Background Collecting duct carcinoma (CDC) is a rare but very aggressive variant of kidney carcinoma that arises from the epithelium of Bellini's ducts, in the distal portion of the nephron. In order to gain an insight into the biology of this tumor we evaluated the expression of five genes involved in the development of renal cancer ( FEZ1/LZTS1 ,  FHIT ,  TP53 ,  P27 kip 1 , and  BCL2 ).
Background
Collecting duct carcinoma (CDC) is a rare but very aggressive variant of kidney carcinoma that arises from the epithelium of Bellini's ducts, in the distal portion of the nephron. In order to gain an insight into the biology of this tumor we evaluated the expression of five genes involved in the development of renal cancer ( FEZ1/LZTS1 ,  FHIT ,  TP53 ,  P27 kip 1 , and  BCL2 ).FEZ1/LZTS1FHITTP53P27kipBCL2Methods We studied eleven patients who underwent radical nephrectomy for primary CDC. All patients had an adequate clinical follow-up and none of them received any systemic therapy before surgery. The expression of the five markers for tumor initiation and/or progression were assessed by immunohistochemistry and correlated to the clinicopathological parameters, and survival by univariate analysis.
Methods
We studied eleven patients who underwent radical nephrectomy for primary CDC. All patients had an adequate clinical follow-up and none of them received any systemic therapy before surgery. The expression of the five markers for tumor initiation and/or progression were assessed by immunohistochemistry and correlated to the clinicopathological parameters, and survival by univariate analysis.Results Results showed that Fez1 protein expression was undetectable or substantially reduced in 7 of the 11 (64%) cases. Fhit protein was absent in three cases (27%). The overexpression of p53 protein was predominantly nuclear and detected in 4 of 11 cases (36%). Immunostaining for p27 was absent in 5 of 11 cases (45.5%). Five of the six remaining cases (90%) showed exclusively cytoplasmic protein expression, where, in the last case, p27 protein was detected in both nucleus and cytoplasm. Bcl2 expression with 100% of the tumor cells positive was observed in 4 of 11 (36%) cases. Statistical analysis showed a statistical trend (P = 0.06) between loss and reduction of Fez1 and presence of lymph node metastases.
Results
Results showed that Fez1 protein expression was undetectable or substantially reduced in 7 of the 11 (64%) cases. Fhit protein was absent in three cases (27%). The overexpression of p53 protein was predominantly nuclear and detected in 4 of 11 cases (36%). Immunostaining for p27 was absent in 5 of 11 cases (45.5%). Five of the six remaining cases (90%) showed exclusively cytoplasmic protein expression, where, in the last case, p27 protein was detected in both nucleus and cytoplasm. Bcl2 expression with 100% of the tumor cells positive was observed in 4 of 11 (36%) cases. Statistical analysis showed a statistical trend (P = 0.06) between loss and reduction of Fez1 and presence of lymph node metastases.Conclusions These findings suggest that Fez1 may represent not only a molecular diagnostic marker but also a prognostic marker in CDC.
Conclusions
These findings suggest that Fez1 may represent not only a molecular diagnostic marker but also a prognostic marker in CDC.

Background Renal cancer accounts for 3% of adult malignancies. An estimated 35,710 new cases are expected to occur in the U.S. in the current year and 12,480 patients will die of this disease [ 1 ]. The majority of renal cancers arises from the proximal tubular epithelium, with a characteristic clear or granular cell appearance by light microscopy, and is referred to as renal cell carcinoma (RCC). Recent evidence suggests that solid renal parenchymal tumors arising in the distal portion of the nephron, such as oncocytomas, chromophobe renal cell carcinomas and collecting duct carcinomas represent an heterogeneous group of neoplasm from both clinical and biological perspectives [ 2 ]. Collecting duct carcinoma (CDC), also known as Bellini duct carcinoma, is a rare but highly aggressive renal neoplasm arising from the distal portion of the nephron, and represents approximately 2% of all the RCCs [ 3 , 4 ]. Clinically, CDC appears as a renal mass often accompanied by flank pain and hematuria and is frequently mistaken for RCC or transitional cell carcinoma of the renal pelvis [ 3 , 5 , 6 ]. CDC, however, can be identified based on gross, microscopic, histochemical, and immunohistochemical features. Macroscopically, CDCs are often located at the confluence of the medulla and renal pelvis, and show a characteristic gray-white-tan color, with absence of foci of necrosis and hemorrhage [ 7 ]. Histologically, CDC presents a tubulo-papillary morphology, often accompanied by desmoplasia, atypia in collecting ducts, and intratubular spread [ 7 ], features rarely seen in RCC. Histochemically, CDC cells contain intracytoplasmic mucicarminophilic material, where RCCs do not [ 7 ]. CDCs are also positive by immunohistochemical staining with high-molecular-weight keratin and lectin, proteins typically expressed in the epithelium of the distal tubules [ 8 , 9 ]. Conversely, almost all other renal carcinomas express antigens widely expressed in the cells of the proximal tubules, such as low molecular weight cytokeratins and vimentin [ 9 ]. Although little is known about the genetic profile of CDCs, a DNA flow cytometry study has demonstrated aneuploidy in 90% of these tumors [ 5 ], and cytogenetics has shown frequent monosomy of chromosomes 1, 6, 8, 14, 15, and 22 [ 10 , 11 ]. Loss of heterozygosity (LOH) analysis in six CDCs revealed allelic loss at chromosomes 8p and 13q in 50% of the tumors, and rarely at the short arm of chromosome 3 [ 12 , 13 ]. This data suggests that distinct genetic alterations from those observed in RCC, in which 3p LOH is common [ 14 ], occur in the development of this rare renal tumor. To improve our understanding of the biology of CDC and to explore the possibility that different genes may be involved in the etiology and prognosis of this neoplasm, we analyzed by immunohistochemistry eleven cases of CDC for the expression of five genes (Fez1, Fhit, p53, p27, and bcl2) often involved in the development of many common cancers. These findings were correlated with conventional clinical-pathological features including clinical outcome.
Background
Renal cancer accounts for 3% of adult malignancies. An estimated 35,710 new cases are expected to occur in the U.S. in the current year and 12,480 patients will die of this disease [ 1 ]. The majority of renal cancers arises from the proximal tubular epithelium, with a characteristic clear or granular cell appearance by light microscopy, and is referred to as renal cell carcinoma (RCC).Recent evidence suggests that solid renal parenchymal tumors arising in the distal portion of the nephron, such as oncocytomas, chromophobe renal cell carcinomas and collecting duct carcinomas represent an heterogeneous group of neoplasm from both clinical and biological perspectives [ 2 ]. Collecting duct carcinoma (CDC), also known as Bellini duct carcinoma, is a rare but highly aggressive renal neoplasm arising from the distal portion of the nephron, and represents approximately 2% of all the RCCs [ 3 , 4 ]. Clinically, CDC appears as a renal mass often accompanied by flank pain and hematuria and is frequently mistaken for RCC or transitional cell carcinoma of the renal pelvis [ 3 , 5 , 6 ]. CDC, however, can be identified based on gross, microscopic, histochemical, and immunohistochemical features. Macroscopically, CDCs are often located at the confluence of the medulla and renal pelvis, and show a characteristic gray-white-tan color, with absence of foci of necrosis and hemorrhage [ 7 ]. Histologically, CDC presents a tubulo-papillary morphology, often accompanied by desmoplasia, atypia in collecting ducts, and intratubular spread [ 7 ], features rarely seen in RCC.Histochemically, CDC cells contain intracytoplasmic mucicarminophilic material, where RCCs do not [ 7 ]. CDCs are also positive by immunohistochemical staining with high-molecular-weight keratin and lectin, proteins typically expressed in the epithelium of the distal tubules [ 8 , 9 ]. Conversely, almost all other renal carcinomas express antigens widely expressed in the cells of the proximal tubules, such as low molecular weight cytokeratins and vimentin [ 9 ]. Although little is known about the genetic profile of CDCs, a DNA flow cytometry study has demonstrated aneuploidy in 90% of these tumors [ 5 ], and cytogenetics has shown frequent monosomy of chromosomes 1, 6, 8, 14, 15, and 22 [ 10 , 11 ].Loss of heterozygosity (LOH) analysis in six CDCs revealed allelic loss at chromosomes 8p and 13q in 50% of the tumors, and rarely at the short arm of chromosome 3 [ 12 , 13 ]. This data suggests that distinct genetic alterations from those observed in RCC, in which 3p LOH is common [ 14 ], occur in the development of this rare renal tumor.To improve our understanding of the biology of CDC and to explore the possibility that different genes may be involved in the etiology and prognosis of this neoplasm, we analyzed by immunohistochemistry eleven cases of CDC for the expression of five genes (Fez1, Fhit, p53, p27, and bcl2) often involved in the development of many common cancers. These findings were correlated with conventional clinical-pathological features including clinical outcome.Methods Patients Eleven patients with primary CDC (two women and nine men; age range 40 to 84 years, mean 62) underwent radical nephrectomy between 1983 and 2000 in the Department of Urology, University of Padua, Italy. Tissue specimens from these tumors were registered in the Department of Pathology at the same institution. All eleven patients had an adequate clinical follow up and were included in our study. Eight of the 11 patients had metastatic disease, five with lymph node metastasis and five with distant metastases at the time of surgery. None of these eleven patients received any systemic therapy before surgery. Samples were fixed in 10% buffered formalin for routine histological processing and were stained with hematoxylin and eosin. Pathological study Immunohistochemical reactions using anticytokeratin (Keratins 5, 8, 10, an 18) monoclonal antibodies, and  Ulex Europaeus Lectin , polyclonal antibody were performed as previously described [ 15 ]. The tumors were classified histologically according to standardized criteria [ 3 , 5 - 7 ] and staged according to the guidelines of the tumor-node-metastasis (TNM) classification of malignant tumor [ 16 ]. Immunohistochemistry Paraffin sections containing non-neoplastic kidney as well as neoplastic areas were deparaffinized according to standard procedures followed by rehydration through graded ethanol series, and mounted on positively charged slides. Immunostaining was performed as previously described [ 17 ]. Briefly for Fez1, Fhit, p53, and p27 immunostaining, slides were immersed in citrate buffer [0.01 M sodium citrate (pH6.0)] and heated in a microwave oven at 600 W (three times for 5 min each) to enhance antigen retrieval. For retrieval of Bcl2 oncoprotein, we used the target retrieval solution, high pH from DAKO (Carpinteria, CA, USA) per manufacturers instructions. The primary antibodies used in this study were: anti-Fez1 rabbit polyclonal antibody [ 17 ], anti-Fhit rabbit polyclonal antibody (Zymed Laboratories, San Francisco, CA) at 1:1,000 dilution, anti-p53, anti-p27, anti-Bcl2 (DAKO, Carpinteria, CA, USA) at dilution of 1:25, 1:50, and 1:40 respectively, as specified by the manufacturers. The primary antibodies were omitted and replaced with pre-immune serum in the negative controls. Sections were reacted with biotinylated anti-rabbit or anti mouse antibodies and streptavidin-biotin-peroxidase (Histostain-SP Kit; Zymed laboratories, San Francisco, CA). Diaminobenzidine (DAB) was used as a chromogen substrate to visualize staining. Finally, sections were washed in distilled water and weakly counterstained with Harry's modified hematoxylin. The immunostaining was evaluated by two pathologists in a blinded fashion (A.V.; R.B.). For statistical analysis, cases were scored as positive if they had more than 20% (p53) or more than 40% (Fez1 and p27) of positive cells. Fhit and bcl2 cases showed either all positive or all negative cells and were scored accordingly. Statistical analysis We evaluated the association between each marker's expression (positive or negative) and each clinical-pathological outcome (metastasis and stage) with Fisher's exact test. For survival, we used the Kaplan-Meier method and log-rank test, as well as Cox proportional hazards regression.
Methods
Patients Eleven patients with primary CDC (two women and nine men; age range 40 to 84 years, mean 62) underwent radical nephrectomy between 1983 and 2000 in the Department of Urology, University of Padua, Italy. Tissue specimens from these tumors were registered in the Department of Pathology at the same institution. All eleven patients had an adequate clinical follow up and were included in our study. Eight of the 11 patients had metastatic disease, five with lymph node metastasis and five with distant metastases at the time of surgery. None of these eleven patients received any systemic therapy before surgery. Samples were fixed in 10% buffered formalin for routine histological processing and were stained with hematoxylin and eosin.
Patients
Eleven patients with primary CDC (two women and nine men; age range 40 to 84 years, mean 62) underwent radical nephrectomy between 1983 and 2000 in the Department of Urology, University of Padua, Italy. Tissue specimens from these tumors were registered in the Department of Pathology at the same institution. All eleven patients had an adequate clinical follow up and were included in our study. Eight of the 11 patients had metastatic disease, five with lymph node metastasis and five with distant metastases at the time of surgery. None of these eleven patients received any systemic therapy before surgery. Samples were fixed in 10% buffered formalin for routine histological processing and were stained with hematoxylin and eosin.Pathological study Immunohistochemical reactions using anticytokeratin (Keratins 5, 8, 10, an 18) monoclonal antibodies, and  Ulex Europaeus Lectin , polyclonal antibody were performed as previously described [ 15 ]. The tumors were classified histologically according to standardized criteria [ 3 , 5 - 7 ] and staged according to the guidelines of the tumor-node-metastasis (TNM) classification of malignant tumor [ 16 ].
Pathological study
Immunohistochemical reactions using anticytokeratin (Keratins 5, 8, 10, an 18) monoclonal antibodies, and  Ulex Europaeus Lectin , polyclonal antibody were performed as previously described [ 15 ]. The tumors were classified histologically according to standardized criteria [ 3 , 5 - 7 ] and staged according to the guidelines of the tumor-node-metastasis (TNM) classification of malignant tumor [ 16 ].Ulex Europaeus LectinImmunohistochemistry Paraffin sections containing non-neoplastic kidney as well as neoplastic areas were deparaffinized according to standard procedures followed by rehydration through graded ethanol series, and mounted on positively charged slides. Immunostaining was performed as previously described [ 17 ]. Briefly for Fez1, Fhit, p53, and p27 immunostaining, slides were immersed in citrate buffer [0.01 M sodium citrate (pH6.0)] and heated in a microwave oven at 600 W (three times for 5 min each) to enhance antigen retrieval. For retrieval of Bcl2 oncoprotein, we used the target retrieval solution, high pH from DAKO (Carpinteria, CA, USA) per manufacturers instructions. The primary antibodies used in this study were: anti-Fez1 rabbit polyclonal antibody [ 17 ], anti-Fhit rabbit polyclonal antibody (Zymed Laboratories, San Francisco, CA) at 1:1,000 dilution, anti-p53, anti-p27, anti-Bcl2 (DAKO, Carpinteria, CA, USA) at dilution of 1:25, 1:50, and 1:40 respectively, as specified by the manufacturers. The primary antibodies were omitted and replaced with pre-immune serum in the negative controls. Sections were reacted with biotinylated anti-rabbit or anti mouse antibodies and streptavidin-biotin-peroxidase (Histostain-SP Kit; Zymed laboratories, San Francisco, CA). Diaminobenzidine (DAB) was used as a chromogen substrate to visualize staining. Finally, sections were washed in distilled water and weakly counterstained with Harry's modified hematoxylin. The immunostaining was evaluated by two pathologists in a blinded fashion (A.V.; R.B.). For statistical analysis, cases were scored as positive if they had more than 20% (p53) or more than 40% (Fez1 and p27) of positive cells. Fhit and bcl2 cases showed either all positive or all negative cells and were scored accordingly.
Immunohistochemistry
Paraffin sections containing non-neoplastic kidney as well as neoplastic areas were deparaffinized according to standard procedures followed by rehydration through graded ethanol series, and mounted on positively charged slides. Immunostaining was performed as previously described [ 17 ]. Briefly for Fez1, Fhit, p53, and p27 immunostaining, slides were immersed in citrate buffer [0.01 M sodium citrate (pH6.0)] and heated in a microwave oven at 600 W (three times for 5 min each) to enhance antigen retrieval. For retrieval of Bcl2 oncoprotein, we used the target retrieval solution, high pH from DAKO (Carpinteria, CA, USA) per manufacturers instructions. The primary antibodies used in this study were: anti-Fez1 rabbit polyclonal antibody [ 17 ], anti-Fhit rabbit polyclonal antibody (Zymed Laboratories, San Francisco, CA) at 1:1,000 dilution, anti-p53, anti-p27, anti-Bcl2 (DAKO, Carpinteria, CA, USA) at dilution of 1:25, 1:50, and 1:40 respectively, as specified by the manufacturers. The primary antibodies were omitted and replaced with pre-immune serum in the negative controls. Sections were reacted with biotinylated anti-rabbit or anti mouse antibodies and streptavidin-biotin-peroxidase (Histostain-SP Kit; Zymed laboratories, San Francisco, CA). Diaminobenzidine (DAB) was used as a chromogen substrate to visualize staining. Finally, sections were washed in distilled water and weakly counterstained with Harry's modified hematoxylin. The immunostaining was evaluated by two pathologists in a blinded fashion (A.V.; R.B.). For statistical analysis, cases were scored as positive if they had more than 20% (p53) or more than 40% (Fez1 and p27) of positive cells. Fhit and bcl2 cases showed either all positive or all negative cells and were scored accordingly.Statistical analysis We evaluated the association between each marker's expression (positive or negative) and each clinical-pathological outcome (metastasis and stage) with Fisher's exact test. For survival, we used the Kaplan-Meier method and log-rank test, as well as Cox proportional hazards regression.
Statistical analysis
We evaluated the association between each marker's expression (positive or negative) and each clinical-pathological outcome (metastasis and stage) with Fisher's exact test. For survival, we used the Kaplan-Meier method and log-rank test, as well as Cox proportional hazards regression.Results Pathological study All cases showed several common microscopic features. The main tumor consisted of a tubulo-papillary carcinoma showing pleomorphism and a high mitotic rate with several bizarre mitotic figures, and presence of small cystic spaces surrounded by a highly desmoplastic stroma. Two cases showed a predominant papillary pattern accompanied with dilated and atypical changes in the epithelium of the collecting ducts in the adjacent renal medulla, which was the most convincing feature supporting a collecting duct origin of these tumors. Metastases, when available for examination, were histologically similar to the infiltrating part of the primary tumors. Immunocytochemically, tumor cells from all cases examined did not express keratin 10, while strong positivity to anti-keratin 5, 8, and 18 antibodies, and anti-UEL antibody was observed. The positivity to high molecular weight keratin and UEL further substantiate the diagnosis of CDC. The clinical-pathological features and protein expression of the study's 11 cases are shown in Table  1 . Fez1 expression Our results showed that Fez1 protein expression was undetectable in six of the eleven (55%) cases. One case showed a substantial reduction of expression with 60% of the tumor cells negative, while the remaining four showed diffuse positivity for Fez1 immunostaining (Figures  1A  and  1B ). Overall 64% of the cases showed absence or reduction of Fez1 expression. Loss and reduction of Fez1 were correlated with a higher prevalence of lymph node metastases (71% vs. 0%, p = 0.061), although the same was not true for distant metastases or tumor stage. Fez1-negative tumors also tended to have higher mortality than Fez1-positive tumors. Median survival time was estimated to be 17 months for the former group, while median survival was not reached for the latter group during the course of the study (i.e., fewer than half of the patients died) (p = 0.078; Figure  2A ). This corresponds to an estimated 5-fold increase in mortality risk for the Fez1-negative CDCs (hazard ratio of 5.5). Fhit expression Fhit protein was absent in three of eleven cases (27%), and the remaining eight showed diffuse immunoreactivity (Figure  1C ). Reduction of Fhit protein expression did not correlate with any of the clinicopathological features of the tumors. There was a tendency for Fhit-negative cases to have worse survival than Fhit-positive cases (median survival times of 17 vs. 35 months, respectively; hazard ratio of 2.7), although the difference was not statistically significant (p = 0.206: Figure  2B ). p53 expression p53 protein was found to be overexpressed mainly in the nucleus in four of eleven cases (36%) (Figure  1D ). One of the four positive cases showed overexpression in 85% of the cancer cells, whereas the remaining three showed 20%, 30% and 50% of positive cancer cells, respectively. p53 was not detectable in seven of eleven cases (63%) as well as in the normal renal epithelium adjacent to tumor. Overexpression of p53 was not related to histologic grade, tumor stage, lymph node status, and survival. p27 expression Immunoreactivity for p27 was observed in the nuclei of most glomerular and tubular cells in the normal kidney. p27 immunostaining in CDC showed high variability. Protein expression was absent in five of eleven cases (45.5%). All the remaining six carcinomas showed a high percentage (> 40%) of p27 positive cells. The expression of p27 protein was detected exclusively in the cytoplasm in five of the six positive cases (90%) while a mixture of nuclear and cytoplasmic protein staining was observed in the last case (Figure  1E ). Overall, we observed lack or subcellular compartmentalization of p27 in 90% of the cases. The status of p27 did not correlate to any of the clinical-pathological parameters tested. Bcl2 expression Bcl2 protein expression in 100% of the CDC tumor cells was observed in four of eleven cases (36%) (Figure  1F ), while the remaining seven (64%) were negative. Statistical analysis did not reveal any significant correlation between Bcl2 expression and other clinical-pathological parameters. Clinical-pathological features as well as immunohistochemistry results are listed in Table  1 . The proportion of marker expression ranged from 36% (Fez1 and Bcl2) to 73% (Fhit). Marker expression tended to be positively correlated, with the correlation between Fhit and p27 being the strongest (0.67), followed by that between Fhit and Fez1 and Fhit and Bcl2 (both 0.46).
Results
Pathological study All cases showed several common microscopic features. The main tumor consisted of a tubulo-papillary carcinoma showing pleomorphism and a high mitotic rate with several bizarre mitotic figures, and presence of small cystic spaces surrounded by a highly desmoplastic stroma. Two cases showed a predominant papillary pattern accompanied with dilated and atypical changes in the epithelium of the collecting ducts in the adjacent renal medulla, which was the most convincing feature supporting a collecting duct origin of these tumors. Metastases, when available for examination, were histologically similar to the infiltrating part of the primary tumors. Immunocytochemically, tumor cells from all cases examined did not express keratin 10, while strong positivity to anti-keratin 5, 8, and 18 antibodies, and anti-UEL antibody was observed. The positivity to high molecular weight keratin and UEL further substantiate the diagnosis of CDC. The clinical-pathological features and protein expression of the study's 11 cases are shown in Table  1 .
Pathological study
All cases showed several common microscopic features. The main tumor consisted of a tubulo-papillary carcinoma showing pleomorphism and a high mitotic rate with several bizarre mitotic figures, and presence of small cystic spaces surrounded by a highly desmoplastic stroma.Two cases showed a predominant papillary pattern accompanied with dilated and atypical changes in the epithelium of the collecting ducts in the adjacent renal medulla, which was the most convincing feature supporting a collecting duct origin of these tumors. Metastases, when available for examination, were histologically similar to the infiltrating part of the primary tumors. Immunocytochemically, tumor cells from all cases examined did not express keratin 10, while strong positivity to anti-keratin 5, 8, and 18 antibodies, and anti-UEL antibody was observed. The positivity to high molecular weight keratin and UEL further substantiate the diagnosis of CDC. The clinical-pathological features and protein expression of the study's 11 cases are shown in Table  1 .Fez1 expression Our results showed that Fez1 protein expression was undetectable in six of the eleven (55%) cases. One case showed a substantial reduction of expression with 60% of the tumor cells negative, while the remaining four showed diffuse positivity for Fez1 immunostaining (Figures  1A  and  1B ). Overall 64% of the cases showed absence or reduction of Fez1 expression. Loss and reduction of Fez1 were correlated with a higher prevalence of lymph node metastases (71% vs. 0%, p = 0.061), although the same was not true for distant metastases or tumor stage. Fez1-negative tumors also tended to have higher mortality than Fez1-positive tumors. Median survival time was estimated to be 17 months for the former group, while median survival was not reached for the latter group during the course of the study (i.e., fewer than half of the patients died) (p = 0.078; Figure  2A ). This corresponds to an estimated 5-fold increase in mortality risk for the Fez1-negative CDCs (hazard ratio of 5.5).
Fez1 expression
Our results showed that Fez1 protein expression was undetectable in six of the eleven (55%) cases. One case showed a substantial reduction of expression with 60% of the tumor cells negative, while the remaining four showed diffuse positivity for Fez1 immunostaining (Figures  1A  and  1B ). Overall 64% of the cases showed absence or reduction of Fez1 expression. Loss and reduction of Fez1 were correlated with a higher prevalence of lymph node metastases (71% vs. 0%, p = 0.061), although the same was not true for distant metastases or tumor stage. Fez1-negative tumors also tended to have higher mortality than Fez1-positive tumors. Median survival time was estimated to be 17 months for the former group, while median survival was not reached for the latter group during the course of the study (i.e., fewer than half of the patients died) (p = 0.078; Figure  2A ). This corresponds to an estimated 5-fold increase in mortality risk for the Fez1-negative CDCs (hazard ratio of 5.5).Fhit expression Fhit protein was absent in three of eleven cases (27%), and the remaining eight showed diffuse immunoreactivity (Figure  1C ). Reduction of Fhit protein expression did not correlate with any of the clinicopathological features of the tumors. There was a tendency for Fhit-negative cases to have worse survival than Fhit-positive cases (median survival times of 17 vs. 35 months, respectively; hazard ratio of 2.7), although the difference was not statistically significant (p = 0.206: Figure  2B ).
Fhit expression
Fhit protein was absent in three of eleven cases (27%), and the remaining eight showed diffuse immunoreactivity (Figure  1C ). Reduction of Fhit protein expression did not correlate with any of the clinicopathological features of the tumors. There was a tendency for Fhit-negative cases to have worse survival than Fhit-positive cases (median survival times of 17 vs. 35 months, respectively; hazard ratio of 2.7), although the difference was not statistically significant (p = 0.206: Figure  2B ).p53 expression p53 protein was found to be overexpressed mainly in the nucleus in four of eleven cases (36%) (Figure  1D ). One of the four positive cases showed overexpression in 85% of the cancer cells, whereas the remaining three showed 20%, 30% and 50% of positive cancer cells, respectively. p53 was not detectable in seven of eleven cases (63%) as well as in the normal renal epithelium adjacent to tumor. Overexpression of p53 was not related to histologic grade, tumor stage, lymph node status, and survival.
p53 expression
p53 protein was found to be overexpressed mainly in the nucleus in four of eleven cases (36%) (Figure  1D ). One of the four positive cases showed overexpression in 85% of the cancer cells, whereas the remaining three showed 20%, 30% and 50% of positive cancer cells, respectively. p53 was not detectable in seven of eleven cases (63%) as well as in the normal renal epithelium adjacent to tumor. Overexpression of p53 was not related to histologic grade, tumor stage, lymph node status, and survival.p27 expression Immunoreactivity for p27 was observed in the nuclei of most glomerular and tubular cells in the normal kidney. p27 immunostaining in CDC showed high variability. Protein expression was absent in five of eleven cases (45.5%). All the remaining six carcinomas showed a high percentage (> 40%) of p27 positive cells. The expression of p27 protein was detected exclusively in the cytoplasm in five of the six positive cases (90%) while a mixture of nuclear and cytoplasmic protein staining was observed in the last case (Figure  1E ). Overall, we observed lack or subcellular compartmentalization of p27 in 90% of the cases. The status of p27 did not correlate to any of the clinical-pathological parameters tested.
p27 expression
Immunoreactivity for p27 was observed in the nuclei of most glomerular and tubular cells in the normal kidney. p27 immunostaining in CDC showed high variability. Protein expression was absent in five of eleven cases (45.5%). All the remaining six carcinomas showed a high percentage (> 40%) of p27 positive cells. The expression of p27 protein was detected exclusively in the cytoplasm in five of the six positive cases (90%) while a mixture of nuclear and cytoplasmic protein staining was observed in the last case (Figure  1E ). Overall, we observed lack or subcellular compartmentalization of p27 in 90% of the cases. The status of p27 did not correlate to any of the clinical-pathological parameters tested.Bcl2 expression Bcl2 protein expression in 100% of the CDC tumor cells was observed in four of eleven cases (36%) (Figure  1F ), while the remaining seven (64%) were negative. Statistical analysis did not reveal any significant correlation between Bcl2 expression and other clinical-pathological parameters. Clinical-pathological features as well as immunohistochemistry results are listed in Table  1 . The proportion of marker expression ranged from 36% (Fez1 and Bcl2) to 73% (Fhit). Marker expression tended to be positively correlated, with the correlation between Fhit and p27 being the strongest (0.67), followed by that between Fhit and Fez1 and Fhit and Bcl2 (both 0.46).
Bcl2 expression
Bcl2 protein expression in 100% of the CDC tumor cells was observed in four of eleven cases (36%) (Figure  1F ), while the remaining seven (64%) were negative. Statistical analysis did not reveal any significant correlation between Bcl2 expression and other clinical-pathological parameters.Clinical-pathological features as well as immunohistochemistry results are listed in Table  1 . The proportion of marker expression ranged from 36% (Fez1 and Bcl2) to 73% (Fhit). Marker expression tended to be positively correlated, with the correlation between Fhit and p27 being the strongest (0.67), followed by that between Fhit and Fez1 and Fhit and Bcl2 (both 0.46).Discussion The application of the most recent molecular cytogenetic techniques revealed that renal parenchymal tumors can be classified into distinct subtypes based on the combination of specific genetic alterations [ 18 ]. The pathological and immunohistochemical description as well as the cytogenetic abnormalities support the hypothesis that CDC is more similar to urothelial carcinoma than to clear cell carcinoma of the kidney. Indeed, whereas allelic deletion of the short arm of chromosome 3 is considered a genetic hallmark of clear cell carcinoma, LOH at chromosomes 8p, 9p, and 17p has been frequently described in both transitional cell carcinoma and CDC. Here, we have reported the results of our immunohistochemical analysis of the expression of five genes ( FEZ1 ,  FHIT ,  P53 ,  P27 kip 1 , and  BCL2 ) in a relatively large series of CDCs (eleven cases), considering the rarity of this tumor. FEZ1/LZTS1  (leucine zipper tumor suppressor 1) is a putative tumor suppressor gene located at 8p22 [ 19 ]. Studies have indicated this chromosomal region is the location of an important tumor suppressor gene (TSG) [ 20 ]. LOH at 8p has been described in 50% of the CDCs studied [ 12 ], suggesting that a TSG in this region may also play a role in the development of this rare tumor. In our study, we found loss of Fez1 expression in the majority of CDCs studied and a correlation with the presence of lymph node metastasis. Furthermore, lack of Fez1 protein correlated with a poorer prognosis in 90% of patients with median survival of 17 months. FEZ1  encodes a 67-kDa leucine-zipper protein with a region of similarity to cAMP-dependent activated protein [ 19 ]. Mutations of  FEZ1  gene have been reported in several solid tumors, including prostate, breast, esophageal, and gastric carcinomas [ 17 , 19 ]. In addition, reduced Fez1 expression is associated with high-grade transitional cell carcinoma of the bladder [ 21 ]. Recent studies have shown that the introduction of  FEZ1  into Fez1 null cancer cells reduced cell growth with the accumulation of cells at late S to G 2 /M phase of the cell cycle. Conversely, inhibition of Fez1 expression stimulated cells growth [ 22 ]. Furthermore, LOH at the chromosomal region where the  FEZ1  gene lies (8p21-22) has been also associated with the invasive behavior of breast cancer [ 23 ] and with prostate cancer progression [ 24 ]. These data are consistent with an important role of  FEZ1  in several human cancers including CDC. The tumor suppressor gene  FHIT  maps to the short arm of chromosome 3 (3p14.2), encompasses the common  FRA3B  fragile region, and encodes for a protein of 16.8 kDa, with diadenosine triphosphate hydrolase activity [ 25 ]. Reduction of Fhit protein expression as consequence of alteration of the  FHIT  gene has been observed by immunohistochemistry in many types of cancers [ 26 , 27 ]. Although Shoemberg et al. did not detect LOH involving chromosome 3p [ 12 ], Hadaczek et al. reported LOH at 3p in two cases of CDC [ 13 ]. The same authors described a correlation between reduced Fhit expression and 3p allelic loss in renal carcinomas, particularly in CDCs [ 28 ]. While in our study Fhit inactivation does not seem to be a common event in CDC, an involvement of the  FHIT  gene in tumorigenesis of this rare tumor cannot be excluded. The  TP53  tumor suppressor gene maps to chromosome 17p13.1 and plays a major role in DNA transcription, cell growth, proliferation and apoptosis process [ 29 ]. In normal cells, expression of wild type p53 protein is generally below the detection level when studied by immunohistochemical method. However, p53 gene point mutations occur frequently (22–76%) in different solid neoplasms. Mutated p53 protein, being more resistant to degradation, accumulates in the cells and can be detected by immunohistochemistry. Although an association between p53 protein overexpression and tumor stage, grade and survival has been observed in RCC [ 30 ] our data suggest that involvement of p53 alterations does not occur with the same frequency in CDC. P27 is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. The expression of this important protein is regulated by cell to cell contact inhibition as well as by specific growth factors, such as transforming growth factor (TGF-β). In addition to its role as a CDKI, p27 is considered a putative tumor suppressor gene, a major regulator of drug resistance in solid tumors, and a promoter of apoptosis. p27 acts also as a safeguard against inflammatory injury and has a role in cell differentiation [ 31 ]. It has been suggested that loss of the p27 negative cell cycle regulation may contribute to oncogenesis and tumor progression in several tumor types. In renal cell carcinoma, Kamai et al. reported that low level of p27 protein was associated with tumor invasion and unfavorable prognosis, suggesting p27 as a powerful prognostic marker for survival in urinary tract cancer [ 32 ]. Masuda et al. indicated that p27 has an independent predictive prognostic value for transitional cell carcinoma of the renal pelvis [ 33 ]. Our results show that p27 loss or subcellular compartmentalization represents a frequent feature in CDCs. Previous studies have noted that cytoplasmic localization of p27 lead to an inactivation of its normal function as negative cell cycle regulator [ 34 ]. Nevertheless, we did not find statistical correlation to assess its involvement in CDC biology, possibly due to the limited number of tumors studied. The proto-oncogene  BCL2 , implicated in the regulation of cell death by inhibiting apoptosis, seems to be vital in normal kidney morphogenesis. In fact, Bcl2 deficient mice develop polycystic kidneys characterized by dilated proximal and distal tubules [ 35 ]. High levels of Bcl2 protein expression have been found in many different types of cancer, suggesting a possible role for Bcl2 deregulation of apoptosis and malignant tissue transformation. Expression of Bcl2 has also been associated with poor prognosis in patients with various cancers including prostate cancer [ 36 ]. In the present study, Bcl2 expression was not associated with any clinical-pathological variables. Our results suggest a potential association between FEZ1 expression and CDC pathology and prognosis. No similar patterns were seen for any of the other markers studied. Even so, the statistical power of the study was limited and negative findings should not be construed as evidence that these markers are not important. Rather, a larger study would need to be carried out to further investigate their role in CDC.
Discussion
The application of the most recent molecular cytogenetic techniques revealed that renal parenchymal tumors can be classified into distinct subtypes based on the combination of specific genetic alterations [ 18 ]. The pathological and immunohistochemical description as well as the cytogenetic abnormalities support the hypothesis that CDC is more similar to urothelial carcinoma than to clear cell carcinoma of the kidney. Indeed, whereas allelic deletion of the short arm of chromosome 3 is considered a genetic hallmark of clear cell carcinoma, LOH at chromosomes 8p, 9p, and 17p has been frequently described in both transitional cell carcinoma and CDC. Here, we have reported the results of our immunohistochemical analysis of the expression of five genes ( FEZ1 ,  FHIT ,  P53 ,  P27 kip 1 , and  BCL2 ) in a relatively large series of CDCs (eleven cases), considering the rarity of this tumor.FEZ1FHITP53P27kipBCL2FEZ1/LZTS1  (leucine zipper tumor suppressor 1) is a putative tumor suppressor gene located at 8p22 [ 19 ]. Studies have indicated this chromosomal region is the location of an important tumor suppressor gene (TSG) [ 20 ]. LOH at 8p has been described in 50% of the CDCs studied [ 12 ], suggesting that a TSG in this region may also play a role in the development of this rare tumor. In our study, we found loss of Fez1 expression in the majority of CDCs studied and a correlation with the presence of lymph node metastasis. Furthermore, lack of Fez1 protein correlated with a poorer prognosis in 90% of patients with median survival of 17 months.FEZ1/LZTS1FEZ1  encodes a 67-kDa leucine-zipper protein with a region of similarity to cAMP-dependent activated protein [ 19 ]. Mutations of  FEZ1  gene have been reported in several solid tumors, including prostate, breast, esophageal, and gastric carcinomas [ 17 , 19 ]. In addition, reduced Fez1 expression is associated with high-grade transitional cell carcinoma of the bladder [ 21 ]. Recent studies have shown that the introduction of  FEZ1  into Fez1 null cancer cells reduced cell growth with the accumulation of cells at late S to G 2 /M phase of the cell cycle. Conversely, inhibition of Fez1 expression stimulated cells growth [ 22 ]. Furthermore, LOH at the chromosomal region where the  FEZ1  gene lies (8p21-22) has been also associated with the invasive behavior of breast cancer [ 23 ] and with prostate cancer progression [ 24 ]. These data are consistent with an important role of  FEZ1  in several human cancers including CDC.FEZ1FEZ1FEZ1FEZ1FEZ1The tumor suppressor gene  FHIT  maps to the short arm of chromosome 3 (3p14.2), encompasses the common  FRA3B  fragile region, and encodes for a protein of 16.8 kDa, with diadenosine triphosphate hydrolase activity [ 25 ]. Reduction of Fhit protein expression as consequence of alteration of the  FHIT  gene has been observed by immunohistochemistry in many types of cancers [ 26 , 27 ]. Although Shoemberg et al. did not detect LOH involving chromosome 3p [ 12 ], Hadaczek et al. reported LOH at 3p in two cases of CDC [ 13 ]. The same authors described a correlation between reduced Fhit expression and 3p allelic loss in renal carcinomas, particularly in CDCs [ 28 ]. While in our study Fhit inactivation does not seem to be a common event in CDC, an involvement of the  FHIT  gene in tumorigenesis of this rare tumor cannot be excluded.FHITFRA3BFHITFHITThe  TP53  tumor suppressor gene maps to chromosome 17p13.1 and plays a major role in DNA transcription, cell growth, proliferation and apoptosis process [ 29 ]. In normal cells, expression of wild type p53 protein is generally below the detection level when studied by immunohistochemical method. However, p53 gene point mutations occur frequently (22–76%) in different solid neoplasms. Mutated p53 protein, being more resistant to degradation, accumulates in the cells and can be detected by immunohistochemistry. Although an association between p53 protein overexpression and tumor stage, grade and survival has been observed in RCC [ 30 ] our data suggest that involvement of p53 alterations does not occur with the same frequency in CDC.TP53P27 is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. The expression of this important protein is regulated by cell to cell contact inhibition as well as by specific growth factors, such as transforming growth factor (TGF-β). In addition to its role as a CDKI, p27 is considered a putative tumor suppressor gene, a major regulator of drug resistance in solid tumors, and a promoter of apoptosis. p27 acts also as a safeguard against inflammatory injury and has a role in cell differentiation [ 31 ]. It has been suggested that loss of the p27 negative cell cycle regulation may contribute to oncogenesis and tumor progression in several tumor types. In renal cell carcinoma, Kamai et al. reported that low level of p27 protein was associated with tumor invasion and unfavorable prognosis, suggesting p27 as a powerful prognostic marker for survival in urinary tract cancer [ 32 ]. Masuda et al. indicated that p27 has an independent predictive prognostic value for transitional cell carcinoma of the renal pelvis [ 33 ]. Our results show that p27 loss or subcellular compartmentalization represents a frequent feature in CDCs. Previous studies have noted that cytoplasmic localization of p27 lead to an inactivation of its normal function as negative cell cycle regulator [ 34 ].Nevertheless, we did not find statistical correlation to assess its involvement in CDC biology, possibly due to the limited number of tumors studied.The proto-oncogene  BCL2 , implicated in the regulation of cell death by inhibiting apoptosis, seems to be vital in normal kidney morphogenesis. In fact, Bcl2 deficient mice develop polycystic kidneys characterized by dilated proximal and distal tubules [ 35 ]. High levels of Bcl2 protein expression have been found in many different types of cancer, suggesting a possible role for Bcl2 deregulation of apoptosis and malignant tissue transformation. Expression of Bcl2 has also been associated with poor prognosis in patients with various cancers including prostate cancer [ 36 ]. In the present study, Bcl2 expression was not associated with any clinical-pathological variables.BCL2Our results suggest a potential association between FEZ1 expression and CDC pathology and prognosis. No similar patterns were seen for any of the other markers studied. Even so, the statistical power of the study was limited and negative findings should not be construed as evidence that these markers are not important. Rather, a larger study would need to be carried out to further investigate their role in CDC.Conclusions Our results suggest that Fez1 expression may be associated to both clinical-pathological features and survival in patients with CDC.  FEZ1  gene alterations may be linked to the high frequency of LOH found at 8p22, where  FEZ1  resides. The lack of similar association for the other four genes studied may be due to the low statistical power of the study.
Conclusions
Our results suggest that Fez1 expression may be associated to both clinical-pathological features and survival in patients with CDC.  FEZ1  gene alterations may be linked to the high frequency of LOH found at 8p22, where  FEZ1  resides. The lack of similar association for the other four genes studied may be due to the low statistical power of the study.FEZ1FEZ1Competing interests None declared.
Competing interests
None declared.Abbreviations CDC, Collecting Duct Carcinoma; RCC, renal cell carcinoma; LOH, loss of heterozygosity
Abbreviations
CDC, Collecting Duct Carcinoma; RCC, renal cell carcinoma; LOH, loss of heterozygosityAuthors' contributions A.V. carried out the immunohistochemical analysis and reviewed the slides, he also contributed to the draft of the manuscript. T.P.G. was responsible for the clinical study. M.G. carried out the original histopathological diagnosis. H.I. participated in the immunohistochemical analysis and statistical analysis. E.G. participated in the immunohistochemical analysis and statistical analysis. F.P. was responsible for the clinical study. L.G.G. participated in designing the study and in drafting the manuscript. C.M.C. participated in designing the study. R.B. participated in the original design and coordination of the study, and in writing the manuscript
Authors' contributions
A.V. carried out the immunohistochemical analysis and reviewed the slides, he also contributed to the draft of the manuscript. T.P.G. was responsible for the clinical study. M.G. carried out the original histopathological diagnosis. H.I. participated in the immunohistochemical analysis and statistical analysis. E.G. participated in the immunohistochemical analysis and statistical analysis. F.P. was responsible for the clinical study. L.G.G. participated in designing the study and in drafting the manuscript. C.M.C. participated in designing the study. R.B. participated in the original design and coordination of the study, and in writing the manuscriptPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background Pelvic lymph nodes metastasis is an important prognostic factor for patients with cervical carcinoma. However, the relationships between the number of positive nodes, site of metastases nodes, adjuvant therapy and the prognosis is controversial. The purpose of this study was to investigate the influence of positive lymph nodes on the prognosis of Chinese women with stage IB1-IIB cervical carcinoma.
Background
Pelvic lymph nodes metastasis is an important prognostic factor for patients with cervical carcinoma. However, the relationships between the number of positive nodes, site of metastases nodes, adjuvant therapy and the prognosis is controversial. The purpose of this study was to investigate the influence of positive lymph nodes on the prognosis of Chinese women with stage IB1-IIB cervical carcinoma.Patients and methods Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery in Cancer Hospital, Fudan University. Of these sixty-six patients (16.6%) who were histologically confirmed to have positive pelvic lymph nodes were analyzed retrospectively. The survival was estimated using Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model.
Patients and methods
Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery in Cancer Hospital, Fudan University. Of these sixty-six patients (16.6%) who were histologically confirmed to have positive pelvic lymph nodes were analyzed retrospectively. The survival was estimated using Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model.Results The 5-year survival of the patients with pelvic lymph nodes metastases was 40.7%. Cox proportional hazard model analysis showed that cellular differentiation, the number of positive nodes and adjuvant therapy to be the independent prognostic factors ( P  < 0.05). The 5-year survival of patients with one positive node was higher than that of those with two or more positive nodes (56.5% vs. 36.4%,  P  < 0.05). The distant metastasis rate in the former group (5.9%) was lower than the latter's (32.7%) ( P  = 0.05). However, there was no significant difference of pelvic recurrence between the two groups ( P  > 0.05). The number of positive nodes positively correlated with the level of positive nodes ( P  < 0.01). The 5-year survival of the patients who had no adjuvant therapy (12.6%) was much lower than that (53.7%) of those with adjuvant therapy ( P  < 0.05). However, there was no obvious difference between adjuvant radiotherapy, chemotherapy and chemo-radiotherapy ( P  > 0.05).
Results
The 5-year survival of the patients with pelvic lymph nodes metastases was 40.7%. Cox proportional hazard model analysis showed that cellular differentiation, the number of positive nodes and adjuvant therapy to be the independent prognostic factors ( P  < 0.05). The 5-year survival of patients with one positive node was higher than that of those with two or more positive nodes (56.5% vs. 36.4%,  P  < 0.05). The distant metastasis rate in the former group (5.9%) was lower than the latter's (32.7%) ( P  = 0.05). However, there was no significant difference of pelvic recurrence between the two groups ( P  > 0.05). The number of positive nodes positively correlated with the level of positive nodes ( P  < 0.01). The 5-year survival of the patients who had no adjuvant therapy (12.6%) was much lower than that (53.7%) of those with adjuvant therapy ( P  < 0.05). However, there was no obvious difference between adjuvant radiotherapy, chemotherapy and chemo-radiotherapy ( P  > 0.05).PPPPPPPConclusions The prognosis of patients with stage IB1-IIB node-positive cervical carcinoma who underwent radical surgery alone was very poor. Adjuvant therapy increases the survival rate, decreases the pelvic recurrence and distant metastasis.
Conclusions
The prognosis of patients with stage IB1-IIB node-positive cervical carcinoma who underwent radical surgery alone was very poor. Adjuvant therapy increases the survival rate, decreases the pelvic recurrence and distant metastasis.

Background Although radical radiotherapy (RT) and radical surgery can be the proper choices for patients with early stage cervical cancer, most of the patients in China prefer the radical surgery to RT. Hence, in China the radical surgery has been widely used as first-line therapy for this group of women. Some poor prognostic subgroups have been identified, among these the pelvic lymph node status has been considered as the most important prognostic factor. Radical hysterectomy with bilateral pelvic lymphadenectomy produces an expected 85–90% survival in women with stage IB and IIA cervical carcinoma without lymphatic spread. However, once tumors involve regional lymph nodes, 5-year survival has been reported to be only 30–60% [ 1 ]. In most of the studies the presence of pelvic lymph node metastases has been associated with increased pelvic recurrence and distance metastases, and a decrease in overall survival [ 2 - 7 ]. However, many questions such as the relationship between the numbers, the site of positive nodes, the modality of postoperative multidisciplinary therapy and the prognosis is not yet clear. This study investigated the factors that could predict the prognosis of the patients with stage IB1-IIB node-positive cervical carcinoma.
Background
Although radical radiotherapy (RT) and radical surgery can be the proper choices for patients with early stage cervical cancer, most of the patients in China prefer the radical surgery to RT. Hence, in China the radical surgery has been widely used as first-line therapy for this group of women. Some poor prognostic subgroups have been identified, among these the pelvic lymph node status has been considered as the most important prognostic factor. Radical hysterectomy with bilateral pelvic lymphadenectomy produces an expected 85–90% survival in women with stage IB and IIA cervical carcinoma without lymphatic spread. However, once tumors involve regional lymph nodes, 5-year survival has been reported to be only 30–60% [ 1 ]. In most of the studies the presence of pelvic lymph node metastases has been associated with increased pelvic recurrence and distance metastases, and a decrease in overall survival [ 2 - 7 ]. However, many questions such as the relationship between the numbers, the site of positive nodes, the modality of postoperative multidisciplinary therapy and the prognosis is not yet clear. This study investigated the factors that could predict the prognosis of the patients with stage IB1-IIB node-positive cervical carcinoma.Patients and methods Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery at the Department of Gynecologic Oncology, Cancer Hospital of Fudan University. Of these 66 patients who had undergone Wertheims-Meigs' surgery (radical hysterectomy and pelvic lymphadenectomy) and were histologically confirmed to harbor positive pelvic lymph node were included in this study. The median age at diagnosis was 49 years (range 21 to71). Out of 66, 8 patients were in stage IB1 (12.1%), 37 patients (56.1%) in stage IIA and 21 patients (31.8%) in stage IIB. Histologically 41 women (62.1%) had squamous carcinoma, 20 (30.3%) had adenocarcinoma, 4 (6.1%) adenosquamous carcinoma and 1 patient (1.5%) had small cell carcinoma. The tumors in 4 patients (6.1%) were well differentiation, 46 cases (69.7%) moderately differentiated and 16 (24.2%) poorly differentiated. The average lymph nodes resected were 14.8 per patient while the average positive lymph nodes resected were 3.7 (1~28) per patient. The average diameter of the cervical tumors was 3.6 cm (1~7 cm). The details of the patients' clinical characteristics are listed in Table  1 . Table 1 Clinico-pathologic characteristics of patients with node-positive cervical carcinoma after radical surgery Factors n Percentage (%) Age(yrs) <40 15 22.7 ≥40 51 77.3 Stage IB 8 12.1 IIA 37 56.1 IIB 21 31.8 Tumor size(cm) <4 31 47.0 ≥4 35 53.0 Histology Squamous 41 62.1 Adenocarcinoma 20 30.3 Adenosquamous 4 6.1 Others 1 1.5 Differentiation Poor 16 24.2 Moderate 46 69.7 Well 4 6.1 Pelvic lymph node metastases 1 17 25.8 ≥2 49 74.2 Parametrial extension Negative 58 87.9 Positive 8 12.1 Vaginal margin involved Negative 64 97.0 Positive 2 3.0 Depth of stromal invasion ≤2/3 19 28.8 ≤2/3 47 71.2 Lymphvascular permeation Negative 47 71.2 Positive 19 28.8 Sixty four of these women (97.0%) had brachytherapy in either three or four fractions with a total dose of 15~20 Gy at point A, two weeks prior to radical surgery because of bulky tumor or vaginal vault involvement. Intra-arterial chemotherapy was administered to 11 patients (16.7%) before surgery because of bulky tumor or parametrial extension. The regimen based on cisplatin (CDDP) + 5-Fluorouracil(5-FU) with 2~3 cycles at 3-weeks intervals was used. All patients underwent Wertheims-Meigs' procedure that included radical hysterectomy and bilateral pelvic lymphadenectomy. Three patients had para-aortic lymph node (PALN) sampling because there was suspicion of metastasis. Postoperative external beam irradiation was delivered to 18 patients (27.3%) with 6 MV X-ray routine anterior and posterior portal at a dose of 35~45 Gy at point B with 1.8 Gy daily fraction. Two patients with positive vaginal margin were given extra brachytherapy with 15 Gy 0.5 cm below the vaginal mucosa. 12 cases with positive common iliac node and 3 with PALN metastasis received an additional 40 Gy to the PALN chain area. 10 patients (15.2%) were given postoperative chemotherapy. The regimen consisted CDDP + 5-FU + Cyclophosphamide (CTX) for squamous carcinoma, and CDDP + 5-FU + Mitomycin (MMC) for adenocarcinoma with 2~6 cycles at 3~4-weeks intervals. A total of 19 patients (28.8%) received adjuvant radiotherapy and chemotherapy as mentioned above. Follow-up Patients were evaluated every two months for the first two years and then six monthly for the additional years by clinical interview, or telephone, or letters. Disease-free survival (DFS) was defined as the time from the date of surgery to local or nodal recurrence or metastasis. Overall survival (OS) was calculated from the date of surgery to the date of death. Recurrences were defined as local if they were detected in the pelvis or vagina and distant metastases as detected in extra-pelvic locations. The median follow-up time was 32 months (range 2~108 months). Statistical analysis Data analysis was performed with Statistical package for social sciences (SPSS) version 10.0 statistical package. The survival was calculated by Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model. The correlation analysis was performed by Kendall's method. Pearson's chi-square or Fisher's exact test was used to compare the difference of proportions. A probability value of  P  < 0.05 was considered significant.
Patients and methods
Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery at the Department of Gynecologic Oncology, Cancer Hospital of Fudan University. Of these 66 patients who had undergone Wertheims-Meigs' surgery (radical hysterectomy and pelvic lymphadenectomy) and were histologically confirmed to harbor positive pelvic lymph node were included in this study. The median age at diagnosis was 49 years (range 21 to71). Out of 66, 8 patients were in stage IB1 (12.1%), 37 patients (56.1%) in stage IIA and 21 patients (31.8%) in stage IIB. Histologically 41 women (62.1%) had squamous carcinoma, 20 (30.3%) had adenocarcinoma, 4 (6.1%) adenosquamous carcinoma and 1 patient (1.5%) had small cell carcinoma. The tumors in 4 patients (6.1%) were well differentiation, 46 cases (69.7%) moderately differentiated and 16 (24.2%) poorly differentiated. The average lymph nodes resected were 14.8 per patient while the average positive lymph nodes resected were 3.7 (1~28) per patient. The average diameter of the cervical tumors was 3.6 cm (1~7 cm). The details of the patients' clinical characteristics are listed in Table  1 .Clinico-pathologic characteristics of patients with node-positive cervical carcinoma after radical surgeryFactorsnPercentage (%)Sixty four of these women (97.0%) had brachytherapy in either three or four fractions with a total dose of 15~20 Gy at point A, two weeks prior to radical surgery because of bulky tumor or vaginal vault involvement. Intra-arterial chemotherapy was administered to 11 patients (16.7%) before surgery because of bulky tumor or parametrial extension. The regimen based on cisplatin (CDDP) + 5-Fluorouracil(5-FU) with 2~3 cycles at 3-weeks intervals was used. All patients underwent Wertheims-Meigs' procedure that included radical hysterectomy and bilateral pelvic lymphadenectomy. Three patients had para-aortic lymph node (PALN) sampling because there was suspicion of metastasis.Postoperative external beam irradiation was delivered to 18 patients (27.3%) with 6 MV X-ray routine anterior and posterior portal at a dose of 35~45 Gy at point B with 1.8 Gy daily fraction. Two patients with positive vaginal margin were given extra brachytherapy with 15 Gy 0.5 cm below the vaginal mucosa. 12 cases with positive common iliac node and 3 with PALN metastasis received an additional 40 Gy to the PALN chain area. 10 patients (15.2%) were given postoperative chemotherapy. The regimen consisted CDDP + 5-FU + Cyclophosphamide (CTX) for squamous carcinoma, and CDDP + 5-FU + Mitomycin (MMC) for adenocarcinoma with 2~6 cycles at 3~4-weeks intervals. A total of 19 patients (28.8%) received adjuvant radiotherapy and chemotherapy as mentioned above.Follow-up Patients were evaluated every two months for the first two years and then six monthly for the additional years by clinical interview, or telephone, or letters. Disease-free survival (DFS) was defined as the time from the date of surgery to local or nodal recurrence or metastasis. Overall survival (OS) was calculated from the date of surgery to the date of death. Recurrences were defined as local if they were detected in the pelvis or vagina and distant metastases as detected in extra-pelvic locations. The median follow-up time was 32 months (range 2~108 months).
Follow-up
Patients were evaluated every two months for the first two years and then six monthly for the additional years by clinical interview, or telephone, or letters. Disease-free survival (DFS) was defined as the time from the date of surgery to local or nodal recurrence or metastasis. Overall survival (OS) was calculated from the date of surgery to the date of death. Recurrences were defined as local if they were detected in the pelvis or vagina and distant metastases as detected in extra-pelvic locations. The median follow-up time was 32 months (range 2~108 months).Statistical analysis Data analysis was performed with Statistical package for social sciences (SPSS) version 10.0 statistical package. The survival was calculated by Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model. The correlation analysis was performed by Kendall's method. Pearson's chi-square or Fisher's exact test was used to compare the difference of proportions. A probability value of  P  < 0.05 was considered significant.
Statistical analysis
Data analysis was performed with Statistical package for social sciences (SPSS) version 10.0 statistical package. The survival was calculated by Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model. The correlation analysis was performed by Kendall's method. Pearson's chi-square or Fisher's exact test was used to compare the difference of proportions. A probability value of  P  < 0.05 was considered significant.PResults The 5-year overall survival of the patients with pelvic lymph node metastasis was 40.7%. The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes ( P  < 0.05). The former's distant metastasis rate (5.9%) was lower than the latter's (32.7%) ( P  = 0.05). However, there was no significant difference of pelvic recurrence between them ( P  > 0.05) (Table  2 , Figure  1 ) Table 2 Relationship between the number of positive nodes and prognosis Number of positive nodes n 5-year survival (%) P Recurrence rate (%) P Metastasis rate (%) P 1 17 56.5 0.033 23.5 0.807 5.9 0.050 ≥2 49 36.4 26.5 32.7 Figure 1 Overall survival according to one vs. two or more positive lymph nodes. The 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was less than that (43.1%) of   patients with lower than common iliac node involvement. However, the difference was not significant (P > 0.05). The former's pelvic recurrence rate (13.3%) was lower than the latter's (29.4%) (P > 0.05). However, the patients with common iliac or paraaortic nodes had significantly higher distant metastasis (53.3%) than that of the patients with lower nodes (17.6%) (P < 0.01). The number of the positive lymph nodes was closely correlated with the level of lymph node metastasis. The relative coefficient was 0.557 (P < 0.01) (Table  3 ). Table 3 Relationship between the site of positive nodes and prognosis Site n 5-year survival rate (%) P Recurrence rate (%) P Metastasis rate (%) P Common iliac or above 15 33.3 0.086 13.3 0.318 53.3 0.005 Lower than common iliac 51 43.1 29.4 17.6 The 5-year survival (12.6%) of the   patients who had no adjuvant therapy was much lower than that (53.7%) of   those with adjuvant therapy (P<0.05). The 5-year survival rates of the adjuvant radiotherapy, adjuvant chemotherapy, and adjuvant radio-chemotherapy groups were 53.5%, 49.2% and 56.1% respectively (P > 0.05). The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than those with adjuvant therapeutic groups. However, the differences were not significant (P > 0.05) (Table  4 , Figure  2 ) Table 4 Relationship between adjuvant therapy and prognosis n 5-year survival (%) Recurrence rate (%) Metastasis rate (%) No adjuvant therapy 19 12.6 42.1 31.6 Radiotherapy 18 53.5 22.2 22.2 Chemotherapy 10 49.2 20.0 10.0 Radiochemotherapy 19 56.1 15.8 21.1 Figure 2 Overall survival according to adjuvant therapy [radiotherapy (RT) vs. chemotherapy (CT) vs. radiochemotherapy (RT+CT) vs. no adjuvant therapy (No)]. Multivariate analysis of prognostic factors Cox proportional hazard model analysis showed that cellular differentiation, number of positive nodes and adjuvant therapy to be the independent prognostic factors for survival ( P  < 0.05) (Table  5 ) Table 5 Cox proportional hazard model analysis of variables in predicting overall survival Factors Coefficient RR 95%CI P Age -0.031 0.970 0.932~1.010 0.136 Stage 0.283 1.327 0.379~4.644 0.658 Tumor size 0.332 1.394 0.984~1.975 0.061 Histology -0.035 0.966 0.563~1.659 0.900 Differentiation 0.787 2.196 1.104~4.370 0.025* Number of positive nodes 0.076 1.079 1.006~1.158 0.034* Parametrial extension 0.484 1.623 0.584~4.508 0.353 Vaginal margin involved -1.007 0.365 0.029~4.655 0.438 Depth of stromal invasion 0.423 1.526 0.856~2.722 0.152 Lymphvascular permeation 0.270 1.311 0.500~3.437 0.582 Nerve invasion 0.101 1.106 0.092~13.337 0.937 Adjuvant therapy -1.684 0.186 0.075~0.459 0.000* *  P  < 0.05
Results
The 5-year overall survival of the patients with pelvic lymph node metastasis was 40.7%. The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes ( P  < 0.05). The former's distant metastasis rate (5.9%) was lower than the latter's (32.7%) ( P  = 0.05). However, there was no significant difference of pelvic recurrence between them ( P  > 0.05) (Table  2 , Figure  1 )PPPRelationship between the number of positive nodes and prognosisNumber of positive nodesn5-year survival (%)PPRecurrence rate (%)PPMetastasis rate (%)PPThe 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was less than that (43.1%) of   patients with lower than common iliac node involvement. However, the difference was not significant (P > 0.05). The former's pelvic recurrence rate (13.3%) was lower than the latter's (29.4%) (P > 0.05). However, the patients with common iliac or paraaortic nodes had significantly higher distant metastasis (53.3%) than that of the patients with lower nodes (17.6%) (P < 0.01). The number of the positive lymph nodes was closely correlated with the level of lymph node metastasis. The relative coefficient was 0.557 (P < 0.01) (Table  3 ).Relationship between the site of positive nodes and prognosisSiten5-year survival rate (%)PRecurrence rate (%)PMetastasis rate (%)PThe 5-year survival (12.6%) of the   patients who had no adjuvant therapy was much lower than that (53.7%) of   those with adjuvant therapy (P<0.05). The 5-year survival rates of the adjuvant radiotherapy, adjuvant chemotherapy, and adjuvant radio-chemotherapy groups were 53.5%, 49.2% and 56.1% respectively (P > 0.05). The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than those with adjuvant therapeutic groups. However, the differences were not significant (P > 0.05) (Table  4 , Figure  2 )Relationship between adjuvant therapy and prognosisn5-year survival (%)Recurrence rate (%)Metastasis rate (%)Multivariate analysis of prognostic factors Cox proportional hazard model analysis showed that cellular differentiation, number of positive nodes and adjuvant therapy to be the independent prognostic factors for survival ( P  < 0.05) (Table  5 ) Table 5 Cox proportional hazard model analysis of variables in predicting overall survival Factors Coefficient RR 95%CI P Age -0.031 0.970 0.932~1.010 0.136 Stage 0.283 1.327 0.379~4.644 0.658 Tumor size 0.332 1.394 0.984~1.975 0.061 Histology -0.035 0.966 0.563~1.659 0.900 Differentiation 0.787 2.196 1.104~4.370 0.025* Number of positive nodes 0.076 1.079 1.006~1.158 0.034* Parametrial extension 0.484 1.623 0.584~4.508 0.353 Vaginal margin involved -1.007 0.365 0.029~4.655 0.438 Depth of stromal invasion 0.423 1.526 0.856~2.722 0.152 Lymphvascular permeation 0.270 1.311 0.500~3.437 0.582 Nerve invasion 0.101 1.106 0.092~13.337 0.937 Adjuvant therapy -1.684 0.186 0.075~0.459 0.000* *  P  < 0.05
Multivariate analysis of prognostic factors
Cox proportional hazard model analysis showed that cellular differentiation, number of positive nodes and adjuvant therapy to be the independent prognostic factors for survival ( P  < 0.05) (Table  5 )PCox proportional hazard model analysis of variables in predicting overall survival*  P  < 0.05PDiscussion Cervical cancer remains the third common cancer in women around the world, although its incidence is on the decline in North American and in Europe. It is estimated that there will be 10,370 new cases in 2005 in North America [ 8 ]. In many developing countries, not only is cervical cancer the most frequently occurring cancer among middle-aged women, but it is also a leading cause of death, partly due to the poor access to medical care and the unavailability of routine screening in many of these countries [ 9 ]. In Shanghai, the biggest city in China, there were 150 new cases in 2000. The standard incidence rate was 2.9 per 100 000 [ 10 ]. Radical surgery has been found to be very effective in patients with early stage (IB-IIA) cervical carcinoma [ 11 - 13 ]. However, the 5-year survival has been lingering at 50%-90% during the past twenty years [ 14 ]. The reported risk factors of cervical carcinoma included clinical stage, bulky tumor, histological types, cellular differentiation, deep stromal invasion, parametrial extension, vaginal margin involved, lymphvascular permeation, lymph node metastasis, race and age [ 13 , 15 - 18 ]. In most studies the presence of pelvic lymph node metastases has been associated with increased pelvic recurrence and distance metastases, and a decrease in overall survival. Our Cox proportional hazard model analysis showed cellular differentiation, number of positive nodes and adjuvant therapy to be the independent prognostic factors ( P  < 0.05). The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes ( P  < 0.05). There was no significant difference of pelvic recurrence between them. However, the former's distant metastases rate (5.9%) was lower than the latter's (32.7%) ( P  = 0.05). In the analysis of the site of lymph node metastases, the 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was lower than that (43.1%) of patients with lower than common iliac node involvement. Unlike the series of Tsai the difference in our series was not statistically significant [ 19 ]. This is probably due to limited number of cases with paraaortic or common iliac nodes. The number of positive nodes correlated with the height of positive nodes ( P  < 0.01). The result suggested that in patients with more positive pelvic nodes there is higher chance of nodal metastasis at higher level node basins, which supported the external irradiation of PALN chain area for the multiple pelvic nodes involvement. The distant metastasis rate was 32.7% in patients with multiple positive lymph nodes, which showed the limitation of adjuvant radiotherapy and theoretically supported the postoperative combined chemotherapy. The prognosis of patients with positive lymph node was poor because of local recurrence and distant metastasis. How to improve the prognosis of these patients has been the focus of gynecological oncology. Stock  et al  [ 7 ] compared postoperative whole pelvic irradiation with those treated with radical hysterectomy alone, and showed a significantly improved pelvic control rate, 5-year disease-free survival (DFS) and overall survival (OS) (78% Vs 45%, 65% Vs 41%, 58% Vs 46%, respectively). Peters' study [ 20 ] demonstrated that postoperative radio-chemotherapy could greatly improve the 4-year DFS and OS compared to the adjuvant radiotherapy alone. In our study, adjuvant therapy improved the 5-year survival than the surgery alone (53.7% vs. 12.6%), decreased the pelvic recurrence and distant metastasis, which suggested the clinical importance of adjuvant therapy in patients with positive nodes. In this study the 5-year survival of adjuvant radio-chemotherapy arm was higher than adjuvant radiotherapy alone or chemotherapy alone. The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than the other three therapeutic arms. However, the differences were not significant and the limited number of cases in each arm could be the reason. It is necessary that these results are verified in prospective randomized control trials. Several randomized clinical trials [ 20 - 25 ] performed by the Gynecologic Oncology Group (GOG), the Radiation Therapy Oncology Group (RTOG) and the Southwest Oncology Group (SWOG) have demonstrated a significant advantage both in DFS and OS when cisplatin-based chemotherapy was administered during radiation for advanced stages of cervical cancer and early stage disease with poor prognostic factors. Green  et al  [ 26 ] did a systemic review of all known randomized controlled trials published between 1981 and 2000, 2865–3611 patients were available for analysis. The findings suggested that concomitant chemotherapy (CT) and radiotherapy (RT) improves OS and DFS, and reduces local and distant recurrence. Based on results, the US National Cancer Institute (NCI) released a clinical announcement supporting the concurrent use of cisplatin-based chemotherapy with RT for high-risk early stage and locally advanced stage cervical cancer [ 9 , 27 ]. Recently a prospective randomized trial performed by the National Cancer Institute (NCI) of Canada [ 25 ] failed to show benefit with the use of chemoradiation compared with RT alone. The potential inclusion of paraaortic nodes positive patients and the significant difference in anemia raises question about this trial result [ 28 ]. The therapy of patients with stage IIB cervical carcinoma is still a controversy [ 29 ]. Although radical radiotherapy (RT) is the proper choice for patients with stage IIB cervical cancer in general, the therapeutic effect is not as good as expected if the tumor is too bulky or is histologically adenocarcinoma. Hence a few gynecology oncologists tried brachytherapy and/or neoadjuvant chemotherapy to decrease the lesion, then performed radical surgery for some stage IIB patients with bulky tumor (>4 cm) or slight (less than 1/2) parametrial extension [ 30 , 31 ]. Postoperative adjuvant therapy would then be recommended according to risk factors [ 9 , 17 , 20 , 32 ]. In our study, the 5-year survival of the patients with stage IIB cervical carcinoma was only 54.3%, which was not at all satisfactory compared with the reported 58.9%–80.1% [ 33 - 35 ]. So we advocate that radical surgery should be taken cautiously for this group of patients. Any attempt to improve their prognosis by means of adjuvant therapy is not recommended if the parametrium can not be thoroughly dissected from the pelvic wall.
Discussion
Cervical cancer remains the third common cancer in women around the world, although its incidence is on the decline in North American and in Europe. It is estimated that there will be 10,370 new cases in 2005 in North America [ 8 ]. In many developing countries, not only is cervical cancer the most frequently occurring cancer among middle-aged women, but it is also a leading cause of death, partly due to the poor access to medical care and the unavailability of routine screening in many of these countries [ 9 ]. In Shanghai, the biggest city in China, there were 150 new cases in 2000. The standard incidence rate was 2.9 per 100 000 [ 10 ].Radical surgery has been found to be very effective in patients with early stage (IB-IIA) cervical carcinoma [ 11 - 13 ]. However, the 5-year survival has been lingering at 50%-90% during the past twenty years [ 14 ]. The reported risk factors of cervical carcinoma included clinical stage, bulky tumor, histological types, cellular differentiation, deep stromal invasion, parametrial extension, vaginal margin involved, lymphvascular permeation, lymph node metastasis, race and age [ 13 , 15 - 18 ]. In most studies the presence of pelvic lymph node metastases has been associated with increased pelvic recurrence and distance metastases, and a decrease in overall survival. Our Cox proportional hazard model analysis showed cellular differentiation, number of positive nodes and adjuvant therapy to be the independent prognostic factors ( P  < 0.05). The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes ( P  < 0.05). There was no significant difference of pelvic recurrence between them. However, the former's distant metastases rate (5.9%) was lower than the latter's (32.7%) ( P  = 0.05). In the analysis of the site of lymph node metastases, the 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was lower than that (43.1%) of patients with lower than common iliac node involvement. Unlike the series of Tsai the difference in our series was not statistically significant [ 19 ]. This is probably due to limited number of cases with paraaortic or common iliac nodes. The number of positive nodes correlated with the height of positive nodes ( P  < 0.01). The result suggested that in patients with more positive pelvic nodes there is higher chance of nodal metastasis at higher level node basins, which supported the external irradiation of PALN chain area for the multiple pelvic nodes involvement. The distant metastasis rate was 32.7% in patients with multiple positive lymph nodes, which showed the limitation of adjuvant radiotherapy and theoretically supported the postoperative combined chemotherapy.PPPPThe prognosis of patients with positive lymph node was poor because of local recurrence and distant metastasis. How to improve the prognosis of these patients has been the focus of gynecological oncology. Stock  et al  [ 7 ] compared postoperative whole pelvic irradiation with those treated with radical hysterectomy alone, and showed a significantly improved pelvic control rate, 5-year disease-free survival (DFS) and overall survival (OS) (78% Vs 45%, 65% Vs 41%, 58% Vs 46%, respectively).et alPeters' study [ 20 ] demonstrated that postoperative radio-chemotherapy could greatly improve the 4-year DFS and OS compared to the adjuvant radiotherapy alone. In our study, adjuvant therapy improved the 5-year survival than the surgery alone (53.7% vs. 12.6%), decreased the pelvic recurrence and distant metastasis, which suggested the clinical importance of adjuvant therapy in patients with positive nodes. In this study the 5-year survival of adjuvant radio-chemotherapy arm was higher than adjuvant radiotherapy alone or chemotherapy alone. The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than the other three therapeutic arms. However, the differences were not significant and the limited number of cases in each arm could be the reason. It is necessary that these results are verified in prospective randomized control trials.Several randomized clinical trials [ 20 - 25 ] performed by the Gynecologic Oncology Group (GOG), the Radiation Therapy Oncology Group (RTOG) and the Southwest Oncology Group (SWOG) have demonstrated a significant advantage both in DFS and OS when cisplatin-based chemotherapy was administered during radiation for advanced stages of cervical cancer and early stage disease with poor prognostic factors. Green  et al  [ 26 ] did a systemic review of all known randomized controlled trials published between 1981 and 2000, 2865–3611 patients were available for analysis. The findings suggested that concomitant chemotherapy (CT) and radiotherapy (RT) improves OS and DFS, and reduces local and distant recurrence. Based on results, the US National Cancer Institute (NCI) released a clinical announcement supporting the concurrent use of cisplatin-based chemotherapy with RT for high-risk early stage and locally advanced stage cervical cancer [ 9 , 27 ]. Recently a prospective randomized trial performed by the National Cancer Institute (NCI) of Canada [ 25 ] failed to show benefit with the use of chemoradiation compared with RT alone. The potential inclusion of paraaortic nodes positive patients and the significant difference in anemia raises question about this trial result [ 28 ].et alThe therapy of patients with stage IIB cervical carcinoma is still a controversy [ 29 ]. Although radical radiotherapy (RT) is the proper choice for patients with stage IIB cervical cancer in general, the therapeutic effect is not as good as expected if the tumor is too bulky or is histologically adenocarcinoma. Hence a few gynecology oncologists tried brachytherapy and/or neoadjuvant chemotherapy to decrease the lesion, then performed radical surgery for some stage IIB patients with bulky tumor (>4 cm) or slight (less than 1/2) parametrial extension [ 30 , 31 ]. Postoperative adjuvant therapy would then be recommended according to risk factors [ 9 , 17 , 20 , 32 ]. In our study, the 5-year survival of the patients with stage IIB cervical carcinoma was only 54.3%, which was not at all satisfactory compared with the reported 58.9%–80.1% [ 33 - 35 ]. So we advocate that radical surgery should be taken cautiously for this group of patients. Any attempt to improve their prognosis by means of adjuvant therapy is not recommended if the parametrium can not be thoroughly dissected from the pelvic wall.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors Contributions XC  collected the data, patients' follow-up and drafted the article. SC, ZL, MT, MX  collectively designed the study, participated in collection of data and revising the article. RZ  participated in collection and analysis of data, revising the article
Authors Contributions
XC  collected the data, patients' follow-up and drafted the article.XCSC, ZL, MT, MX  collectively designed the study, participated in collection of data and revising the article.SC, ZL, MT, MXRZ  participated in collection and analysis of data, revising the articleRZFunding Source None
Funding Source
None