Background We have previously shown that irregular lifestyle of young Japanese female students are significantly related to their desire to be thinner. In the present study, we examined the nutritional knowledge and food habits of Chinese university students and compared them with those of other Asian populations.
Background
We have previously shown that irregular lifestyle of young Japanese female students are significantly related to their desire to be thinner. In the present study, we examined the nutritional knowledge and food habits of Chinese university students and compared them with those of other Asian populations.Methods A self-reported questionnaire was administered to 540 students, ranging in age from 19-24 years. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. The parametric variables were analyzed using the Student's  t -test. Chi-square analyses were conducted for non-parametric variables
Methods
A self-reported questionnaire was administered to 540 students, ranging in age from 19-24 years. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. The parametric variables were analyzed using the Student's  t -test. Chi-square analyses were conducted for non-parametric variablestResults Our results showed that 80.5% of students had a normal BMI and 16.6 % of students were underweight with the prevalence of BMI>30 obesity being very low in this study sample. Young Chinese female students had a greater desire to be thinner (62.0%) than males (47.4%). Habits involving regular eating patterns and vegetable intake were reported and represent practices that ought to be encouraged.
Results
Our results showed that 80.5% of students had a normal BMI and 16.6 % of students were underweight with the prevalence of BMI>30 obesity being very low in this study sample. Young Chinese female students had a greater desire to be thinner (62.0%) than males (47.4%). Habits involving regular eating patterns and vegetable intake were reported and represent practices that ought to be encouraged.Conclusions The university and college arenas represent the final opportunity for the health and nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition.
Conclusions
The university and college arenas represent the final opportunity for the health and nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition.

Background The increasing problem of obesity has been observed in many lower-income countries during the last decades. China has adopted an open-market policy and experienced explosive economic growth, which has led to less food scarcity at the national level and to a remarkable transition in the structure of the diet of Chinese [ 1 ]. The composition of the Chinese diet has been shifting towards a diet higher in fat and meat, and lower in carbohydrates and fiber [ 2 ]. Additionally, decreased levels of physical activity and leisure are linked to increases in the prevalence of an overweight condition, obesity and diet-related non-communicable diseases [ 3 ]. In previous reports, we examined eating habits and dietary knowledge of female students in Japan. Our results showed that irregular lifestyle was significantly related to indefinite complaint, with the majority of students having a desire to be thinner although the prevalence of students who were overweight was very low in this study sample [ 4 ]. Universities and colleges are potentially important targets for the promotion of healthy lifestyles of the adult population. However, little is known concerning the body mass index (BMI) distribution and nutritional and health-related behavior of Chinese university students. The purpose of this study was to obtain a preliminary understanding of the relative level of BMI distribution of Chinese university students and to determine the nutritional knowledge and body-shape perceptions.
Background
The increasing problem of obesity has been observed in many lower-income countries during the last decades. China has adopted an open-market policy and experienced explosive economic growth, which has led to less food scarcity at the national level and to a remarkable transition in the structure of the diet of Chinese [ 1 ]. The composition of the Chinese diet has been shifting towards a diet higher in fat and meat, and lower in carbohydrates and fiber [ 2 ]. Additionally, decreased levels of physical activity and leisure are linked to increases in the prevalence of an overweight condition, obesity and diet-related non-communicable diseases [ 3 ].In previous reports, we examined eating habits and dietary knowledge of female students in Japan. Our results showed that irregular lifestyle was significantly related to indefinite complaint, with the majority of students having a desire to be thinner although the prevalence of students who were overweight was very low in this study sample [ 4 ]. Universities and colleges are potentially important targets for the promotion of healthy lifestyles of the adult population. However, little is known concerning the body mass index (BMI) distribution and nutritional and health-related behavior of Chinese university students. The purpose of this study was to obtain a preliminary understanding of the relative level of BMI distribution of Chinese university students and to determine the nutritional knowledge and body-shape perceptions.Material and Methods This study was carried out between February 2001 and April 2002. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. A sample of 540 students aged 19 – 24 years were administered a self-reported questionnaire. The questionnaire consisted of 21 questions regarding eating, drinking and smoking habits (19 questions), with 2 questions related to dieting (trying to lose weight). Self-reported height and weight were used to calculate BMI (kg/m 2 ). The questionnaire was designed by the authors and based on a national dietary survey held by the Health and Labor Ministry of Japan. Some of the authors also traveled to China to investigate the dietary life of Chinese to facilitate questionnaire design. The questionnaire was first written in Japanese and then translated to Chinese utilizing fluent bilingual linguistic services. The translated Chinese version was back-translated to insure the original meaning was not lost. Informed consent was obtained from all participants of this study according to the Declaration of Helsinki. The statistical software package SPSS 10.0 was used for the analysis of data [ 5 ]. In this study, parametric variables were analyzed using the Student's  t -test. Chi-square analyses were conducted for non-parametric variables. All analyses were two-tailed, and a 'p' value less than 0.05 was considered statistically significant.
Material and Methods
This study was carried out between February 2001 and April 2002. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. A sample of 540 students aged 19 – 24 years were administered a self-reported questionnaire. The questionnaire consisted of 21 questions regarding eating, drinking and smoking habits (19 questions), with 2 questions related to dieting (trying to lose weight). Self-reported height and weight were used to calculate BMI (kg/m 2 ). The questionnaire was designed by the authors and based on a national dietary survey held by the Health and Labor Ministry of Japan. Some of the authors also traveled to China to investigate the dietary life of Chinese to facilitate questionnaire design. The questionnaire was first written in Japanese and then translated to Chinese utilizing fluent bilingual linguistic services. The translated Chinese version was back-translated to insure the original meaning was not lost. Informed consent was obtained from all participants of this study according to the Declaration of Helsinki. The statistical software package SPSS 10.0 was used for the analysis of data [ 5 ]. In this study, parametric variables were analyzed using the Student's  t -test. Chi-square analyses were conducted for non-parametric variables. All analyses were two-tailed, and a 'p' value less than 0.05 was considered statistically significant.tResults Characteristics of the sample and BMI categories The response rate was 96% (512 / 540). The characteristics of the subjects are shown in Table  1 . A total of 212 men and 300 women, with a mean age of 20 ± 1.9 years, participated in this study. The average height was 165.8 ± 7.8 cm, while the average weight was 56.9 ± 9.2 kg. Mean BMI was 20.6 ± 2.2. To analyze the distribution of BMI and health-related behavior, BMI was categorized into 4 groups according to mean BMI of ± 1 standard deviation (SD) (Figure  1 ). The average BMI for male students was 21.4 ± 2.5 and was highest in the categories 18.9≤BMI<21.4 (37.7%) and 21.4≤BMI<23.9 (32.5%). The average BMI for female students was 20.0 ± 1.8, with the categories 18.2≤BMI<20.0 (37.5%) and 20.0≤BMI<21.8 (31.4%) displaying high values. According to WHO BMI classifications [ 6 ], 97.1% of students were classified into the underweight or normal weight categories. 2.5% (13/512) students were overweight (BMI>25) and 0.4% (2/512) of students were obese (BMI>30). BMI values of deviations from the average sample show the presence of few extreme values. Eating habit The life style practices were compared by gender (see  additional file 1 ). The majority of students (83.6 %) reported taking meals regularly, with 79.0% eating meals 3 times per day; there were no gender differences. However, a significant gender difference was found in the response relating to breakfast intake, with 66.8% of males and 82.3% of females reporting eating breakfast regularly (p < 0.0006). The frequency of snacking rate was significantly higher in females (31.1%) than in males (11.5%; p < 0.0001). The present sample demonstrated high consumption of vegetable and fruits. A total of 47.9% of students reported the consumption of colored vegetables such as spinach and carrots, and 32.5% of subjects reported eating fruit daily. Female students tend to eat more fruit than males (p < 0.0001). In addition, female students tend to eat with friends and family more frequently than males (p < 0.01). Few subjects smoke or drink alcohol. When the students eat out, female students are more likely to consider the calorie content of the menu than males (data not shown). Although 85.6% of students are aware of the concept of nutritionally balanced food, only a small number of students (7%) apply this concept when selecting food from a menu. Moreover, only 51% of students showed a desire to learn about healthy diets. Body image and health consciousness When subjects were asked about their history of dieting, 22.7% of respondents reported that they had dieted (see  additional file 2 ). The proportion of female students having a dieting experience (29.8%) was more than twice as great as that of male subjects (12.7%; p < 0.0006). In total, 56% of the students selected 'thin or slim is beautiful'. The percentage by gender was, 47.4% for male and 62.0% for female students. Female students have a significantly greater desire to be thinner than males (p < 0.001). More than half of the respondents reported a desire to adopt healthier dietary habits. Moreover, a question regarding the degree of consciousness pertaining to health and diet was asked; 45.2% of male students and 48.3% of female students wish to learn about health and diet. Among female subjects, BMI<18.2 strongly showed their consciousness of health and diet (p < 0.03).
Results
Characteristics of the sample and BMI categories The response rate was 96% (512 / 540). The characteristics of the subjects are shown in Table  1 . A total of 212 men and 300 women, with a mean age of 20 ± 1.9 years, participated in this study. The average height was 165.8 ± 7.8 cm, while the average weight was 56.9 ± 9.2 kg. Mean BMI was 20.6 ± 2.2. To analyze the distribution of BMI and health-related behavior, BMI was categorized into 4 groups according to mean BMI of ± 1 standard deviation (SD) (Figure  1 ). The average BMI for male students was 21.4 ± 2.5 and was highest in the categories 18.9≤BMI<21.4 (37.7%) and 21.4≤BMI<23.9 (32.5%). The average BMI for female students was 20.0 ± 1.8, with the categories 18.2≤BMI<20.0 (37.5%) and 20.0≤BMI<21.8 (31.4%) displaying high values. According to WHO BMI classifications [ 6 ], 97.1% of students were classified into the underweight or normal weight categories. 2.5% (13/512) students were overweight (BMI>25) and 0.4% (2/512) of students were obese (BMI>30). BMI values of deviations from the average sample show the presence of few extreme values.
Characteristics of the sample and BMI categories
The response rate was 96% (512 / 540). The characteristics of the subjects are shown in Table  1 . A total of 212 men and 300 women, with a mean age of 20 ± 1.9 years, participated in this study. The average height was 165.8 ± 7.8 cm, while the average weight was 56.9 ± 9.2 kg. Mean BMI was 20.6 ± 2.2. To analyze the distribution of BMI and health-related behavior, BMI was categorized into 4 groups according to mean BMI of ± 1 standard deviation (SD) (Figure  1 ). The average BMI for male students was 21.4 ± 2.5 and was highest in the categories 18.9≤BMI<21.4 (37.7%) and 21.4≤BMI<23.9 (32.5%). The average BMI for female students was 20.0 ± 1.8, with the categories 18.2≤BMI<20.0 (37.5%) and 20.0≤BMI<21.8 (31.4%) displaying high values. According to WHO BMI classifications [ 6 ], 97.1% of students were classified into the underweight or normal weight categories. 2.5% (13/512) students were overweight (BMI>25) and 0.4% (2/512) of students were obese (BMI>30). BMI values of deviations from the average sample show the presence of few extreme values.Eating habit The life style practices were compared by gender (see  additional file 1 ). The majority of students (83.6 %) reported taking meals regularly, with 79.0% eating meals 3 times per day; there were no gender differences. However, a significant gender difference was found in the response relating to breakfast intake, with 66.8% of males and 82.3% of females reporting eating breakfast regularly (p < 0.0006). The frequency of snacking rate was significantly higher in females (31.1%) than in males (11.5%; p < 0.0001). The present sample demonstrated high consumption of vegetable and fruits. A total of 47.9% of students reported the consumption of colored vegetables such as spinach and carrots, and 32.5% of subjects reported eating fruit daily. Female students tend to eat more fruit than males (p < 0.0001). In addition, female students tend to eat with friends and family more frequently than males (p < 0.01). Few subjects smoke or drink alcohol. When the students eat out, female students are more likely to consider the calorie content of the menu than males (data not shown). Although 85.6% of students are aware of the concept of nutritionally balanced food, only a small number of students (7%) apply this concept when selecting food from a menu. Moreover, only 51% of students showed a desire to learn about healthy diets.
Eating habit
The life style practices were compared by gender (see  additional file 1 ). The majority of students (83.6 %) reported taking meals regularly, with 79.0% eating meals 3 times per day; there were no gender differences. However, a significant gender difference was found in the response relating to breakfast intake, with 66.8% of males and 82.3% of females reporting eating breakfast regularly (p < 0.0006). The frequency of snacking rate was significantly higher in females (31.1%) than in males (11.5%; p < 0.0001). The present sample demonstrated high consumption of vegetable and fruits. A total of 47.9% of students reported the consumption of colored vegetables such as spinach and carrots, and 32.5% of subjects reported eating fruit daily. Female students tend to eat more fruit than males (p < 0.0001). In addition, female students tend to eat with friends and family more frequently than males (p < 0.01). Few subjects smoke or drink alcohol. When the students eat out, female students are more likely to consider the calorie content of the menu than males (data not shown). Although 85.6% of students are aware of the concept of nutritionally balanced food, only a small number of students (7%) apply this concept when selecting food from a menu. Moreover, only 51% of students showed a desire to learn about healthy diets.Body image and health consciousness When subjects were asked about their history of dieting, 22.7% of respondents reported that they had dieted (see  additional file 2 ). The proportion of female students having a dieting experience (29.8%) was more than twice as great as that of male subjects (12.7%; p < 0.0006). In total, 56% of the students selected 'thin or slim is beautiful'. The percentage by gender was, 47.4% for male and 62.0% for female students. Female students have a significantly greater desire to be thinner than males (p < 0.001). More than half of the respondents reported a desire to adopt healthier dietary habits. Moreover, a question regarding the degree of consciousness pertaining to health and diet was asked; 45.2% of male students and 48.3% of female students wish to learn about health and diet. Among female subjects, BMI<18.2 strongly showed their consciousness of health and diet (p < 0.03).
Body image and health consciousness
When subjects were asked about their history of dieting, 22.7% of respondents reported that they had dieted (see  additional file 2 ). The proportion of female students having a dieting experience (29.8%) was more than twice as great as that of male subjects (12.7%; p < 0.0006). In total, 56% of the students selected 'thin or slim is beautiful'. The percentage by gender was, 47.4% for male and 62.0% for female students. Female students have a significantly greater desire to be thinner than males (p < 0.001). More than half of the respondents reported a desire to adopt healthier dietary habits. Moreover, a question regarding the degree of consciousness pertaining to health and diet was asked; 45.2% of male students and 48.3% of female students wish to learn about health and diet. Among female subjects, BMI<18.2 strongly showed their consciousness of health and diet (p < 0.03).Discussion This study aimed to determine the health, nutritional knowledge and dietary behavior of university students in China. As a result, we recorded the distribution of BMI among Chinese students and found a low prevalence of obesity, a finding that is consistent with a study of Japanese female students (BMI≥25 overweight was 5.8%, BMI>30 of obesity was 0%) [ 4 ]. In the United States, 35% of the college students are reported to be overweight or obese (BMI≥25) [ 7 ]. According to the WHO definition of obesity, BMI>30 is the cut-off point [ 6 ]. The definition is based on research of Caucasian populations. Asian populations are reported to have a higher body fat (%) at a lower BMI compared to Caucasians [ 8 ]. The WHO expert consultation reported that BMI in Asian populations is related to disease at a lower level [ 9 ]. In order to compare obesity prevalence between ethnic groups, BMI cut-off points for Asians need to be considered by well constructed and standardized body composition studies. It is notable that in China, the prevalence of overweight individuals increased from 1991 to 1997, with the increasing rate changing from 6.4 to 7.7 [ 10 ]. The proportion of energy derived from the fat of both vegetable and animal sources increased each year. A recent study revealed that energy derived from dietary fat accounted for more than 30% of the total energy [ 11 ]. Changes in dietary composition, which correspond to socioeconomic growth, may accelerate the prevalence of obesity in China. The results of our study show that the majority of students regularly eat three times per day, and almost 80% of students eat vegetables and fruit twice per day. These eating habits ought to be encouraged. The traditional Chinese diet contains plenty of vegetables and is rice-based. The present study reported a high proportion of Chinese students eat breakfast daily. In contrast, a dietary survey of young Japanese subjects revealed a low rate of individuals engaged in regular eating patterns [ 12 ]. The skipping of breakfast has been associated with lower nutritional status and the risk of cardiovascular diseases [ 13 ]. It has also been reported that less adequate breakfast habits may contribute to the appearance and further development of obesity [ 14 ]. Therefore the importance of regular eating patterns cannot be overemphasized in nutritional education. Our results showed that body figure perception was significantly different between female and male students. A number of researchers have investigated the relationship of body image and gender role. Women tend to desire a thinner figure, express more anxiety about becoming fat, and are more likely to diet than men [ 15 , 16 ]. In contrast, men have reported a desire for a heavier physique and muscularity [ 17 ]. In recent years, eating disorders have been increasing dramatically among young women. The results of our study did not confirm this suggestion to the level of statistical significance; however, it is worth pointing out that 65.0% of female students with BMI<20, which is under to normal weight range, indicated a desire to be thin. Dissatisfaction with body figure and eating disorders are closely related [ 18 - 20 ]. Being young, female, and dieting are identified risk factors that have been reliably linked to the development of eating disorders [ 21 ]. It was speculated that some of the students who were preoccupied with a thin body may develop eating disturbances. Thus, the promotion of healthy weight management practices should be considered when developing health education programs.
Discussion
This study aimed to determine the health, nutritional knowledge and dietary behavior of university students in China. As a result, we recorded the distribution of BMI among Chinese students and found a low prevalence of obesity, a finding that is consistent with a study of Japanese female students (BMI≥25 overweight was 5.8%, BMI>30 of obesity was 0%) [ 4 ]. In the United States, 35% of the college students are reported to be overweight or obese (BMI≥25) [ 7 ]. According to the WHO definition of obesity, BMI>30 is the cut-off point [ 6 ]. The definition is based on research of Caucasian populations. Asian populations are reported to have a higher body fat (%) at a lower BMI compared to Caucasians [ 8 ]. The WHO expert consultation reported that BMI in Asian populations is related to disease at a lower level [ 9 ]. In order to compare obesity prevalence between ethnic groups, BMI cut-off points for Asians need to be considered by well constructed and standardized body composition studies. It is notable that in China, the prevalence of overweight individuals increased from 1991 to 1997, with the increasing rate changing from 6.4 to 7.7 [ 10 ]. The proportion of energy derived from the fat of both vegetable and animal sources increased each year. A recent study revealed that energy derived from dietary fat accounted for more than 30% of the total energy [ 11 ]. Changes in dietary composition, which correspond to socioeconomic growth, may accelerate the prevalence of obesity in China.The results of our study show that the majority of students regularly eat three times per day, and almost 80% of students eat vegetables and fruit twice per day. These eating habits ought to be encouraged. The traditional Chinese diet contains plenty of vegetables and is rice-based. The present study reported a high proportion of Chinese students eat breakfast daily. In contrast, a dietary survey of young Japanese subjects revealed a low rate of individuals engaged in regular eating patterns [ 12 ]. The skipping of breakfast has been associated with lower nutritional status and the risk of cardiovascular diseases [ 13 ]. It has also been reported that less adequate breakfast habits may contribute to the appearance and further development of obesity [ 14 ]. Therefore the importance of regular eating patterns cannot be overemphasized in nutritional education.Our results showed that body figure perception was significantly different between female and male students. A number of researchers have investigated the relationship of body image and gender role. Women tend to desire a thinner figure, express more anxiety about becoming fat, and are more likely to diet than men [ 15 , 16 ]. In contrast, men have reported a desire for a heavier physique and muscularity [ 17 ]. In recent years, eating disorders have been increasing dramatically among young women. The results of our study did not confirm this suggestion to the level of statistical significance; however, it is worth pointing out that 65.0% of female students with BMI<20, which is under to normal weight range, indicated a desire to be thin. Dissatisfaction with body figure and eating disorders are closely related [ 18 - 20 ]. Being young, female, and dieting are identified risk factors that have been reliably linked to the development of eating disorders [ 21 ]. It was speculated that some of the students who were preoccupied with a thin body may develop eating disturbances. Thus, the promotion of healthy weight management practices should be considered when developing health education programs.Conclusions In conclusion, our findings reveal that the majority of students were classified into the normal BMI group, with the prevalence of BMI >30 obesity being very low in this study sample. Young female students had a greater desire to be thinner than male students. Habits involving regular eating patterns and vegetable intake were found and represent practices that ought to be encouraged. The meal and snack patterns in Chinese students were very similar to the traditional eating pattern model, although diets are changing rapidly in China and other low-income countries. The university and college arenas represent the final opportunity for nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition, especially with respect to information relating to sources of nutrition and healthy weight management. Furthermore, public demand for health and nutritional information should be taken into consideration when implementing strategies aimed at improving the nutritional well-being of individuals.
Conclusions
In conclusion, our findings reveal that the majority of students were classified into the normal BMI group, with the prevalence of BMI >30 obesity being very low in this study sample. Young female students had a greater desire to be thinner than male students. Habits involving regular eating patterns and vegetable intake were found and represent practices that ought to be encouraged. The meal and snack patterns in Chinese students were very similar to the traditional eating pattern model, although diets are changing rapidly in China and other low-income countries. The university and college arenas represent the final opportunity for nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition, especially with respect to information relating to sources of nutrition and healthy weight management. Furthermore, public demand for health and nutritional information should be taken into consideration when implementing strategies aimed at improving the nutritional well-being of individuals.Authors' contributions R.S carried out questionnaire design, manuscript drafting and total coordination of the study. K.T has been involved in drafting and revision of the article. R.A contributed to the data entry and its analysis. L.CJ contributed to the questionnaire design, data collection and language translations. N.S contributed to final approval of the manuscript.
Authors' contributions
R.S carried out questionnaire design, manuscript drafting and total coordination of the study. K.T has been involved in drafting and revision of the article. R.A contributed to the data entry and its analysis. L.CJ contributed to the questionnaire design, data collection and language translations. N.S contributed to final approval of the manuscript.Supplementary Material Additional File 1 Table 2 containing the results of questions related to lifestyle practices with special reference to food habit. The meal patterns, consumption of fruits and vegetables, consumption of fried foods, consumption of alcohol were assessed in male and female students. The Chi-square analyses were employed to compare the behavioral differences by gender. The evaluations of statistical significance were made at the p < 0.05. Click here for file Additional File 2 Table 3 contains the results of body shape perception and health consciousness of male and female students. Male and female respondents were categorized in to 4 groups respectively, according to mean BMI of ± 1 standard deviation (SD). Analyses were made between BMI groups using Chi-square analysis. The evaluations of statistical significance were made at the p < 0.05. Click here for file
Supplementary Material
Additional File 1
Table 2 containing the results of questions related to lifestyle practices with special reference to food habit. The meal patterns, consumption of fruits and vegetables, consumption of fried foods, consumption of alcohol were assessed in male and female students. The Chi-square analyses were employed to compare the behavioral differences by gender. The evaluations of statistical significance were made at the p < 0.05.Click here for fileAdditional File 2
Table 3 contains the results of body shape perception and health consciousness of male and female students. Male and female respondents were categorized in to 4 groups respectively, according to mean BMI of ± 1 standard deviation (SD). Analyses were made between BMI groups using Chi-square analysis. The evaluations of statistical significance were made at the p < 0.05.Click here for file


Background Trastuzumab, a humanized monoclonal antibody against the HER2 receptor is currently being used in breast and other tumor types. Early studies have shown that a variable proportion of cervical carcinoma tumors overexpress the HER2 receptor as evaluated by diverse techniques and antibodies. Currently it is known that a tumor response to trastuzumab strongly correlates with the level of HER2 expression evaluated by the Hercep Test, thus, it seems desirable to evaluate the status of expression of this receptor using the FDA-approved Hercep Test and grading system to gain insight in the feasibility of using trastuzumab in cervical cancer patients.
Background
Trastuzumab, a humanized monoclonal antibody against the HER2 receptor is currently being used in breast and other tumor types. Early studies have shown that a variable proportion of cervical carcinoma tumors overexpress the HER2 receptor as evaluated by diverse techniques and antibodies. Currently it is known that a tumor response to trastuzumab strongly correlates with the level of HER2 expression evaluated by the Hercep Test, thus, it seems desirable to evaluate the status of expression of this receptor using the FDA-approved Hercep Test and grading system to gain insight in the feasibility of using trastuzumab in cervical cancer patients.Methods We analyzed a series of cervical cancer cell lines, the primary tumors of 35 cases of cervical cancer patients and four recurrent cases, with the Hercep Test in order to establish whether this tumor type overexpress HER2 at level of 2+/3+ as trastuzumab is currently approved for breast cancer having such level of expression.
Methods
We analyzed a series of cervical cancer cell lines, the primary tumors of 35 cases of cervical cancer patients and four recurrent cases, with the Hercep Test in order to establish whether this tumor type overexpress HER2 at level of 2+/3+ as trastuzumab is currently approved for breast cancer having such level of expression.Results The results indicate that only 1 out of 35 primary tumors cases overexpress the receptor at this level, however, two out of four recurrent tumors that tested negative at diagnosis shifted to Hercep Test 2+ and 3+ respectively.
Results
The results indicate that only 1 out of 35 primary tumors cases overexpress the receptor at this level, however, two out of four recurrent tumors that tested negative at diagnosis shifted to Hercep Test 2+ and 3+ respectively.Conclusions The low frequency of expression in primary cases suggests that trastuzumab could have a limited value for the primary management of cervical cancer patients, however, the finding of "conversion" to Hercep Test 2+ and 3+ of recurrent tumors indicates the need to further evaluate the expression of HER2 in the metastatic and recurrent cases.
Conclusions
The low frequency of expression in primary cases suggests that trastuzumab could have a limited value for the primary management of cervical cancer patients, however, the finding of "conversion" to Hercep Test 2+ and 3+ of recurrent tumors indicates the need to further evaluate the expression of HER2 in the metastatic and recurrent cases.

Background Cervical carcinoma is a leading cause of death in women of reproductive age worldwide, particularly in developing countries. While curable in early stages, the treatment results of locally advanced disease are unsatisfactory. The current standard of treatment -cisplatin-based chemoradiation- fails to cure at least 15% to 45% of bulky IB to IIIB patients, and in addition, multimodality treatment incorporating chemotherapy, surgery and radiation at its best is unlikely to substantially increase the cure rate. Because of this, the logical step to follow is the testing of molecular targeted therapies trying to improve the prognosis of cervical cancer patients [ 1 ]. Human papillomavirus infection is recognized as the stronger etiological factor for the development of this tumor; however, overexpression of the epidermal growth factor receptor family members is also common and seems to play an important oncogenic role [ 2 ]. HER2 (also known as c-erbB-2) is a transmembrane receptor protein with tyrosine kinase activity that belongs to this family and it is overexpressed in a number of solid tumors. Its overexpression and prognostic significance in breast cancer led to the development and approval of the use of trastuzumab (Trastuzumab, Genentech, South San Francisco, CA), a recombinant monoclonal antibody to HER2, for the treatment of patients with metastatic breast carcinomas overexpressing HER2 [ 3 ]. Until more recently, poor standardization in HER2 status evaluation precluded reliable comparison of overexpression rates in different tumors. A source of variability in results not only comes from methodological variations in tissue processing (time to fixation, duration of fixation, denaturation, heating, antigen retrieval, the staining procedure) and grading scores but also from the antibody used. This issue was addressed by Press et al., who showed extremely variable results in 187 breast cancer specimens evaluated with 7 polyclonal and 21 monoclonal antibodies [ 4 ]. However, standardized methodologies have been introduced recently for these analyses, and have identified frequencies of 51%, 44%, 26% and 25% in Wilm's tumor, bladder, pancreatic and breast carcinoma, respectively. Other tumors tested had frequencies below 20% [ 5 ]. Before the introduction of the Hercep Test, it was known that a variable subset of cervical carcinomas ranging from 8% to 77% express HER2 as evaluated by diverse methods [ 6 - 14 ] and that in some studies its overexpression has shown to confer a worse prognosis [ 7 - 9 , 13 ]. Because these results on HER2 expression in cervical cancer were obtained before the standardization required in breast cancer, we wanted to investigate the expression status of HER2 using the Hercep Test in a series of cervical carcinoma cell lines, primary tumors of locally advanced cervical cancer cases and in four recurrent tumors of these patients.
Background
Cervical carcinoma is a leading cause of death in women of reproductive age worldwide, particularly in developing countries. While curable in early stages, the treatment results of locally advanced disease are unsatisfactory. The current standard of treatment -cisplatin-based chemoradiation- fails to cure at least 15% to 45% of bulky IB to IIIB patients, and in addition, multimodality treatment incorporating chemotherapy, surgery and radiation at its best is unlikely to substantially increase the cure rate. Because of this, the logical step to follow is the testing of molecular targeted therapies trying to improve the prognosis of cervical cancer patients [ 1 ].Human papillomavirus infection is recognized as the stronger etiological factor for the development of this tumor; however, overexpression of the epidermal growth factor receptor family members is also common and seems to play an important oncogenic role [ 2 ]. HER2 (also known as c-erbB-2) is a transmembrane receptor protein with tyrosine kinase activity that belongs to this family and it is overexpressed in a number of solid tumors. Its overexpression and prognostic significance in breast cancer led to the development and approval of the use of trastuzumab (Trastuzumab, Genentech, South San Francisco, CA), a recombinant monoclonal antibody to HER2, for the treatment of patients with metastatic breast carcinomas overexpressing HER2 [ 3 ].Until more recently, poor standardization in HER2 status evaluation precluded reliable comparison of overexpression rates in different tumors. A source of variability in results not only comes from methodological variations in tissue processing (time to fixation, duration of fixation, denaturation, heating, antigen retrieval, the staining procedure) and grading scores but also from the antibody used. This issue was addressed by Press et al., who showed extremely variable results in 187 breast cancer specimens evaluated with 7 polyclonal and 21 monoclonal antibodies [ 4 ]. However, standardized methodologies have been introduced recently for these analyses, and have identified frequencies of 51%, 44%, 26% and 25% in Wilm's tumor, bladder, pancreatic and breast carcinoma, respectively. Other tumors tested had frequencies below 20% [ 5 ].Before the introduction of the Hercep Test, it was known that a variable subset of cervical carcinomas ranging from 8% to 77% express HER2 as evaluated by diverse methods [ 6 - 14 ] and that in some studies its overexpression has shown to confer a worse prognosis [ 7 - 9 , 13 ]. Because these results on HER2 expression in cervical cancer were obtained before the standardization required in breast cancer, we wanted to investigate the expression status of HER2 using the Hercep Test in a series of cervical carcinoma cell lines, primary tumors of locally advanced cervical cancer cases and in four recurrent tumors of these patients.Methods Tumor specimens Thirty-five paraffin-embedded tumor tissues from patients FIGO staged as IB2 to IIIB, treated with standard radiation concurrent with weekly cisplatin. Diagnosis was made on the basis of routine hematoxilin-eosin examination under light microscopy according to the World Health Organization criteria. Tumor specimens at diagnosis were taken before any treatment was instituted whereas the tumors samples from the four recurrent cases were also taken before patients received any second line therapy. Cell lines and reagents DMEM culture media and Fetal Calf Serum were purchased from Gibco BRL Life Technologies (Grand Island, New York). HeLa, CasKi, SiHa and C33A carcinoma cell lines were obtained from the ATCC. The cell line ViBo established from a Mexican patient with cervical cancer was kindly provided by Dr. Monroy (FES Zaragoza, UNAM, Mexico City). Cells were grown in DMEM supplemented with 10% FCS at 37°C and 5% CO 2 . Cell lines were grown on two-chamber polysterene vessel Falcon ®  (Becton Dickinson, NJ.) and subsequently formalin-fixed for 24 hrs at room temperature, then rehydrated in graded ethanol. Afterwards immunochemistry was performed as below described. Hercep test Hercep Test was performed following the manufacturer's guidelines of HER2 protein expression as follows. Sections were deparaffinized in xylene and rehydrated through graded ethanols to distilled water. The sections were immersed in Dako Epitope Retrieval Solution (10 mM citrate buffer, pH6) that had been preheated to 95°C in a water bath and then heat-treated at 95°C for 40 min. After a 20-minute cooldown period at room temperature, the sections were washed with Dako Wash Buffer, a procedure that followed every subsequent incubation. Endogenous peroxidase was blocked with Dako Blocking Buffer (0.3% hydrogen peroxide containing 15 mM sodium azide) for 5 min at room temperature. The sections were incubated with the primary polyclonal antibody, an affinity-purified rabbit antihuman HER2 antibody supplied in the kit, for 30 min at room temperature. Bound primary antibody was labeled by incubating the slides with the Dako Visualization reagent (horseradish peroxidase-labeled dextran polymer conjugated to affinity-purified goat antirabbit immunoglobulins in Tris-HCl) for 30 min. Color development was achieved with 3,3'-diaminobenzidine (DAB) for 10 min. The sections were counterstained with hematoxylin and eosin. To confirm validation of the staining run, control cell slides, which were provided in the kit and consisted of three pelleted, formalin-fixed, paraffin-embedded human breast cell lines with known HER2 positivity (MDA-231: 0; MDA-175: 1+; SK-BR-3: 3+), were also stained simultaneously. In the negative controls, the primary antibody was replaced by normal rabbit serum (Dako Negative Control Reagent) for the HER2 primary antibody. The antibody used in Hercep Test did not exhibit cross-reactivity to HER3 and HER4 in western blot analysis. Following the FDA scoring guidelines for breast carcinomas, only membrane staining intensity and pattern were evaluated using the 0–3+ scale as illustrated in the Hercep Test kit scoring guidelines (0 for no staining at all or membrane staining in less than 10% of the tumor cells; 1+ for only partial, weak staining of the cell membrane of more than 10% of the tumor cells; 2+ for moderate staining of the complete cell membrane in more than 10% of the tumor cells; 3+ for intense staining of the complete membrane in more than 10% of the tumor cells). The analysis was performed by a pathologist (VP-S) familiarized in the use of Hercep Test for breast cancer patients. In accordance with the Hercep Test kit guide, HER2 overexpression was assessed as negative for scores of 0 or 1+ and positive for scores of 2+ and 3+. FISH in the four recurrent cases Amplification of  Her-2/neu  was evaluated using the Path-Vysion DNA Probe Kit (Vysis), which uses a dual-color probe for determining the number of copies of both  Her-2/neu  (orange) and the chromosome 17 centromeres (green). The kit was used following the manufacturer's instructions with a few minor modifications. Slides containing 5μ thick paraffin-embedded tissue sections of studied cervical tumor cases and a known Her-2/neu amplified breast tumor were placed on a slide warmer overnight at 58°C, followed by deparaffinization in Xilol, dehydration in 100 ethanol, and drying on a slide warmer at 45 to 50°C. Slides were then pretreated with 0.2 N hydrochloric acid for 20 minutes, followed by washes in purified water and immersion in Vysis wash buffer. They were subsequently immersed in Vysis protease solution at 37°C for 10 minutes, washed in Vysis wash buffer, and dried on the slide warmer. The slides were then immersed in 10% buffered formalin at room temperature for 10 minutes, immersed in Vysis wash buffer, and dried on the slide warmer. Sections were denatured by placing the slides in formamide for 5 minutes at 72°C followed by dehydration in 70, 85 and then 100% ethanol. Slides were then dried on a slide warmer, and 10 μl of probe was applied. They were then coverslipped, sealed and placed in a prewarmed humid incubation chamber at 37°C for 21 hours. This was followed by immersion in prewarmed postwash solution at 72°C for 2 minutes. The slides were air-dried, and a 4',6-diamidino-2-phenylindole (DAPI) counterstain was applied. The scoring system used is described in detail in the manufacturer's instruction. A minimum of 60 nuclei were scored by each of 2 observers using a Zeiss Axioskop-2 fluorescent microscope with V.2 filter. The ratio of  Her-2/neu  signals (orange) to chromosome 17 centromere signals (green) was determined with ratios of <2.0 considered nonamplified and those ≥ 2.0 amplified.
Methods
Tumor specimens Thirty-five paraffin-embedded tumor tissues from patients FIGO staged as IB2 to IIIB, treated with standard radiation concurrent with weekly cisplatin. Diagnosis was made on the basis of routine hematoxilin-eosin examination under light microscopy according to the World Health Organization criteria. Tumor specimens at diagnosis were taken before any treatment was instituted whereas the tumors samples from the four recurrent cases were also taken before patients received any second line therapy.
Tumor specimens
Thirty-five paraffin-embedded tumor tissues from patients FIGO staged as IB2 to IIIB, treated with standard radiation concurrent with weekly cisplatin. Diagnosis was made on the basis of routine hematoxilin-eosin examination under light microscopy according to the World Health Organization criteria. Tumor specimens at diagnosis were taken before any treatment was instituted whereas the tumors samples from the four recurrent cases were also taken before patients received any second line therapy.Cell lines and reagents DMEM culture media and Fetal Calf Serum were purchased from Gibco BRL Life Technologies (Grand Island, New York). HeLa, CasKi, SiHa and C33A carcinoma cell lines were obtained from the ATCC. The cell line ViBo established from a Mexican patient with cervical cancer was kindly provided by Dr. Monroy (FES Zaragoza, UNAM, Mexico City). Cells were grown in DMEM supplemented with 10% FCS at 37°C and 5% CO 2 . Cell lines were grown on two-chamber polysterene vessel Falcon ®  (Becton Dickinson, NJ.) and subsequently formalin-fixed for 24 hrs at room temperature, then rehydrated in graded ethanol. Afterwards immunochemistry was performed as below described.
Cell lines and reagents
DMEM culture media and Fetal Calf Serum were purchased from Gibco BRL Life Technologies (Grand Island, New York). HeLa, CasKi, SiHa and C33A carcinoma cell lines were obtained from the ATCC. The cell line ViBo established from a Mexican patient with cervical cancer was kindly provided by Dr. Monroy (FES Zaragoza, UNAM, Mexico City). Cells were grown in DMEM supplemented with 10% FCS at 37°C and 5% CO 2 . Cell lines were grown on two-chamber polysterene vessel Falcon ®  (Becton Dickinson, NJ.) and subsequently formalin-fixed for 24 hrs at room temperature, then rehydrated in graded ethanol. Afterwards immunochemistry was performed as below described.Hercep test Hercep Test was performed following the manufacturer's guidelines of HER2 protein expression as follows. Sections were deparaffinized in xylene and rehydrated through graded ethanols to distilled water. The sections were immersed in Dako Epitope Retrieval Solution (10 mM citrate buffer, pH6) that had been preheated to 95°C in a water bath and then heat-treated at 95°C for 40 min. After a 20-minute cooldown period at room temperature, the sections were washed with Dako Wash Buffer, a procedure that followed every subsequent incubation. Endogenous peroxidase was blocked with Dako Blocking Buffer (0.3% hydrogen peroxide containing 15 mM sodium azide) for 5 min at room temperature. The sections were incubated with the primary polyclonal antibody, an affinity-purified rabbit antihuman HER2 antibody supplied in the kit, for 30 min at room temperature. Bound primary antibody was labeled by incubating the slides with the Dako Visualization reagent (horseradish peroxidase-labeled dextran polymer conjugated to affinity-purified goat antirabbit immunoglobulins in Tris-HCl) for 30 min. Color development was achieved with 3,3'-diaminobenzidine (DAB) for 10 min. The sections were counterstained with hematoxylin and eosin. To confirm validation of the staining run, control cell slides, which were provided in the kit and consisted of three pelleted, formalin-fixed, paraffin-embedded human breast cell lines with known HER2 positivity (MDA-231: 0; MDA-175: 1+; SK-BR-3: 3+), were also stained simultaneously. In the negative controls, the primary antibody was replaced by normal rabbit serum (Dako Negative Control Reagent) for the HER2 primary antibody. The antibody used in Hercep Test did not exhibit cross-reactivity to HER3 and HER4 in western blot analysis. Following the FDA scoring guidelines for breast carcinomas, only membrane staining intensity and pattern were evaluated using the 0–3+ scale as illustrated in the Hercep Test kit scoring guidelines (0 for no staining at all or membrane staining in less than 10% of the tumor cells; 1+ for only partial, weak staining of the cell membrane of more than 10% of the tumor cells; 2+ for moderate staining of the complete cell membrane in more than 10% of the tumor cells; 3+ for intense staining of the complete membrane in more than 10% of the tumor cells). The analysis was performed by a pathologist (VP-S) familiarized in the use of Hercep Test for breast cancer patients. In accordance with the Hercep Test kit guide, HER2 overexpression was assessed as negative for scores of 0 or 1+ and positive for scores of 2+ and 3+.
Hercep test
Hercep Test was performed following the manufacturer's guidelines of HER2 protein expression as follows. Sections were deparaffinized in xylene and rehydrated through graded ethanols to distilled water. The sections were immersed in Dako Epitope Retrieval Solution (10 mM citrate buffer, pH6) that had been preheated to 95°C in a water bath and then heat-treated at 95°C for 40 min. After a 20-minute cooldown period at room temperature, the sections were washed with Dako Wash Buffer, a procedure that followed every subsequent incubation. Endogenous peroxidase was blocked with Dako Blocking Buffer (0.3% hydrogen peroxide containing 15 mM sodium azide) for 5 min at room temperature. The sections were incubated with the primary polyclonal antibody, an affinity-purified rabbit antihuman HER2 antibody supplied in the kit, for 30 min at room temperature. Bound primary antibody was labeled by incubating the slides with the Dako Visualization reagent (horseradish peroxidase-labeled dextran polymer conjugated to affinity-purified goat antirabbit immunoglobulins in Tris-HCl) for 30 min. Color development was achieved with 3,3'-diaminobenzidine (DAB) for 10 min. The sections were counterstained with hematoxylin and eosin. To confirm validation of the staining run, control cell slides, which were provided in the kit and consisted of three pelleted, formalin-fixed, paraffin-embedded human breast cell lines with known HER2 positivity (MDA-231: 0; MDA-175: 1+; SK-BR-3: 3+), were also stained simultaneously. In the negative controls, the primary antibody was replaced by normal rabbit serum (Dako Negative Control Reagent) for the HER2 primary antibody. The antibody used in Hercep Test did not exhibit cross-reactivity to HER3 and HER4 in western blot analysis.Following the FDA scoring guidelines for breast carcinomas, only membrane staining intensity and pattern were evaluated using the 0–3+ scale as illustrated in the Hercep Test kit scoring guidelines (0 for no staining at all or membrane staining in less than 10% of the tumor cells; 1+ for only partial, weak staining of the cell membrane of more than 10% of the tumor cells; 2+ for moderate staining of the complete cell membrane in more than 10% of the tumor cells; 3+ for intense staining of the complete membrane in more than 10% of the tumor cells). The analysis was performed by a pathologist (VP-S) familiarized in the use of Hercep Test for breast cancer patients. In accordance with the Hercep Test kit guide, HER2 overexpression was assessed as negative for scores of 0 or 1+ and positive for scores of 2+ and 3+.FISH in the four recurrent cases Amplification of  Her-2/neu  was evaluated using the Path-Vysion DNA Probe Kit (Vysis), which uses a dual-color probe for determining the number of copies of both  Her-2/neu  (orange) and the chromosome 17 centromeres (green). The kit was used following the manufacturer's instructions with a few minor modifications. Slides containing 5μ thick paraffin-embedded tissue sections of studied cervical tumor cases and a known Her-2/neu amplified breast tumor were placed on a slide warmer overnight at 58°C, followed by deparaffinization in Xilol, dehydration in 100 ethanol, and drying on a slide warmer at 45 to 50°C. Slides were then pretreated with 0.2 N hydrochloric acid for 20 minutes, followed by washes in purified water and immersion in Vysis wash buffer. They were subsequently immersed in Vysis protease solution at 37°C for 10 minutes, washed in Vysis wash buffer, and dried on the slide warmer. The slides were then immersed in 10% buffered formalin at room temperature for 10 minutes, immersed in Vysis wash buffer, and dried on the slide warmer. Sections were denatured by placing the slides in formamide for 5 minutes at 72°C followed by dehydration in 70, 85 and then 100% ethanol. Slides were then dried on a slide warmer, and 10 μl of probe was applied. They were then coverslipped, sealed and placed in a prewarmed humid incubation chamber at 37°C for 21 hours. This was followed by immersion in prewarmed postwash solution at 72°C for 2 minutes. The slides were air-dried, and a 4',6-diamidino-2-phenylindole (DAPI) counterstain was applied. The scoring system used is described in detail in the manufacturer's instruction. A minimum of 60 nuclei were scored by each of 2 observers using a Zeiss Axioskop-2 fluorescent microscope with V.2 filter. The ratio of  Her-2/neu  signals (orange) to chromosome 17 centromere signals (green) was determined with ratios of <2.0 considered nonamplified and those ≥ 2.0 amplified.
FISH in the four recurrent cases
Amplification of  Her-2/neu  was evaluated using the Path-Vysion DNA Probe Kit (Vysis), which uses a dual-color probe for determining the number of copies of both  Her-2/neu  (orange) and the chromosome 17 centromeres (green). The kit was used following the manufacturer's instructions with a few minor modifications. Slides containing 5μ thick paraffin-embedded tissue sections of studied cervical tumor cases and a known Her-2/neu amplified breast tumor were placed on a slide warmer overnight at 58°C, followed by deparaffinization in Xilol, dehydration in 100 ethanol, and drying on a slide warmer at 45 to 50°C. Slides were then pretreated with 0.2 N hydrochloric acid for 20 minutes, followed by washes in purified water and immersion in Vysis wash buffer. They were subsequently immersed in Vysis protease solution at 37°C for 10 minutes, washed in Vysis wash buffer, and dried on the slide warmer. The slides were then immersed in 10% buffered formalin at room temperature for 10 minutes, immersed in Vysis wash buffer, and dried on the slide warmer. Sections were denatured by placing the slides in formamide for 5 minutes at 72°C followed by dehydration in 70, 85 and then 100% ethanol. Slides were then dried on a slide warmer, and 10 μl of probe was applied. They were then coverslipped, sealed and placed in a prewarmed humid incubation chamber at 37°C for 21 hours. This was followed by immersion in prewarmed postwash solution at 72°C for 2 minutes. The slides were air-dried, and a 4',6-diamidino-2-phenylindole (DAPI) counterstain was applied.Her-2/neuHer-2/neuThe scoring system used is described in detail in the manufacturer's instruction. A minimum of 60 nuclei were scored by each of 2 observers using a Zeiss Axioskop-2 fluorescent microscope with V.2 filter. The ratio of  Her-2/neu  signals (orange) to chromosome 17 centromere signals (green) was determined with ratios of <2.0 considered nonamplified and those ≥ 2.0 amplified.Her-2/neuResults The immunohistochemical expression of HER2 in the primary tumors of 35 patients at diagnosis was evaluated. Accordingly, these patients had no received any previous anticancer therapy; their mean age was 40.8 years; 5 were staged as IB2-IIA, 14 as IIB and 16 as IIIB; 31 and 4 were histologically classified as squamous and adeno/adenosquamous respectively. Overexpression of HER2 was demonstrated in only one out of 35 cases, which had a score of 3+ (not shown). The remaining cases were interpreted as negative [score of 0]. The case with HER2 overexpression at diagnosis was a 56-year old woman diagnosed with a FIGO stage IIB large cell poorly differentiated squamous cell carcinoma, who received treatment with 6 weekly courses of cisplatin concurrent with external beam radiation and brachytherapy. She is currently free of disease at 44 months of follow-up. At a median follow-up time 40 months, seven patients have relapsed. HER2 expression at recurrence could only be analyzed in four of these seven relapsed patients in whom whose recurrent disease was histopathologically confirmed. Two of these four tested positive with a staining intensity of 2+ and 3+ respectively, (Figures  1  and  2 ), both cases were squamous cell carcinomas and tested negative in the pretreatment surgical specimen The five cervical cancer cell lines were negative. None of the four recurrent cases tested by FISH were HER2 amplified (Figure  3 ).
Results
The immunohistochemical expression of HER2 in the primary tumors of 35 patients at diagnosis was evaluated. Accordingly, these patients had no received any previous anticancer therapy; their mean age was 40.8 years; 5 were staged as IB2-IIA, 14 as IIB and 16 as IIIB; 31 and 4 were histologically classified as squamous and adeno/adenosquamous respectively. Overexpression of HER2 was demonstrated in only one out of 35 cases, which had a score of 3+ (not shown). The remaining cases were interpreted as negative [score of 0]. The case with HER2 overexpression at diagnosis was a 56-year old woman diagnosed with a FIGO stage IIB large cell poorly differentiated squamous cell carcinoma, who received treatment with 6 weekly courses of cisplatin concurrent with external beam radiation and brachytherapy. She is currently free of disease at 44 months of follow-up. At a median follow-up time 40 months, seven patients have relapsed.HER2 expression at recurrence could only be analyzed in four of these seven relapsed patients in whom whose recurrent disease was histopathologically confirmed. Two of these four tested positive with a staining intensity of 2+ and 3+ respectively, (Figures  1  and  2 ), both cases were squamous cell carcinomas and tested negative in the pretreatment surgical specimenThe five cervical cancer cell lines were negative.None of the four recurrent cases tested by FISH were HER2 amplified (Figure  3 ).Discussion Molecular targeted therapies are currently being tested in a variety of tumor types with promising results. Because HER2 overexpression occurs and is related to a worse prognosis in cervical cancer, [ 7 - 9 , 13 ], its blockade with trastuzumab could potentially have therapeutic value. This monoclonal antibody is currently widely used in metastatic breast cancer and is being evaluated in an adjuvant setting as well as in a variety of tumor types [ 3 , 15 - 17 ], Based on the fact that the efficacy of this antibody is strongly associated to the level of HER2 expression in the primary tumor, the FDA approved the Hercep Test in the aim to grade the expression level so that only those patients whose tumors exhibit a 2+/3+ levels are candidates to trastuzumab therapy, though currently in most centers, tumors expression of 2+ is considered undeterminate therefore these cases are also evaluated by FISH analysis [ 18 ]. Previous reports on cervical cancer using non-standardized methods for HER2 expression showed that up to 77% of cases express the receptor and that in general HER2 expression predominates in adenocarcinoma and adenosquamous carcinoma histologies [ 6 - 14 ] In this work, using the Hercep Test with its corresponding guidelines for evaluation, we found contrastating results as none of the cell lines expressed HER2 and only a single tumor of squamous histology (1 out of 35) expresses this oncoprotein at a level of 3+. Such a discrepancy does not seem to be limited to this tumor type. For instances, in ovarian clear cell carcinoma a 43% of overexpression was reported utilizing systems other than Hercep Test [ 19 ], however, when evaluated with this standardized test, only 1 out of 17 tumors expressed 3+ [ 20 ]. Likewise, the proportion of patients with 2+/3+ expression level of HER2 with the Hercep Test is uniformly low in tumor such as lung [ 21 ], colorectal carcinomas [ 22 ], gallbladder [ 23 ], and melanoma [ 24 ]. A variety of factors such as the kind of antibody used, the technique per se, and scoring criteria may explain such phenomenon and its clarification requires further studies. A recent paper by Bellone et al., have reported that a substantially higher proportion of cervical cancer cells lines either established from fresh tumors or commercial ones (including CasKi, SiHa, HeLa and C33A which are negative by immunochemistry) express the receptor when evaluated by flow-cytometry and are growth inhibited when incubated with trastuzumab or trastuzumab plus IL-2 [ 25 ]. These data led them to suggest the targeting the HER in cervical cancer could be more effective than the indicated by low immunohistochemical expression [ 25 ]. However, it is largely known that in breast [ 26 ] and more recently in lung cancer [ 27 ], tumor responses are almost confined to those with a 3+ level of expression. For instances, in a recent published study in 111 assessable breast cancer patients, the response rate to single agent trastuzumab for those expressing 3+ versus 2+ was 35% and 0% respectively [ 26 ], while in lung cancer, a phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive patients, yet there was not overall differences in response between both arms, the benefit was limited to those with 3+ of expression with the Hercep Test. Accordingly, five out of six patients with such level of expression receiving trastuzumab plus gemcitabine-cisplatin showed response [ 27 ]. These data argue against the potential usefulness of trastuzumab in cervical cancer patients with HER expression that can only be detected by flow cytometry [ 25 ]. On the other hand, the HER2 expression in breast cancer is relatively stable, with 95% concordance between the HER2 status of primary and metastatic lesions, being rare a shift from positivity in the primary to negativity in the metastases [ 5 ]. Conversely, 6% of breast cancer patients whose primary tumors are HER2 negative, convert to high expression (Hercep Test 3+) in their metastases [ 28 ]. Such behavior is in line with experimental observations that receptor activation potentiates tumor cell motility, protease secretion and invasion, and also modulates cell cycle checkpoint function, DNA repair, apoptotic responses and multidrug resistance [ 29 , 30 ]. The findings of "conversion to positive" in cells of recurrent cervical tumors showed by us and other authors [ 25 , 31 ] strongly suggest that expression of HER2 may have a role in tumor resistance and progression as shown in experimental models, and therefore its targeting in recurrent cervical cancer could have therapeutic value. It is remarkable the finding that none of the four recurrent cases, including the two that converted to IHC positive analyzed by FISH showed HER2 gene amplification. This result is unlikely to be a false negative as the green signal was perfectly observed in most of the cells. Previous studies in cervical cancer have shown that the frequency of gene amplification as determined by FISH is low irrespective of tumor histology. Mark et al., reported only 2 out of 23 cases amplified using the Her-2/neu FISH probe (Vysis, Inc., Downers Grove, IL) both of which were adenocarcinomas [ 32 ]. In a more recent study looking at DNA copy number of cervical adenocarcinomas, it was found that despite more than 50% of patients had chromosome 17q copy number gains, only 9% (2 out of 22) of these tumors showed an HER-2/neu protein over-expression at the level of 2+ with the Hercep test. These findings suggest that amplification of HER-2/ neu  is rare in cervical adenocarcinomas and that low level chromosome 17q copy number gains are not associated with HER-2/ neu  overexpression [ 33 ]. Such overexpression without gene amplification could result from transcriptional deregulation leading to increased receptor expression [ 34 ] and is not a rare phenomenon in breast carcinoma [ 35 ].
Discussion
Molecular targeted therapies are currently being tested in a variety of tumor types with promising results. Because HER2 overexpression occurs and is related to a worse prognosis in cervical cancer, [ 7 - 9 , 13 ], its blockade with trastuzumab could potentially have therapeutic value. This monoclonal antibody is currently widely used in metastatic breast cancer and is being evaluated in an adjuvant setting as well as in a variety of tumor types [ 3 , 15 - 17 ], Based on the fact that the efficacy of this antibody is strongly associated to the level of HER2 expression in the primary tumor, the FDA approved the Hercep Test in the aim to grade the expression level so that only those patients whose tumors exhibit a 2+/3+ levels are candidates to trastuzumab therapy, though currently in most centers, tumors expression of 2+ is considered undeterminate therefore these cases are also evaluated by FISH analysis [ 18 ].Previous reports on cervical cancer using non-standardized methods for HER2 expression showed that up to 77% of cases express the receptor and that in general HER2 expression predominates in adenocarcinoma and adenosquamous carcinoma histologies [ 6 - 14 ] In this work, using the Hercep Test with its corresponding guidelines for evaluation, we found contrastating results as none of the cell lines expressed HER2 and only a single tumor of squamous histology (1 out of 35) expresses this oncoprotein at a level of 3+. Such a discrepancy does not seem to be limited to this tumor type. For instances, in ovarian clear cell carcinoma a 43% of overexpression was reported utilizing systems other than Hercep Test [ 19 ], however, when evaluated with this standardized test, only 1 out of 17 tumors expressed 3+ [ 20 ]. Likewise, the proportion of patients with 2+/3+ expression level of HER2 with the Hercep Test is uniformly low in tumor such as lung [ 21 ], colorectal carcinomas [ 22 ], gallbladder [ 23 ], and melanoma [ 24 ]. A variety of factors such as the kind of antibody used, the technique per se, and scoring criteria may explain such phenomenon and its clarification requires further studies. A recent paper by Bellone et al., have reported that a substantially higher proportion of cervical cancer cells lines either established from fresh tumors or commercial ones (including CasKi, SiHa, HeLa and C33A which are negative by immunochemistry) express the receptor when evaluated by flow-cytometry and are growth inhibited when incubated with trastuzumab or trastuzumab plus IL-2 [ 25 ]. These data led them to suggest the targeting the HER in cervical cancer could be more effective than the indicated by low immunohistochemical expression [ 25 ]. However, it is largely known that in breast [ 26 ] and more recently in lung cancer [ 27 ], tumor responses are almost confined to those with a 3+ level of expression. For instances, in a recent published study in 111 assessable breast cancer patients, the response rate to single agent trastuzumab for those expressing 3+ versus 2+ was 35% and 0% respectively [ 26 ], while in lung cancer, a phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive patients, yet there was not overall differences in response between both arms, the benefit was limited to those with 3+ of expression with the Hercep Test. Accordingly, five out of six patients with such level of expression receiving trastuzumab plus gemcitabine-cisplatin showed response [ 27 ]. These data argue against the potential usefulness of trastuzumab in cervical cancer patients with HER expression that can only be detected by flow cytometry [ 25 ].On the other hand, the HER2 expression in breast cancer is relatively stable, with 95% concordance between the HER2 status of primary and metastatic lesions, being rare a shift from positivity in the primary to negativity in the metastases [ 5 ]. Conversely, 6% of breast cancer patients whose primary tumors are HER2 negative, convert to high expression (Hercep Test 3+) in their metastases [ 28 ]. Such behavior is in line with experimental observations that receptor activation potentiates tumor cell motility, protease secretion and invasion, and also modulates cell cycle checkpoint function, DNA repair, apoptotic responses and multidrug resistance [ 29 , 30 ]. The findings of "conversion to positive" in cells of recurrent cervical tumors showed by us and other authors [ 25 , 31 ] strongly suggest that expression of HER2 may have a role in tumor resistance and progression as shown in experimental models, and therefore its targeting in recurrent cervical cancer could have therapeutic value.It is remarkable the finding that none of the four recurrent cases, including the two that converted to IHC positive analyzed by FISH showed HER2 gene amplification. This result is unlikely to be a false negative as the green signal was perfectly observed in most of the cells. Previous studies in cervical cancer have shown that the frequency of gene amplification as determined by FISH is low irrespective of tumor histology. Mark et al., reported only 2 out of 23 cases amplified using the Her-2/neu FISH probe (Vysis, Inc., Downers Grove, IL) both of which were adenocarcinomas [ 32 ]. In a more recent study looking at DNA copy number of cervical adenocarcinomas, it was found that despite more than 50% of patients had chromosome 17q copy number gains, only 9% (2 out of 22) of these tumors showed an HER-2/neu protein over-expression at the level of 2+ with the Hercep test. These findings suggest that amplification of HER-2/ neu  is rare in cervical adenocarcinomas and that low level chromosome 17q copy number gains are not associated with HER-2/ neu  overexpression [ 33 ]. Such overexpression without gene amplification could result from transcriptional deregulation leading to increased receptor expression [ 34 ] and is not a rare phenomenon in breast carcinoma [ 35 ].neuneuConclusions In conclusion, our study suggest that the clinical usefulness of anti-HER2 antibodies in the primary treatment of cervical cancer patients may be limited due to the low frequency of HER overexpression, nevertheless, it is desirable to further test a larger number of recurrent cervical cancer patients by IHC and FISH analyses regardless of the histological type, as a start point for clinical trials design using trastuzumab.
Conclusions
In conclusion, our study suggest that the clinical usefulness of anti-HER2 antibodies in the primary treatment of cervical cancer patients may be limited due to the low frequency of HER overexpression, nevertheless, it is desirable to further test a larger number of recurrent cervical cancer patients by IHC and FISH analyses regardless of the histological type, as a start point for clinical trials design using trastuzumab.Competing interests None declared.
Competing interests
None declared.Authors'contributions A C-B, AG-F, carried out the tissue culture work and immunohistochemical analysis; VP-S and T V-C interpreted the histological data; MC critically analyzed and participated in manuscript; CP, contribute with the clinical data; SV performed the FISH analysis; and AD-G conceived the study and wrote the manuscript. All authors read and approved the final manuscript.
Authors'contributions
A C-B, AG-F, carried out the tissue culture work and immunohistochemical analysis; VP-S and T V-C interpreted the histological data; MC critically analyzed and participated in manuscript; CP, contribute with the clinical data; SV performed the FISH analysis; and AD-G conceived the study and wrote the manuscript. All authors read and approved the final manuscript.Pre-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: