Angioli,
Sergio Pecorelli,
Alessandro D Santin
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ABSTRACT: Carcinosarcomas of the female genital tract are rare tumors with an aggressive clinical behavior. Trastuzumab, a humanized monoclonal antibody, acts by binding to HER2/neu extracellular domain and exhibits therapeutic efficacy in HER2/neu-overexpressing cancers. Two uterine carcinosarcomas (UMMT-ARK-1, UMMT-ARK-2) and 2 ovarian carcinosarcomas (OMMT-ARK-1, OMMT-ARK-2) were established as primary tumor cell lines in vitro and evaluated for HER2/neu expression by immunohistochemistry, fluorescent in situ hybridization analysis, quantitative real-time polymerase chain reaction, and for membrane-bound complement regulatory proteins CD46, CD55, and CD59 by flow cytometry. Sensitivity to trastuzumab-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was studied in 5-hr chromium release assays. HER2/neu expression was demonstrated in OMMT-ARK-1 and OMMT-ARK-2. OMMT-ARK-2 demonstrated an amplification of the c-erbB2 gene by fluorescent in situ hybridization analysis and a high sensitivity to ADCC (mean killing, 45.6%; range, 32.3%-72.6%). A lower level of killing was detected against the fluorescent in situ hybridization analysis-negative OMMT-ARK-1 cell line (mean, 26.5%; range, 21.0%-31.8%). CD46, CD55, and CD59 membrane-bound complement regulatory proteins were expressed at high levels in all primary mixed müllerian tumor cell lines, and all these tumors were found to be highly resistant to complement-dependent cytotoxicity with or without trastuzumab. Addition of untreated and heat-inactivated plasma did not significantly decrease ADCC against OMMT-ARK-2 cell line, suggesting that while the cell line is highly resistant to complement, irrelevant IgG does not significantly alter the ability of trastuzumab to mediate ADCC. Our results suggest that HER2/neu may represent a novel target for the immunotherapy of a subset of human carcinosarcomas refractory to salvage chemotherapy.
International journal of gynecological pathology: official journal of the International Society of Gynecological Pathologists 04/2012; 31(3):211-21. · 2.07 Impact Factor
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ABSTRACT: To identify the practices and attitudes of gynecologic oncologists regarding the end-of-life discussion.
A pilot survey was sent to 1105 members of the Society of Gynecologic Oncologists (SGO). The survey consisted of 20 questions and was sent via the website Survey Monkey.
Response rate was 12.8%. Sixty percent of respondents were male, most ranged between 30 and 60 years of age and more than half performed 5-10 major surgeries per week. More than half of respondents (53.9%) deferred the End of Life discussion until the patient had sustained a major change in functional and/or medical status. Thirty percent initiated it at the first recurrence or progression of disease. Forty three percent of respondents characterized the discussion as an on-going process. Patients' age, social support, health insurance, and co-morbidities had no influence on the discussion, and neither did the tumor's site of origin or grade. More respondents initiated the discussion in advanced stage cancer (57%) and after salvage chemotherapy institution (54%). Forty four percent of respondents reported that "understanding and acceptance" was the initial response by patient when counseled about withdrawal of care. This increased to 86% when the issue was revisited. Confusion or reluctance to discuss the subject were initially reported to be 12% and 19%, respectively, but decreased to 2% and 3%, respectively, when withdrawal of care was subsequently addressed with the patient.
This pilot survey sheds a light on attitudes and practices about the end-of-life discussion that deserve to be further studied.
Gynecologic Oncology 03/2012; 124(3):471-3. · 3.89 Impact Factor
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ABSTRACT: Different equations used to estimate creatinine clearance (Cl(cr)) in obese oncology patients can produce divergent estimated creatinine clearance values, which in turn can result in significantly different calculated carboplatin doses. Standardization of the calculation of creatinine clearance in patients of all body types is a desirable goal. The objective of our study was to examine the impact of increasing body mass index on the accuracy of creatinine clearance estimation methods and to determine the optimal equation for creatinine clearance estimation in the obese adult female cancer patient.
Retrospective data analysis.
We compared the estimated creatinine clearance values produced by each of 11 equations to 24-hour creatinine clearance values measured in 119 adult female patients with gynecologic cancers grouped according to body composition.
We applied simple linear regression and Tukey mean-difference analysis to assess the relationship between estimated creatinine clearance values produced by these equations and measured creatinine clearance values for each patient. The relationship between measured creatinine clearance and estimated creatinine clearance produced by all equations displayed lower linear regression R (2) values and higher limits of agreement in obese patients than in nonobese groups. Agreement between measured and estimated creatinine clearance produced by the Cockcroft-Gault equation is sensitive to the particular weight parameter incorporated and is lowest using ideal weight or actual body weight. The Cockcroft-Gault equation incorporating an intermediate weight value reduced estimation bias. The Jelliffe equation produced the lowest R (2) values.
Available model equations are less reliable for predicting creatinine clearance in obese female cancer patients (body mass index >30) than in nonobese patients. A measured glomerular filtration rate or creatinine clearance value is most accurate in obese female cancer patients. When using Cockcroft-Gault equation for estimation in this patient population, however, an intermediate weight value (adjusted or modified-adjusted) rather than ideal or actual body weight should be used.
Journal of Oncology Pharmacy Practice 02/2012; 18(3):323-32.
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ABSTRACT: To compare the outcomes of 155 cases of endometrial cancer who had robot-assisted surgical staging to 150 open cases.
Retrospective chart review of cases of endometrial cancer that underwent staging two different ways by two surgeons at an academic institution.
Mean age was 62.4 years in the robotic arm and 65 (P=0.04) in the open arm. Mean body mass index was 34.5 Kg/m(2) in the robotic arm and 33 Kg/m(2) in the open arm (P=0.2). Pelvic and para-aortic lymph node dissection were performed in 94.8% and 67.7% of the robotic cases versus 95.3% and 74% of the open cases, respectively. Mean operative time was 127 min in the robotic arm, and 141 min in the open arm (P=0.0001). Mean lymph node count was 20.3 in the robotic arm, and 20 in the open arm (P=0.567). Mean estimated blood loss was 119 ml in the robotic arm and 185 in the open arm (P=0.015). Mean hospital stay was 1.5 days in the robotic arm, and 4 days in the open arm (P=0.0001). The incidence of postoperative ileus (0.6% vs. 10.7%, P=0.0001), infections (5.2% vs. 24%, P=0.0001), anemia/transfusion (1.3% vs. 7.7%, P=0.005), and cardiopulmonary complications (3.2% vs.14.7%, P=0.003) was significantly lower in the robotic arm vs. the open arm. There was one death in the robotic arm attributed to pre-existing cardiac condition.
Robotic-assisted staging reaps the benefits of minimally invasive surgery without compromising the adequacy of the procedure. Dedication to the technique shortens the operative time.
Gynecologic Oncology 02/2012; 124(2):260-4. · 3.89 Impact Factor
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ABSTRACT: Preeclampsia (gestational proteinuric hypertension) complicates 5% to 8% of all pregnancies, and is a major cause of maternal and perinatal morbidity and mortality. It is a multisystem disorder specific to human pregnancy and the puerperium. Although the etiology is unknown, increasing evidence from both animal and human studies suggests that an imbalance in circulating pro-(vascular endothelial growth factor [VEGF], placental growth factor) and anti-angiogenic factors (soluble fms-like tyrosine kinase 1, soluble endoglin) may be important. Bevacizumab (Avastin®; Genentech, South San Francisco, CA), a humanized recombinant monoclonal IgG antibody that binds VEGF, has been shown to inhibit endothelial cell proliferation, suppress angiogenesis, and shrink a variety of solid tumors. We present two cases of bevacizumab toxicity that mimic preeclampsia with a reversible syndrome characterized by acute-onset severe hypertension, proteinuria, central nervous system irritability (headache, photophobia, blurred vision, seizures), abnormal laboratory tests (elevated liver function tests, thrombocytopenia), and evidence of reversible posterior leukoencephalopathy on neuroimaging. In both cases, the clinical and laboratory manifestations returned to normal with discontinuation of bevacizumab therapy and supportive care. Bevacizumab toxicity can mimic preeclampsia in nonpregnant women. These data suggest that interference with VEGF signaling is sufficient to induce a preeclampsia-like syndrome in nonpregnant patients. VEGF signaling therefore appears to play a central role-perhaps the central role-in the pathogenesis of preeclampsia, and provides a potential biomarker for the prediction, prevention, and treatment of this dangerous disorder.
Reviews in obstetrics and gynecology 01/2012; 5(1):2-8.
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ABSTRACT: To compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine and ovarian carcinosarcomas (CS).
Five primary carcinosarcoma cell lines, two from uterine and three of ovarian origin, were evaluated for growth rate and tested for their in vitro sensitivity/resistance to patupilone versus paclitaxel by MTS assays. To identify potential mechanisms underlying the differential sensitivity/resistance to patupilone, expression levels of β-tubulin III (TUBB3) were determined with quantitative-real-time-polymerase-chain-reaction (q-RT-PCR) in primary uterine and ovarian CS cell lines and in 26 uterine and 9 ovarian CS fresh-frozen-tissues.
No appreciable difference in sensitivity to patupilone versus paclitaxel was noted in ovarian CS cell lines, or when uterine and ovarian CS cell lines were compared in their response to paclitaxel. In contrast, uterine CS cell lines were found to be significantly more sensitive to patupilone than to paclitaxel (P<0.002) and demostrated lower IC(50s) to patupilone (range 0.76-0.93nM) when compared to ovarian CS (range 1.9-3.4 nM, p<0.05). Higher levels of TUBB3 were detected in uterine CS cell lines and fresh frozen tissues when compared to ovarian CS (P<0.05).
Uterine CS cell lines are significantly more sensitive than ovarian CS cell lines to patupilone versus paclitaxel. High expression of TUBB3 is associated with sensitivity to patupilone in primary CS cell lines and may act as a genetic marker to predict chemotherapy efficacy. Patupilone may represent a promising drug in the treatment of this subset of rare but highly aggressive gynecological tumors.
Gynecologic Oncology 12/2011; 125(1):231-6. · 3.89 Impact Factor
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ABSTRACT: Cervical cancer spreads directly and through lymphatic and vascular channels. Perineural invasion is an alternative method of spread. Several risk factors portend poor prognosis and inform management decisions regarding adjuvant therapy.
To evaluate the incidence and significance of PNI in early cervical cancer.
Retrospective chart review of early-stage cervical cancer patients (IA-IIA) from 1994 to 2009.
One hundred ninety two patients were included, 24 with perineural invasion in the cervical stroma (cases) and 168 without (controls). The mean age of the cases was 53 years, versus 45.9 in the controls (P=0.01). PNI was associated with more adjuvant therapy (P=0.0001), a higher stage (P=0.005), a larger tumor size (≥ 4 cm) (P<0.0001), lymphovascular space invasion (P=0.002), parametrial invasion (P<0.0001) and more tumor extension to the uterus (P=0.015). On multivariate analysis using an adjusted hazard ratio, risk factors for recurrence included grade (HR, 95% CI; 3.61, 1.38-9.41) and histopathology (HR, 95% CI; 2.85, 100-8.09). Similarly, risk factors for death included grade (HR, 95% CI; 3.43, 1.24-9.49) and histopathology (HR, 95% CI; 3.71, 1.03-13.33). Perineural invasion was not identified as an independent risk factor for either recurrence or death. The mean follow up time was 56 months. There was no significant difference in recurrence (P=0.601) or over-all survival (P=0.529) between cases and controls.
While perineural invasion was found to be associated with multiple high-risk factors, it was not found to be associated with a worse prognosis in early cervical cancer.
Gynecologic Oncology 12/2011; 123(3):561-4. · 3.89 Impact Factor
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Eliana Bignotti,
Antonella Ravaggi,
Chiara Romani,
Marcella Falchetti,
Silvia Lonardi,
Fabio Facchetti,
Sergio Pecorelli,
Joyce Varughese,
Emiliano Cocco,
Stefania Bellone,
Peter E Schwartz, Thomas J Rutherford,
Alessandro D Santin
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ABSTRACT: We evaluated the expression of human trophoblast cell surface marker (Trop-2) in endometrial endometrioid carcinoma (EEC) and the potential application of hRS7, a humanized monoclonal anti-Trop-2 antibody, as a therapeutic agent against poorly differentiated EEC.
Trop-2 expression was evaluated by immunohistochemistry in 131 EEC with different degrees of differentiation and 32 normal endometrial controls (NEC). Trop-2 expression was also evaluated by quantitative real-time polymerase chain reaction and flow cytometry in 3 primary EEC cell lines derived from patients harboring poorly differentiated EEC. Finally, the sensitivity of grade 3 EEC cell lines to hRS7 antibody-dependent cellular cytotoxicity was tested in standard 5-hour Cr release assays.
Trop-2 expression was detected in 126 (96.2%) of 131 EEC samples. Tumor tissues showed markedly increased Trop-2 positivity compared with NEC (P = 0.001). Trop-2 expression was significantly higher in all grades of EEC versus NEC. Grade 3 tumors displayed significantly stronger Trop-2 immunostaining compared with grade 1 EEC (P = 0.01). High Trop-2 expression by quantitative real-time polymerase chain reaction and flow cytometry was found in 1 grade 3 EEC primary cell line (EEC-ARK-1). Unlike Trop-2-negative EEC cell lines, EEC-ARK-1 was found highly sensitive to hRS7-mediated antibody-dependent cellular cytotoxicity in vitro (range of killing, 33.9%-50.6%; P = 0.004). Human serum did not significantly inhibit hRS7-mediated cytotoxicity against EEC-ARK-1 (P = 0.773).
Trop-2 is highly expressed in EEC, and its expression is significantly higher in poorly differentiated EEC when compared with well-differentiated EEC. Primary grade 3 EECs overexpressing Trop-2 are highly sensitive to hRS7-mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for the treatment of high-grade EEC refractory to standard treatment modalities.
International Journal of Gynecological Cancer 09/2011; 21(9):1613-21. · 1.65 Impact Factor
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Joyce Varughese,
Emiliano Cocco,
Stefania Bellone,
Elena Ratner,
Dan-Arin Silasi,
Masoud Azodi,
Peter E Schwartz, Thomas J Rutherford,
Natalia Buza,
Sergio Pecorelli,
Alessandro D Santin
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ABSTRACT: We evaluated the expression of human trophoblast cell-surface marker (Trop-2) and the potential of hRS7, a humanized monoclonal anti-Trop-2 antibody, against treatment-refractory cervical cancer.
Trop-2 expression was evaluated by immunohistochemistry, real-time polymerase chain reaction, and flow cytometry. Sensitivity to hRS7 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in 5-hour chromium release assays. The effect of interleukin (IL)-2 on hRS7 ADCC was also investigated.
Membrane Trop-2 expression was observed in 8 of 8 (100%) of the cancer samples tested by immunohistochemistry, but not in normal cervix. High messenger RNA expression by real-time polymerase chain reaction and high Trop-2 surface expression by flow cytometry were detected in 80% of cervical cancers (4 of 5 cell lines). Although these tumors were resistant to natural killer cell-dependent cytotoxicity in vitro (mean killing, 6.0%), Trop-2-positive cell lines showed high sensitivity to hRS7 ADCC (range of killing, 30.6-73.2%). Incubation with IL-2 further increased the level of cytotoxicity against Trop-2-positive tumors.
hRS7 may represent a novel treatment option for patients with cervical cancer refractory to conventional treatment modalities.
American journal of obstetrics and gynecology 07/2011; 205(6):567.e1-7. · 3.28 Impact Factor
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Emiliano Cocco,
Joyce Varughese,
Natalia Buza,
Stefania Bellone,
Ken-Yu Lin,
Marta Bellone,
Paola Todeschini,
Dan-Arin Silasi,
Masoud Azodi,
Peter E Schwartz, Thomas J Rutherford,
Luisa Carrara,
Renata Tassi,
Sergio Pecorelli,
Charles J Lockwood,
Alessandro D Santin
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ABSTRACT: We evaluated the expression of tissue factor (TF) in ovarian cancer (EOC) and the potential of hI-con1, an antibody-like molecule targeting TF, as a novel form of therapy against chemotherapy-resistant ovarian disease. We studied the expression of TF in 88 EOC by immunohistochemistry (IHC) and real-time-PCR (qRT-PCR) and the levels of membrane-bound-complement-regulatory-proteins CD46, CD55 and CD59 in primary EOC cell lines by flow-cytometry. Sensitivity to hI-con1-dependent-cell-mediated-cytotoxicity (IDCC), complement-dependent-cell-cytotoxicity and inhibition of IDCC by γ-immunoglobulin were evaluated in 5-h (51)chromium-release-assays. Cytoplasmic and/or membrane TF expression was observed in 24 out of 25 (96%) of the EOC samples tested by IHC, but not in normal ovarian-tissue. EOC with clear cell histology significantly overexpress TF when compared to serous, endometrioid, or undifferentiated tumors by qRT-PCR. With a single exception, all primary EOC that overexpressed TF demonstrated high levels of CD46, CD55 and CD59 and regardless of their histology or resistance to chemotherapy, were highly sensitive to IDCC. The effect of complement and physiologic doses of γ-immunoglobulin on IDCC in ovarian cancer cell lines overexpressing TF was tumor specific and related to the overexpression of CD59 on tumor cells. Small-interfering-RNA-mediated knockdown of CD59 expression in ovarian tumors significantly increased hI-con1-mediated cytotoxic activity in vitro. Finally, low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (P < 0.01). hI-con1 molecule induces strong cytotoxicity against primary chemotherapy-resistant ovarian cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of ovarian tumors refractory to standard treatment modalities.
Clinical and Experimental Metastasis 07/2011; 28(7):689-700. · 3.52 Impact Factor
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Emiliano Cocco,
Joyce Varughese,
Natalia Buza,
Stefania Bellone,
Michelle Glasgow,
Marta Bellone,
Paola Todeschini,
Luisa Carrara,
Dan-Arin Silasi,
Masoud Azodi,
Peter E Schwartz, Thomas J Rutherford,
Sergio Pecorelli,
Charles J Lockwood,
Alessandro D Santin
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ABSTRACT: Cervical cancer continues to be an important worldwide health problem for women. Up to 35% of patients who are diagnosed with and appropriately treated for cervical cancer will recur and treatment results are poor for recurrent disease. Given these sobering statistics, development of novel therapies for cervical cancer remains a high priority. We evaluated the expression of Tissue Factor (TF) in cervical cancer and the potential of hI-con1, an antibody-like-molecule targeted against TF, as a novel form of immunotherapy against multiple primary cervical carcinoma cell lines with squamous- and adenocarcinoma histology.
Because TF is a transmembrane receptor for coagulation factor VII/VIIa (fVII), in this study we evaluated the in vitro expression of TF in cervical carcinoma cell lines by immunohistochemistry (IHC), real time-PCR (qRT-PCR) and flow cytometry. Sensitivity to hI-con1-dependent cell-mediated-cytotoxicity (IDCC) was evaluated in 5-hrs-51chromium-release-assays against cervical cancer cell lines in vitro.
Cytoplasmic and/or membrane TF expression was observed in 8 out of 8 (100%) of the tumor tissues tested by IHC and in 100% (11 out of 11) of the cervical carcinoma cell lines tested by real-time-PCR and flow cytometry but not in normal cervical keratinocytes (p=0.0023 qRT-PCR; p=0.0042 flow cytometry). All primary cervical cancer cell lines tested overexpressing TF, regardless of their histology, were highly sensitive to IDCC (mean killing±SD, 56.2%±15.9%, range, 32.4%-76.9%, p<0.001), while negligible cytotoxicity was seen in the absence of hI-con1 or in the presence of rituximab-control-antibody. Low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (p=0.025) while human serum did not significantly decrease IDCC against cervical cancer cell lines (p=0.597).
TF is highly expressed in squamous and adenocarcinoma of the uterine cervix. hI-con1 induces strong cytotoxicity against primary cervical cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of cervical cancer refractory to standard treatment modalities.
BMC Cancer 06/2011; 11:263. · 3.01 Impact Factor
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Francesca Casagrande,
Emiliano Cocco,
Stefania Bellone,
Christine E Richter,
Marta Bellone,
Paola Todeschini,
Eric Siegel,
Joyce Varughese,
Dan Arin-Silasi,
Masoud Azodi, Thomas J Rutherford,
Sergio Pecorelli,
Peter E Schwartz,
Alessandro D Santin
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ABSTRACT: 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.
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.
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).
CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy-resistant cancer stem cells.
Cancer 06/2011; 117(24):5519-28. · 4.77 Impact Factor
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Marta Bellone,
Emiliano Cocco,
Joyce Varughese,
Stefania Bellone,
Paola Todeschini,
Karim El-Sahwi,
Luisa Carrara,
Federica Guzzo,
Peter E Schwartz, Thomas J Rutherford,
Sergio Pecorelli,
Deborah J Marshall,
Alessandro D Santin
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ABSTRACT: Uterine serous papillary carcinoma (USPC) is an aggressive variant of endometrial cancer characterized by an innate resistance to chemotherapy and poor prognosis. In this study, we evaluated the expression of αV-integrins in primary USPC cell lines and the in vitro ability of intetumumab (CNTO 95), a fully human monoclonal antibody against αV-integrins, to inhibit USPC cell adhesion and migration.
The surface expression of integrins belonging to the αV-family, including αVβ3, αVβ5, and αVβ6, was evaluated in 6 primary USPC cell lines using flow cytometry analysis. To test the ability of intetumumab to inhibit USPC cell adhesion and migration, adhesion assays in the presence of vitronectin and migration assays through an 8.0-μm pore polycarbonate membrane also were performed.
We found high expression of the αV-subunit on the cell surface of all 6 primary USPC cell lines tested (100% positive cells; mean fluorescence intensity range, 13.1-39.5). When the expression of single heterodimeric integrins was evaluated, αVβ3, αVβ5, and αVβ6 were expressed on 37.5%, 32.0%, and 16.3% of cells (mean fluorescence intensity range, 6.5-16.2, 9.2-32.5, and 6.2-11.5, respectively). Importantly, in functional assays, low doses of intetumumab were effective in inhibiting adhesion (0.15 μg/mL, P = 0.003) and migration (1.25 μg/mL P = 0.02) of primary USPC cell lines.
The αV-integrins are overexpressed on the cell surface of primary USPC cell lines. Intetumumab may significantly inhibit USPC cell adhesion and migration pathways and may therefore represent a novel treatment option for patients harboring this rare but highly aggressive variant of endometrial cancer.
International Journal of Gynecological Cancer 05/2011; 21(6):1084-90. · 1.65 Impact Factor
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ABSTRACT: We evaluated the expression of human trophoblast cell-surface marker (Trop-2) and the potential of hRS7, a humanized monoclonal anti-Trop-2 antibody, as a therapeutic agent against chemotherapy-resistant ovarian disease.
Trop-2 expression was evaluated by immunohistochemistry (IHC) in 50 ovarian serous papillary carcinoma specimens. Trop-2 expression was also evaluated by real-time PCR (qRT-PCR) and flow cytometry in a total of 6 primary ovarian cancer cell lines derived from patients with chemotherapy-resistant disease. Sensitivity to hRS7 antibody-dependent cellular cytotoxicity (ADCC) was tested in standard 5-hour ⁵¹Cr-release assays. The effect of serum and interleukin-2 (IL-2) on hRS7-mediated ADCC was also studied.
Trop-2 expression was found in 41 of 50 (82%) tumor tissues tested by IHC. 83% (5 of 6) of the ovarian cancer cell lines tested by qRT-PCR and flow cytometry demonstrated high Trop-2 expression. All primary ovarian cancer cell lines expressing Trop-2 were highly sensitive to hRS7-mediated ADCC in vitro (range of killing: 19.3% to 40.8%) (p<0.001). Negligible cytotoxicity against chemotherapy-resistant ovarian cancers was seen in the absence of hRS7 or in the presence of rituximab control antibody (range of killing: 1.1% to 8.9%). Human serum did not significantly inhibit hRS7-mediated cytotoxicity while incubation with IL-2 in addition to hRS7 further increased the cytotoxic activity (p=0.04).
Trop-2 is highly expressed in chemotherapy-resistant ovarian cancer cell lines at mRNA and protein levels. Primary ovarian carcinoma cell lines are highly sensitive to hRS7-mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for the treatment of high-grade, chemotherapy-resistant ovarian cancer.
Gynecologic Oncology 03/2011; 122(1):171-7. · 3.89 Impact Factor
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ABSTRACT: Chemoresistance is a major limitation in the treatment of ovarian cancer. Phenoxodiol is a novel biomodulator capable of reversing chemoresistance in vitro and in vivo. In this study, we determined the safety and efficacy of intravenous phenoxodiol in combination with cisplatin or paclitaxel in women with platinum/taxane-refractory/resistant ovarian cancers.
Thirty-two patients were randomized to 1 of 2 treatment arms according to their previous responses: (1) platinum refractory/resistant, cisplatin (40 mg/m intravenous) weekly on day 2 + phenoxodiol (3 mg/kg) weekly on days 1 and 2 and (2) taxane refractory/resistant, paclitaxel (80 mg/m IV) weekly on day 2 and phenoxodiol (3 mg/kg) weekly on days 1 and 2. Patients continued on treatment until complete response, disease progression, unacceptable toxicity, or voluntary withdrawal.
There were no treatment-related deaths. There was only one treatment-related hospitalization and 2 grade 4 toxicities. In the cisplatin arm, there were 3 partial responses, 9 patients (56%) achieved stable disease, 4 (25%) progressed, and the overall best response rate was 19%. In the paclitaxel arm, there was one complete response and 2 partial responses, 8 patients (53%) achieved stable disease, 4 patients (27%) progressed, and the overall best response rate was 20%.
The combination of IV phenoxodiol with cisplatin or paclitaxel was well tolerated in this study. Cisplatin-phenoxodiol was particularly active and warrants further study in patients with platinum-resistant ovarian cancer.
International Journal of Gynecological Cancer 03/2011; 21(4):633-9. · 1.65 Impact Factor
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ABSTRACT: Uterine serous papillary carcinoma (USPC) was an aggressive and chemotherapy resistant variant of endometrial cancer. The authors evaluated the expression of human trophoblast-cell-surface-marker (Trop-2) and the potential of hRS7, a humanized anti-Trop-2 monoclonal antibody, as a novel therapeutic strategy against USPC.
Trop-2 expression was evaluated by immunohistochemistry (IHC) in a total of 23 USPC. Six primary USPC cell lines were assessed by flow cytometry and real-time polymerase chain reaction (PCR) for Trop-2 expression. Sensitivity to hRS7 (Immunomedics, Inc.) antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in standard 5-hour ⁵¹Cr-release assays against primary USPC cell lines.
Expression of Trop-2 was found in 15 of 23 (65%) of the tumor tissues tested by IHC and in 50% (3 of 6) of the USPC cell lines tested by real-time PCR and flow-cytometry (Trop-2 expression in USPC versus normal endometrial cells; P < .005). USPC cell lines overexpressing Trop-2, regardless of their intrinsic resistance to natural killer cytotoxicity, were highly sensitive to hRS7-mediated ADCC in vitro (range of killing, 28.2% to 64.4%) (P < .001). Negligible cytotoxicity against USPC was seen in the absence of hRS7 or in the presence of rituximab control antibody (range of killing, 1.1% to 12.4%). Incubation with interleukin-2 (50 IU/mL) in addition to hRS7 further increased the cytotoxic activity against USPC cell lines overexpressing Trop-2 (P = .008).
Trop-2 was highly expressed in uterine serous carcinoma at mRNA and protein levels. Primary USPC cell lines are highly sensitivity to hRS7-mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for USPC refractory to standard treatment modalities.
Cancer 01/2011; 117(14):3163-72. · 4.77 Impact Factor
