erb-b2 Amplification by Fluorescence In Situ Hybridization
erb-b2 Amplification by Fluorescence In Situ Hybridization
We conducted this study to ascertain the prevalence of erb-b2 gene amplification in breast cancer specimens read as 2+ in immunohistochemical analysis. Slides from patients with metastatic or recurrent breast cancer were eligible for fluorescent in situ hybridization (FISH) study if they were read as 2+ immunohisto-chemically for erb-b2 by a certified pathologist. The PathVysion kit (Vysis, Downers Grove, IL) was used for FISH studies. Amplification of the erb-b2 gene was defined as an erb-b2/CEP17 (chromosome 17 centromere) ratio of 2 or more in 30 tumor cells counted. From May 2003 to June 2004, 221 slides were submitted from 24 hospitals around the island. Of 216 successful hybridizations, 96 (44.4%) were determined to be erb-b2 amplified. In addition, the topoisomerase IIα gene was coamplified in 11 (21%) of 53 and deleted in 8 (15%) of 53 erb-b2 amplified cases. The erb-b2 gene amplification rate was very high in cases determined to be 2+ by immunohistochemical analysis; therefore, determination of erb-b2 status by FISH in cases scored 2+ immunohistochemically is strongly recommended.
Breast cancer ranks as the second most common cancer for women in Taiwan. The crude incidence and mortality rates are 42.7 and 10.6 per 100,000 population, respectively, with 4,642 new cases and 1,149 deaths reported in the year 2000. In the same time period, the incidence and mortality rates for whites, African Americans, and Asian/Pacific islanders in the United States were 150 and 26.7, 119 and 35.4, and 97 and 12.6 per 100,000 population, respectively. The local breast cancer incidence peaks between the ages of 40 and 50 years, with a mean age at occurrence of 47 years. Taiwanese women have a lower incidence of breast cancer but a higher probability of dying of it once a diagnosis has been made, and this cancer occurs more commonly in midlife, with significant financial and social implications.
Since the first report of erb-b2 as a poor prognostic factor for breast cancer in 1987, the significance of this oncogene as an adverse prognostic factor has been noted in many other cancers, such as ovary, lung, stomach, and pancreas. Amplification of the erb-b2 gene or overexpression of the erb-b2 protein has been detected in 10% to 30% of breast cancers and, together with the associated topoisomerase IIα (T2α) gene aberrations, shows no change over time, after treatment, and no difference between primary and metastatic lesions. Although no difference in erb-b2 status has been noted between males (15% overexpression in immunohistochemical analysis, 11% amplification in fluorescence in situ hybridization [FISH]) and females or between whites and African Americans, its expression in young Koreans (≤45 years; 47.5%) and in a small study of Chinese women in Taiwan (43.2%) has been somewhat higher.
Analysis of the Cancer and Leukemia Group B 8541 protocol concluded that erb-b2 overexpression identified patients most likely to benefit from high doses of adjuvant doxorubicin, suggesting it to be a predictive marker for anthracycline response. The molecular basis of this phenomenon has been attributed to concurrent amplification of the T2α oncogene, with increased expression of T2α enzymes, a target for the anthracyclines, and this has been confirmed to be the significant target for doxorubicin response in a prospective clinical trial. Molecular genetic studies in erb-b2-amplified primary breast cancers demonstrated coamplification of the T2α gene in 44% and deletion in 42% of cases, with no change in gene copy number in all non-erb-b2-amplified tumors.
The US Food and Drug Administration has approved the HercepTest (DAKO, Carpinteria, CA) and Ventana Pathway (Ventana, Tucson, AZ) as immunohistochemical assays for detection of erb-b2 protein expression in breast cancer and PathVysion (Vysis, Downers Grove, IL) as a FISH method to detect erb-b2 gene amplification. Many studies have since reported more than 90% concordance between the immunohistochemistry and FISH tests, but both tests usually were performed in the same laboratory with a large volume of samples processed annually. In May 2003, the National Comprehensive Cancer Network (Jenkintown, PA) updated its oncology practice guidelines, recommending FISH over immunohistochemical analysis to determine erb-b2 status, based on studies documenting comparable trastuzumab (Herceptin) response and survival duration for FISH-amplified cases, regardless of immunohistochemical status.
Recent reports on very large comparative studies between immunohistochemical analysis and FISH for detection of erb-b2 give a more balanced view on the relationship between the 2 assays. Researchers at the Memorial Sloan-Kettering Cancer Center, New York, NY, found a very high concordance rate between immunohistochemical scores of 0, 1+, and 3+ and FISH data in 2,279 cases studied; however, 25% of cases scored as 2+ immunohistochemically demonstrated gene amplification in FISH studies. Investigators at Impath (Los Angeles, CA) reported 20% of immunohistochemical scores as 3+ in 116,736 specimens and 22.7% FISH amplification in 16,092 specimens, and in 6,556 specimens with both tests done, gene amplification was found in 4.1% of specimens with an immunohistochemical score of 0, 7.4% with a score of 1+, 23% with a score of 2+, and 92% with a score of 3+. An Australian study reporting on 1,536 cancers found immunohistochemical scores of 3+ in 12% of cases, of which 98% were amplified by FISH, and 2+ in 13% of the cases, of which 23% were amplified by FISH. Large-scale studies recommend a FISH assay for specimens with an immunohistochemical score of 2+, because interlaboratory reproducibility of HER-2/neu status is poor by immunohistochemical analysis without standardization of the procedure.
Five years ago, the Taiwan Co-operative Oncology Group (TCOG) launched a correlative study for erb-b2 through its pathology subcommittee. The detection of erb-b2 at member hospitals performed by immunohistochemical analysis was correlated with HercepTest immunohistochemical analysis and PathVysion FISH performed at the molecular genetics laboratory of the National Health Research Institutes (Taipei, Taiwan). Although the data were not published, in 88 specimens (negative, 53; positive, 35) from 11 hospitals, the concordance rate was 100% for all specimens with immunohistochemical scores of 0 (FISH-) and 3+ (FISH+). It was decided to launch a nationwide FISH confirmation study for any case of metastatic breast cancer with erb-b2 with an immunohistochemical score of 2+ determined by a certified pathologist, with the addition of T2α gene evaluation in the latter part of the 1-year study.
We conducted this study to ascertain the prevalence of erb-b2 gene amplification in breast cancer specimens read as 2+ in immunohistochemical analysis. Slides from patients with metastatic or recurrent breast cancer were eligible for fluorescent in situ hybridization (FISH) study if they were read as 2+ immunohisto-chemically for erb-b2 by a certified pathologist. The PathVysion kit (Vysis, Downers Grove, IL) was used for FISH studies. Amplification of the erb-b2 gene was defined as an erb-b2/CEP17 (chromosome 17 centromere) ratio of 2 or more in 30 tumor cells counted. From May 2003 to June 2004, 221 slides were submitted from 24 hospitals around the island. Of 216 successful hybridizations, 96 (44.4%) were determined to be erb-b2 amplified. In addition, the topoisomerase IIα gene was coamplified in 11 (21%) of 53 and deleted in 8 (15%) of 53 erb-b2 amplified cases. The erb-b2 gene amplification rate was very high in cases determined to be 2+ by immunohistochemical analysis; therefore, determination of erb-b2 status by FISH in cases scored 2+ immunohistochemically is strongly recommended.
Breast cancer ranks as the second most common cancer for women in Taiwan. The crude incidence and mortality rates are 42.7 and 10.6 per 100,000 population, respectively, with 4,642 new cases and 1,149 deaths reported in the year 2000. In the same time period, the incidence and mortality rates for whites, African Americans, and Asian/Pacific islanders in the United States were 150 and 26.7, 119 and 35.4, and 97 and 12.6 per 100,000 population, respectively. The local breast cancer incidence peaks between the ages of 40 and 50 years, with a mean age at occurrence of 47 years. Taiwanese women have a lower incidence of breast cancer but a higher probability of dying of it once a diagnosis has been made, and this cancer occurs more commonly in midlife, with significant financial and social implications.
Since the first report of erb-b2 as a poor prognostic factor for breast cancer in 1987, the significance of this oncogene as an adverse prognostic factor has been noted in many other cancers, such as ovary, lung, stomach, and pancreas. Amplification of the erb-b2 gene or overexpression of the erb-b2 protein has been detected in 10% to 30% of breast cancers and, together with the associated topoisomerase IIα (T2α) gene aberrations, shows no change over time, after treatment, and no difference between primary and metastatic lesions. Although no difference in erb-b2 status has been noted between males (15% overexpression in immunohistochemical analysis, 11% amplification in fluorescence in situ hybridization [FISH]) and females or between whites and African Americans, its expression in young Koreans (≤45 years; 47.5%) and in a small study of Chinese women in Taiwan (43.2%) has been somewhat higher.
Analysis of the Cancer and Leukemia Group B 8541 protocol concluded that erb-b2 overexpression identified patients most likely to benefit from high doses of adjuvant doxorubicin, suggesting it to be a predictive marker for anthracycline response. The molecular basis of this phenomenon has been attributed to concurrent amplification of the T2α oncogene, with increased expression of T2α enzymes, a target for the anthracyclines, and this has been confirmed to be the significant target for doxorubicin response in a prospective clinical trial. Molecular genetic studies in erb-b2-amplified primary breast cancers demonstrated coamplification of the T2α gene in 44% and deletion in 42% of cases, with no change in gene copy number in all non-erb-b2-amplified tumors.
The US Food and Drug Administration has approved the HercepTest (DAKO, Carpinteria, CA) and Ventana Pathway (Ventana, Tucson, AZ) as immunohistochemical assays for detection of erb-b2 protein expression in breast cancer and PathVysion (Vysis, Downers Grove, IL) as a FISH method to detect erb-b2 gene amplification. Many studies have since reported more than 90% concordance between the immunohistochemistry and FISH tests, but both tests usually were performed in the same laboratory with a large volume of samples processed annually. In May 2003, the National Comprehensive Cancer Network (Jenkintown, PA) updated its oncology practice guidelines, recommending FISH over immunohistochemical analysis to determine erb-b2 status, based on studies documenting comparable trastuzumab (Herceptin) response and survival duration for FISH-amplified cases, regardless of immunohistochemical status.
Recent reports on very large comparative studies between immunohistochemical analysis and FISH for detection of erb-b2 give a more balanced view on the relationship between the 2 assays. Researchers at the Memorial Sloan-Kettering Cancer Center, New York, NY, found a very high concordance rate between immunohistochemical scores of 0, 1+, and 3+ and FISH data in 2,279 cases studied; however, 25% of cases scored as 2+ immunohistochemically demonstrated gene amplification in FISH studies. Investigators at Impath (Los Angeles, CA) reported 20% of immunohistochemical scores as 3+ in 116,736 specimens and 22.7% FISH amplification in 16,092 specimens, and in 6,556 specimens with both tests done, gene amplification was found in 4.1% of specimens with an immunohistochemical score of 0, 7.4% with a score of 1+, 23% with a score of 2+, and 92% with a score of 3+. An Australian study reporting on 1,536 cancers found immunohistochemical scores of 3+ in 12% of cases, of which 98% were amplified by FISH, and 2+ in 13% of the cases, of which 23% were amplified by FISH. Large-scale studies recommend a FISH assay for specimens with an immunohistochemical score of 2+, because interlaboratory reproducibility of HER-2/neu status is poor by immunohistochemical analysis without standardization of the procedure.
Five years ago, the Taiwan Co-operative Oncology Group (TCOG) launched a correlative study for erb-b2 through its pathology subcommittee. The detection of erb-b2 at member hospitals performed by immunohistochemical analysis was correlated with HercepTest immunohistochemical analysis and PathVysion FISH performed at the molecular genetics laboratory of the National Health Research Institutes (Taipei, Taiwan). Although the data were not published, in 88 specimens (negative, 53; positive, 35) from 11 hospitals, the concordance rate was 100% for all specimens with immunohistochemical scores of 0 (FISH-) and 3+ (FISH+). It was decided to launch a nationwide FISH confirmation study for any case of metastatic breast cancer with erb-b2 with an immunohistochemical score of 2+ determined by a certified pathologist, with the addition of T2α gene evaluation in the latter part of the 1-year study.
