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The zebrafish ( Danio rerio ) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models. Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product. We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex ® and two biosimilars, the erythropoietin alpha Binocrit ® and zeta Retacrit ® . By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4 ± 0.3 fold), embryonic α -Hb (1.9 ± 0.2 fold) and β -Hb (1.6 ± 0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg ( kdrl :EGFP; gata1 :ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8 ± 0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos. Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine and ovarian carcinosarcomas (CS). Methods: 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. Results: 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 IC50s 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). Conclusions: 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. [Copyright &y& Elsevier]

Abstract: Objective. : 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. Methods. : 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. Results. : 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). Conclusions. : 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. [Copyright &y& Elsevier]

Abstract: Objectives. : To evaluate and compare epidermal growth factor type-1 receptor (EGF-R1) expression in short term and established cervical cancer cell lines generated from primary and metastatic/recurrent sites of disease. To evaluate the sensitivity of cervical cancer cell lines to treatment with a chimeric MAb against EGFR-1 (Cetuximab). Methods. : EGFR-1 expression was evaluated by flow cytometry on 22 cervical cancer cell lines including 14 primary cervical cancer cell lines obtained from cervical biopsies (11 patients) and recurrent sites of disease (three patients) as well as eight established cell lines. Tumor cell lines were tested for sensitivity to Cetuximab-mediated complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in 51Cr release assays. Finally, Cetuximab-mediated inhibition of cell proliferation was also tested. Results. : Fourteen out of fourteen (100%) primary tumors and seven out of eight (87.5%) established cervical cancer cell lines expressed EGFR-1 by flow cytometry. Cell lines from recurrent/metastatic sites of disease expressed higher levels of EGFR-1 when compared to those obtained from primary sites (p >0.05). Minimal CDC was detected in the majority of cervical cancer cell lines exposed to complement±Cetuximab in the absence of peripheral blood lymphocytes (PBL). In contrast, cervical tumor cell lines were found highly sensitive to Cetuximab-mediated ADCC when challenged with PBL from either healthy donors or cervical cancer patients. Importantly, ADCC was further increased in the presence of complement. Finally, tumor proliferation was significantly inhibited by Cetuximab in all cervical tumors tested. Conclusions. : EGFR-1 is highly expressed in primary and recurrent cervical tumors. Cetuximab might be a novel and attractive therapeutic strategy in patients harboring chemotherapy-resistant, recurrent, or metastatic cervical cancer. [Copyright &y& Elsevier]

Abstract: Objective : Mammaglobin B is a uteroglobin gene family member recently found highly differentially expressed in serous papillary ovarian cancer by gene expression profiling. In order to evaluate its potential as a novel ovarian cancer biomarker, in this study we quantified and compared Mammaglobin B expression in various histologic types of epithelial ovarian carcinomas (EOC). Methods : Mammaglobin B expression was evaluated by real-time PCR and/or immunohistochemistry in fresh-frozen biopsies and paraffin-embedded tissues derived from a total of 137 patients including 69 primary EOC with different histologies, 28 serous papillary omental metastasis, 8 borderline tumors, 26 benign cystadenomas and 14 normal ovaries. Results : High levels of Mammaglobin B gene expression were detected in 100% (68 out of 68) of the ovarian cancer biopsies tested by real-time PCR. In contrast, normal human ovarian surface epithelium (HOSE) expressed negligible levels of Mammaglobin B mRNA (EOC versus HOSE, p <0.01). Although Mammaglobin B gene expression levels were higher in endometrioid, mucinous and undifferentiated tumors when compared to serous papillary tumors, clear cell tumors and those with mixed histology, these differences were not statistically significant. In agreement with real-time PCR results, EOC were found to express significantly higher levels of Mammaglobin B protein when compared to normal ovaries and benign cystadenomas (p <0.01). However, only 29 out of 68 (42%) of the EOC samples found positive for Mammaglobin B by real-time PCR showed immunoreactivity by IHC. Conclusions : Mammaglobin B gene is highly expressed in EOC and may represent a novel molecular marker for multiple histological types of ovarian cancer. Additional studies to evaluate the clinical utility of Mammaglobin B as a diagnostic and/or therapeutic target in ovarian cancer are warranted. [Copyright &y& Elsevier]

Abstract: Objective.: To identify novel molecular biomarkers useful for the early diagnosis and therapy of ovarian cancer by gene expression profiling. To compare the genetic fingerprints of flash-frozen ovarian serous carcinomas to those of matched highly purified primary tumor cell cultures. Methods.: Gene expression profiles of 19 flash-frozen ovarian serous papillary carcinoma (OSPC) were analyzed and compared to 15 controls (highly purified human ovarian surface epithelium short-term cultures, HOSE) using oligonucleotide microarrays complementary to >14,500 human genes. In addition, gene expression profiling of 5 highly purified primary OSPC cultured in vitro for less than 2 weeks was compared to flash-frozen ovarian carcinoma biopsies obtained from matched samples. Quantitative RT-PCR and IHC staining techniques were used to validate microarray data at RNA and protein levels for some of the differentially expressed genes. Results.: Unsupervised analysis of gene expression data readily distinguished normal tissue from flash-frozen OSPC and identified 901 and 557 genes that exhibited >3-fold up-regulation or down-regulation, respectively, in OSPC when compared to HOSE. Mammaglobin 2, an ovarian secreted protein, was identified as the top differentially expressed gene in OSPC (19 out 19 OSPC versus 0 out of 15 HOSE) with over 827-fold up-regulation relative to HOSE. The claudin and kallikrein family of proteins including the clostridium perfringens enterotoxin receptors claudin 3 and 4, kallikreins 6, 7, 8, 10, 11 and the immunomodulatory molecule B7-H4 were found among the most highly overexpressed genes in OSPC when compared to HOSE. Genetic fingerprints of flash-frozen OSPC were found to have high correlation with those of purified primary OSPC short-term in vitro cultures with only 31 out of 8637 genes (0.35%) differentially expressed between the two groups. Conclusions.: Short-term in vitro culture of primary ovarian carcinomas may greatly increase the purity of ovarian tumor RNA available for gene expression profiling without causing major alteration in OSPC fingerprints. Mammaglobin 2, kallikreins 6, 7, 8, 10, 11, claudin 3 and 4 and B7-H4 gene expression products represent candidate biomarkers endowed with great potential for early screening and therapy of OSPC patients. [Copyright &y& Elsevier]

Abstract: Objective. : To evaluate the potential of human papillomavirus (HPV) type 16 and 18 E7 antigen-loaded autologous dendritic cells (DC) as a therapeutic cellular vaccine in a case series of cervical cancer patients harboring recurrent/metastatic disease refractory to standard treatment modalities. Methods. : Autologous monocyte-derived DC were pulsed with recombinant HPV16 E7 or HPV18 E7 oncoproteins and administered to 4 cervical cancer patients. Vaccinations were followed by subcutaneous administration twice daily of low doses of human recombinant interleukin-2 (1 × 106 IU/m2) from day 3 to day 7. Safety, toxicity, delayed type hypersensitivity reactions (DTH), clinical responses, and induction of serological and cellular immunity against HPV16/18 E7 were monitored. Results. : The vaccine was well-tolerated in all patients and no local or systemic side effects or toxicity were recorded. Three out of four patients were found to be significantly immunocompromised before starting the vaccination treatment, as assessed by DTH with a panel of recall antigens. Specific humoral and cellular CD4+ T cell responses to the E7 vaccine were detected in 2 patients, as detected by ELISA and by IFN-γ ELISpot assays, respectively. Increased numbers of E7-specific IFN-γ secreting CD8+ T cells were detected in all patients after vaccination. Swelling and induration (i.e., a positive DTH response) to the intradermal injection of HPV E7 oncoprotein and/or irradiated autologous tumor cells were detected in two patients after six vaccinations. No objective clinical responses were observed. However, both patients who developed a positive DTH to the vaccine experienced a slow tumor progression (i.e., 13 months survival) while DTH unresponsive patients died within 5 months from the beginning of therapy. Conclusions. : Autologous DC pulsed with HPV16/18 E7 proteins can induce systemic B and T cell responses in patients unresponsive to standard treatment modalities. However, treatment-induced immunosuppression may impose severe limitations on the efficacy of active vaccination strategies in late stage cervical cancer patients. DC-based vaccination trials are warranted in immunocompetent cervical cancer patients with early stage disease and/or limited tumor burden, and at significant risk for tumor recurrence or disease progression. [Copyright &y& Elsevier]

Abstract: Objective. : To evaluate and compare HER2/neu protein overexpression and gene amplification in uterine serous papillary endometrial cancer (USPC). Study design. : Immunohistochemical (IHC) and fluorescent in situ hybridization (FISH) assays were used to analyze and compare HER2/neu protein expression and gene amplification, respectively, in paraffin blocks from 26 women harboring stage IA to IV USPC treated at the University of Arkansas for Medical Sciences from 1997 to 2004. Chromosome 17 polysomy status by FISH was also assessed in all specimens. Results. : Moderate-to-strong expression of HER2/neu protein was noted in 16 (62%) of 26 USPC samples evaluated, with 7 (27%) samples showing moderate staining (2+) and 9 (35%) showing strong staining (3+) for HER2/neu. Amplification of the ERBB2 gene by FISH was observed in 11 of the 26 (42%) cases. Protein overexpression and gene amplification were found to correlate in 100% (9 of 9) of the 3+ positive tumors and 2 out of 7 (29%) of the 2+ positive tumors. Heterogeneity was noted in 3 cases in the amplification of the HER2/neu gene within the same tumor samples with pockets of amplified tumor cells amidst nonamplified tumor cells. None of the 10 USPC cases scored by IHC as 0 or 1+ was found positive for ERBB2 amplification by FISH. Conclusions. : Amplification of the HER2/neu oncogene represents a common finding in USPC. FISH analysis should be used for confirmation of gene amplification in USPC showing 2+ expression of HER2/neu. Prior screening and selection of appropriate immunohistochemistry-positive areas may be beneficial in the selection of some USPC patients undergoing FISH analysis. [Copyright &y& Elsevier]

Abstract: Purpose.: To evaluate and compare autocrine expression and production of interleukin-6 (IL-6), a pleiotropic cytokine involved in the resistance to cytotoxic agents and inhibition of anti-tumor immune function in endometrial carcinoma in vitro as well as in vivo. Patients and methods.: IL-6 gene expression levels were evaluated in twenty-four primary endometrial tumors including 14 endometrioid carcinomas (EC) and 10 uterine serous papillary carcinoma (USPC) as well as in normal control endometrial cells (NEC) by real-time PCR. Secretion of IL-6 protein by 6 primary endometrial tumor cultures including USPC and EC was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) in vitro. Finally, IL-6 concentration in 71 serum samples including 20 apparently healthy women, 19 women with benign abdominal diseases, 19 women with primary EC, and 13 USPC patients was studied. Results.: IL-6 gene expression levels were significantly higher in USPC when compared to EC (mean copy number by RT-PCR = 313 ± 55 vs. 53 ± 11, USPC vs. EC, respectively: P &#60; 0.01). IL-6 serum concentrations between normal healthy females (range 0.01–21.23 pg/ml; mean 3.1 pg/ml) and benign disease patients (range 0.01–95.77 pg/ml; mean 13.07 pg/ml) were not statistically different. In contrast, significantly higher levels of IL-6 were detected in both patients with EC (range 2.86–82.13 pg/ml; mean 20.43 pg/ml) and patients with UPSC (range 16.3–500.1 pg/ml; mean 125.7 pg/ml) when compared to the healthy females (P &#60; 0.01), with a mean serum IL-6 level in USPC patients 6.1-fold higher when compared to EC patients (P &#60; 0.03). Accordingly, higher levels of IL-6 secretion were noted in primary USPC cell lines (mean 3121 pg/ml, range between 1099 and 5017 pg/ml/105 cells/48 h) when compared to primary EC (mean 88, range between 19 and 112 pg/ml/105 cells/48 h) (P &#60; 0.01) in vitro. Conclusions.: IL-6 is highly expressed in USPC, and it is released in high concentration in the serum of USPC patients. IL-6 may be a novel biomarker for USPC. Drugs used to inhibit the expression of IL-6 or the IL-6 signal transduction pathway may potentially be highly beneficial in USPC. [Copyright &y& Elsevier]

Abstract: With the goal of identifying genes with a differential pattern of expression between invasive cervical carcinomas (CVX) and normal cervical keratinocytes (NCK), we used oligonucleotide microarrays to interrogate the expression of 14,500 known genes in 11 primary HPV16 and HPV18-infected stage IB–IIA cervical cancers and four primary normal cervical keratinocyte cultures. Hierarchical cluster analysis of gene expression data identified 240 and 265 genes that exhibited greater than twofold up-regulation and down-regulation, respectively, in primary CVX when compared to NCK. Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), mesoderm-specific transcript, forkhead box M1, v-myb myeloblastosis viral oncogene homolog (avian)-like2 (v-Myb), minichromosome maintenance proteins 2, 4, and 5, cyclin B1, prostaglandin E synthase (PTGES), topoisomerase II alpha (TOP2A), ubiquitin-conjugating enzyme E2C, CD97 antigen, E2F transcription factor 1, and dUTP pyrophosphatase were among the most highly overexpressed genes in CVX when compared to NCK. Down-regulated genes in CVX included transforming growth factor beta 1, transforming growth factor alpha, CFLAR, serine proteinase inhibitors (SERPING1 and SERPINF1), cadherin 13, protease inhibitor 3, keratin 16, and tissue factor pathway inhibitor-2 (TFPI-2). Differential expression of some of these genes including CDKN2A/p16, v-Myb, PTGES, and TOP2A was validated by quantitative real-time PCR. Flow cytometry on primary CVX and NCK and immunohistochemical staining of formalin fixed paraffin-embedded tumor specimens from which primary CVX cultures were derived as well as from a separate set of invasive cervical cancers confirmed differential expression of the CDKN2A/p16 and PTGES markers on CVX versus NCK. These results identify several genes that are coordinately disregulated in cervical cancer, likely representing common signaling pathways triggered by HPV transformation. Moreover, these data obtained with highly purified primary tumor cultures highlight novel molecular features of human cervical cancer and provide a foundation for the development of new type-specific diagnostic and therapeutic strategies for this disease. [Copyright &y& Elsevier]

Objective. To determine whether the Stratum Corneum Chymotryptic Enzyme (SCCE), a novel serine protease known to contribute to the cell shedding process by catalyzing the degradation of intercellular cohesive structures at the skin surface, is overexpressed in human cervical tumors.Methods. SCCE expression was evaluated in 18 cervical cancer cell lines (i.e., 10 primary and 8 established cell lines) as well as in 8 normal cervical keratinocyte cultures by RT-PCR. In addition, SCCE expression was evaluated by immunohistochemistry on paraffin-embedded tumor tissue.Results. Normal cervical keratinocytes did not express SCCE. In contrast, 50% of the primary and 50% of the established cervical cancer cell lines expressed SCCE by RT-PCR. Eighty percent (i.e., four of five) of primary squamous cervical tumors and 20% (i.e., one of five) of primary adenocarcinomas expressed SCCE. Five out of five (100%) of the patients harboring SCCE-positive tumors were found to have metastatic involvement of the pelvic tumor draining lymph nodes. Immunohistochemistry staining of paraffin-embedded cervical cancer specimens confirmed SCCE expression in tumor cells and its absence on normal cervical epithelial cells.Conclusion. Squamous cervical cancer expressed high levels of SCCE, suggesting that this protease may play an important role in invasion and metastasis. Because SCCE appears only in abundance in tumor tissue and contains a secretion signal sequence, suggesting that SCCE is secreted, it may prove to be a useful diagnostic/prognostic tool for the detection of metastatic or recurrent disease or as a novel molecular target for cervical cancer therapy. [Copyright &y& Elsevier]

: ObjectivesThe purpose of this study was to compare HER-2/neu expression on biopsies obtained from early stage cervical cancer, primary cell lines established therefrom, established cervical cancer cell lines, and metastatic or recurrent sites of disease; and to evaluate the sensitivity of primary cervical cancer cell lines to treatment with a humanized MAb against HER-2/neu (Herceptin).: MethodsSurface HER-2/neu expression on 18 cervical cancer cell lines was compared to HER-2/neu detection by immunohistochemistry on biopsies obtained from the original tumors (10 patients) and sites of recurrence (2 patients). Primary cell lines were tested for sensitivity to Herceptin-mediated antibody-dependent cellular cytotoxicity (ADCC) and sensitivity to Herceptin-mediated inhibition of proliferation.: ResultsNine out of 10 primary (90%) and 8 out of 8 (100%) established cervical cancer cell lines expressed HER-2/neu by flow cytometry. Surprisingly, all HER-2/neu-positive primary cell lines were derived from tumor biopsies that scored negative (i.e., 0 to 1+) for HER-2/neu expression by immunohistochemistry. Heavy staining for HER-2/neu (i.e., 3+) was found in the recurrent/metastatic lesions of the two relapsed patients. Importantly, all HER-2/neu-positive primary cell lines were highly sensitive to Herceptin-mediated ADCC, and their proliferation was also significantly inhibited by Herceptin. A significant enhancement of Herceptin-mediated ADCC was demonstrated when effector cells were exposed to low doses of IL-2 in vitro.: ConclusionsEarly stage cervical cancer may develop a population of HER-2/neu-positive cells with a selective growth advantage over HER-2/neu-negative cells. Therapy which targets HER-2/neu may be more effective in patients with cervical cancer than indicated by the commonly low expression of HER-2/neu in tumors removed at the time of primary treatment. [Copyright &y& Elsevier]

: ObjectiveTo evaluate the potential of autologous dendritic cells (DC) pulsed with HPV16 and HPV18 E7 oncoprotein in restoring tumor-specific cytotoxicity in populations of tumor infiltrating lymphocytes (TIL) for adoptive immunotherapy of cervical cancer patients.: MethodsFull-length E7-pulsed DC-stimulated CD8+ T cells derived from peripheral blood (PBL) and from tumor tissues (TIL) were tested and compared for their ability to induce a HLA class-I-restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. In addition, in order to correlate cytotoxic activity by CTL with a particular lymphoid subset, analysis of surface antigens and intracellular CD3 ζ chain and two-color flow cytometric analysis of intracellular cytokine expression (IFN-γ vs IL-4) at the single cell level were performed.: ResultsDC stimulation induced powerful cytotoxicity against autologous tumor target cells by TIL-derived CD8+ T cells from all three cervical cancer patients, while autologous Epstein–Barr virus-transformed lymphoblastoid cell lines were not lysed. Killing of autologous tumor cells was higher by CD8+ T cells from TIL compared to PBL (P > 0.01) and was more strongly inhibited by anti-HLA class I MAb (P > 0.05). Phenotypically, all CTL populations were CD3+/CD8+, with higher levels of CD56 expression by TIL-derived CTL. Finally, although a marked Type 1 cytokine bias (i.e., IFN-γhigh/IL-4low) was observable in both PBL- and TIL-derived DC-stimulated CD8+ T cell populations, TIL-derived CD8+ T cells showed a higher percentage of IFN-γ-positive cells compared to PBL.: ConclusionsFull-length E7-pulsed DC can consistently restore strong CD8+ CTL responses against autologous HPV16- and HPV18-infected cervical cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL compared to PBL for adoptive T cell immunotherapy of patients harboring metastatic or recurrent cervical cancer refractory to standard treatment modalities. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: In early stage cervical carcinoma, most studies of the literature show that adjuvant radiotherapy significantly reduced local relapse; its impact on survival improvement is controversial. In this retrospective study, we analyze the role of adjuvant radiotherapy in negative node patients and the possibility of this treatment to improve survival in selected groups. Methods: Four hundred fifty-four patients with stage IB–IIA carcinoma of the uterine cervix were treated with primary radical hysterectomy and pelvic lymphadenectomy. The patients with negative nodes but with pathologic prognostic factors predictive of a poor outcome, underwent adjuvant radiation therapy, according to personalized indications. Results: Disease-free actuarial 5-year survival (DFS) was 80%: 88% and 57% in patients with negative and positive nodes, respectively. The population of negative node patients was stratified in three risk categories according to the number of worsening prognostic factors: parametrial invasion, depth of stromal invasion (SI) >1/3 and presence of lymph vascular space involvement (LVSI). In the medium risk category (1 or 2 unfavorable prognostic factors), DFS showed significant advantage for patients submitted to post-operative external beam radiation. In the subset of cases without parametrial extension (pT1B) with one or two risk factors on the surgical specimen (LVSI and/or SI >1/3), there was no difference in DFS between the two groups treated or not with adjuvant radiotherapy. Conclusion: Post-operative radiotherapy is controversial in node-negative pathologic stage IB cervical cancer; radical surgery alone has low morbidity, enable more accurate prediction of prognosis and may be sufficient therapy in the majority of patients with lymph node-negative early stage cervical cancer. [Copyright &y& Elsevier]