Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer.

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

Immunocytochemistry and fluorescence in situ hybridization in HER-2/neu status in cell block preparations

OBJECTIVE: Recent studies have validated the use of cytologic materials to determine HER-2/neu status. Good concordance has been shown between results obtained by immunocytochemistry (ICC), immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) on cytologic and surgical specimens. However, the utility of cytologic cell block material in determining HER-2/neu status has not been reported and is the subject of this study. STUDY DESIGN: HER-2/neu status was determined in 25 cases of primary or metastatic breast carcinoma by IHC and FISH. All cases were formalin-fixed, paraffin-embedded (FPPE) cell block preparations. ICC was performed using monoclonal antibodies TAB250 (Zymed) and CB11 (Novacastra Laboratories). FISH analysis was performed using the PathVysion HER-2 probe kit (Vysis, Inc.). Results of ICC and FISH were compared in each case. RESULTS: Of 25 cases studied, 17 showed no protein overexpression or amplification. Five cases showed protein overexpression and amplification. The remaining 3 cases showed 2+ staining intensity by ICC in 10, 20, and 50% of carcinoma cells, respectively, and all demonstrated lack of amplification. CONCLUSION: Immunocytochemistry performed on FFPE cell block material is a reliable method for determining HER-2/neu status in cytologic specimens. We recommend routine preparation of FFPE cell block material in instances of suspected primary of metastatic breast carcinoma.     

Comparison of in situ hybridization methods for the assessment of HER-2/neu gene amplification status in breast cancer using a tissue microarray

BackgroundThis project compared HER-2/neu gene status in breast cancers, as demonstrated by FISH (fluorescent in situ hybridization) and CISH (chromogenic in situ hybridization) and using a tissue microarray (TMA). The study also aimed to show whether the TMA technique could be used in clinical diagnostics, rather than remain a scientific tool.Materials and methodsA TMA was constructed using 121 breast cancer specimens, 6 cores from each specimen. Demonstration and assessment of HER-2/neu gene status was by FISH (Vysis Path) and CISH (DAKO Duo CISH).ResultsThe 121 breast cancer specimens were divided into 3 groups by HER-2 status, as determined by immunohistochemistry. In the HER-2 negative group no amplification was observed in 36 out of 40 cases. 3 cases showed amplification by both methods and one by CISH alone. The equivocal HER-2 group showed no amplification in 30 out of 41 cases and amplification in 9 cases. One case was FISH negative CISH positive and one was discarded. In the HER-2 positive group, amplification was confirmed in 37 of the 40 cases by both methods. 3 cases were unsuitable for assessment.ConclusionsThis study indicated that CISH is a sensitive alternative to FISH in detecting HER2 gene amplification and may replace FISH in HER2 testing. Good agreement was observed between methods (98.5% – 119 out of 121 cases).Furthermore, as only 4 out of 121 cases were unsuitable for assessment (no signal or missing TMA cores) – it may be feasible to use TMA in diagnostics.     

Added Value of HER-2 Amplification Testing by Multiplex Ligation-Dependent Probe Amplification in Invasive Breast Cancer

Background

HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH).

Methods

As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases).

Results

The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001). Overall concordance between IHC and MLPA/CISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining.

Conclusions

MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.     

Quantitative assessment of HER-2/neu protein concentration in breast cancer by enzyme-linked immunosorbent assay

PURPOSE: The HER-2/neu protein (p185) has become a promising target for antibody therapy in breast cancer. We tested the feasibility of a quantitative approach for HER-2/neu testing based on the analysis of tumor tissue extracts by an enzyme-linked immunosorbent assay (ELISA). EXPERIMENTAL DESIGN: Tumor tissue extracts of primary human breast cancers (n=124) were prepared using a triton-based buffer. HER-2/neu concentration was quantified by ELISA. Paraffin-embedded tissue sections of the same tumors were analyzed by immunohistochemical staining applying the monoclonal HER-2/neu antibody TAB 250 (n=124) and by chromogenic in situ hybridization (CISH) (n=73). RESULTS: Concentrations of p185 in tissue extracts determined by ELISA varied from 1 to 927 ng per mg protein with a median of 25 ng/mg protein, whereas normal breast tissue showed values from 0.4 to 5.5 ng/mg with a median of 2.2 ng/mg (p<0.0001, Mann-Whitney U test). A significant correlation between p185 concentration and immunohistochemical staining was observed (p<0.0001, Kruskal-Wallis test). In addition, p185 concentration measured by ELISA was correlated with the degree of HER-2/neu gene amplification determined by CISH. HER-2/neu-amplified tumors had significantly higher p185 concentrations (median value 181 ng/mg protein) than non-amplified tumors (median value 20 ng/mg; p<0.0001, Mann-Whitney U test). CONCLUSIONS: ELISA-based measurement of HER-2/neu protein concentration in breast cancer tissue extracts is feasible and provides quantitative results for p185 protein concentrations that correlate closely with HER-2/neu immunoscore and gene amplification.     

Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

Targeted therapy in breast cancer: the HER-2/neu gene and protein

The HER-2/neu oncogene, a member of the epidermal growth factor receptor or erb gene family, encodes a transmembrane tyrosine kinase receptor that has been linked to prognosis and response to therapy with the anti-HER-2-humanized monoclonal antibody, trastuzumab (Herceptin, Genentech, South San Francisco, CA) in patients with advanced metastatic breast cancer. HER-2/neu status has also been tested for its ability to predict the response of breast cancer to other therapies including hormonal therapies, topoisomerase inhibitors, and anthracyclines. This review includes an analysis of 80 published studies encompassing more than 25,000 patients designed to consider the relative advantages and disadvantages of the various methods of measuring HER-2/neu in clinical breast cancer specimens. Southern blotting, PCR amplification detection, and fluorescence in situ hybridization assays designed to detect HER-2/neu gene amplification are compared with HER-2/neu protein overexpression assays performed by immunohistochemical techniques applied to frozen and paraffin-embedded tissues and enzyme immunoassays performed on tumor cytosols. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The role of HER-2/neu testing for the prediction of response to trastuzumab therapy in breast cancer is reviewed along with the current studies designed to test whether HER-2/neu status can predict the response to standard and newer hormonal therapies, cytotoxic chemotherapy, and radiation. The review will also evaluate the status of serum-based testing for circulating HER-2/neu receptor protein and its ability to predict disease outcome and therapy response.     

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.     

Discordant results obtained for different methods of HER-2/neu testing in breast cancer--a question of standardization, automation and timing

BACKGROUND: HER-2/neu positivity is required for the selection of stage IV breast cancer patients for trastuzumab therapy. We compared the results of the recommended immunohistochemistry (IHC) evaluation with the automated ACIS IHC system and with fluorescence in situ hybridization (FISH). These HER-2/neu tissue results were correlated with the serum HER-2/neu (sHER-2/neu) levels at the time of metastatic spread. PATIENTS AND METHODS: A total of 61 IHC slides from 30 patients were stained using the HercepTest. HER-2/neu gene amplification was determined using the Ventana FISH assay. sHER-2/neu levels were measured with the Oncogene Science" ELISA kit. The concordance of HER-2/neu results was determined using the concordance index Kappa (kappa). RESULTS: The best concordance between any IHC and FISH was found for the automated ACIS system (88.5%, kappa=0.68, category "good"). The comparison between the manual interpretations and the automated IHC was categorized as "very good" (95.1%, kappa=0.85). The median sHER-2/neu level of FISH positive patients was significantly higher (67 ng/mL) than that of FISH negative patients (17 ng/mL, p=0.018). The increase in HER-2/neu positivity comparing tissue to stage IV serum was statistically significant (p=0.001). CONCLUSIONS: The concordance between conventional IHC and computerized analysis was very good. The number of patients with stage IV breast cancer with an elevated sHER-2/neu level was much higher than HER-2/neu positivity in tissue. This discrepancy is only partially explained by the influence of tumor load. Patients with an elevated sHER-2/neu level and no tissue overexpression should be considered for retesting of tissue or a new biopsy.     

Quantitation of HER-2/neu oncoprotein overexpression in invasive breast cancer by image analysis: a study comparing fresh and paraffin-embedded material.

HER-2/neu oncoprotein overexpression was compared in fresh frozen and paraffin-embedded formalin-fixed invasive breast cancer material from the same patients. The HER-2/neu protein was detected by an immunohistochemical staining method, and the average amount of protein staining per cell was measured using the CAS-200 image analysis system and expressed relative to the amount of HER-2/neu protein of calibration cells of the SKBR3 cell line which are known to have amplification of the HER-2/neu gene and overexpression of the HER-2/neu protein. There was a significant correlation between degree of HER-2/neu protein overexpression and DNA-hyperdiploidy (P less than 0.01, chi 2 test). No significant correlation could be demonstrated between degree of HER-2/neu overexpression and tumor size, lymph node status, number of positive nodes or morphometric features. There was in general a good concordance (r = 0.83) in HER-2/neu expression values between fresh and paraffin-embedded material. Pairwise comparison of the two series (Wilcoxon signed ranks test) revealed no significant differences, indicating that there were no systematic differences between HER-2/neu assessments in fresh and paraffin material. When analysing the HER-2/neu expression values according to thresholds used earlier for overexpression, comparable results for fresh and paraffin material were obtained for most cases. In the fresh and paraffin material a different staining pattern was observed (more membrane staining in the fresh material in contrast to a more diffuse staining pattern in the paraffin material). It was concluded that both fresh-frozen and paraffin-embedded, formalin-fixed material is suitable for assessment of HER-2/neu protein overexpression by image analysis and provides comparable HER-2/neu expression values in most cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

HER-2/neu evaluation by fluorescence in situ hybridization on destained cytologic smears from primary and metastatic breast cancer

OBJECTIVE: To evaluate HER-2/neu amplification by fluorescence in situ hybridization (FISH) (HER-2/neu by FISH) on archival cytologic smears stained with May-Grünwald-Giemsa (MGG) stain. STUDY DESIGN: Cytologic specimens from 69 breast cancer lesions (48 primary and 21 metastatic), stained with MGG stain for routine diagnostic cytology, were destained and subjected to HER-2/neu by FISH. Fifteen of the 69 samples were also evaluated by FISH on paired fresh smears. RESULTS: HER-2/neu by FISH was successfully assayed in 25 of the 48 primary tumors and in 15 of the 21 metastatic lesions, corresponding to an overall feasibility of 58%. These cases had been archived between 1 month and 10 years prior to FISH analysis. Eight of the 25 primary and 5 of the 15 metastatic tumors were amplified. In 15 of the 40 evaluable cases, HER-2/neu was also assessed on the corresponding fresh smears: 8 tumors were amplified and 7 unamplified on both destained MGG and fresh smears. CONCLUSION: HER-2/neu can be detected by FISH on routinely MGG-stained cytologic slides. This approach allows HER-2/neu evaluation whenever histologic sections or fresh cytologic material are not available. In these cases, HER-2/neu assessment on destained cytologic smears plays a role in the selection of targeted therapy.     

Routine assessment of hormonal receptor and her-2/neu status underscores the need for more therapeutic targets in Kenyan women with breast cancer

OBJECTIVE: To report the expression of estrogen receptors, progesterone receptors and human epidermal growth factor receptor (Her-2/neu) in 158 Kenyan women with breast cancer and correlation with other prognostic indicators in this high-risk group. This study stressed the importance of routine assessment of the steroid receptors and Her-2/neu as a mode of therapeutic selection of patients for antihormonal or targeting monoclonal antibody (Herceptin) therapy, directed at the juxtamembrane domain of Her-2/neu protein in the developing countries such as Kenya. STUDY DESIGN: The study population consisted of 158 female patients with histologically confirmed breast carcinoma seen at the pathology department of The Nairobi Hospital. An immunohistochemical (IHC) study of ER, PR and Her-2/neu was conducted, followed by fluorescent in situ hybridization (FISH) validation for Her-2/neu gene amplification in cases initially scored as positive 2+ with IHC. Mastectomy samples registered at the pathology department of The Nairobi Hospital were used for this study. The study was approved by the institution's ethical review committee and informed consent obtainedfrom the concerned patients. RESULTS: In the studied cohort, positivity for both hormonal receptors and Her-2/neu was noted in 10 (6.33%) cases and negativity in 44 (27.85%) cases. Conversely, Her-2/neu negativity was noted in 32 (20.25%) cases with both steroid receptors positive and Her-2/neu positivity with both steroid receptors negative in 20 (12.66%) cases. Overall, no predictive factor was found in the Her-2/neu amplified 31/153 (20.26%) cases completely assessed with IHC and FISH. Grade III invasive ductal carcinomas, however, had a high prevalence of Her-2/neu overexpression. Association of both menopausal status (p = 0.044) and progesterone receptor status (p = 0.004) with high grade tumors were found to be statistically significant at 95% CI (p < 0.5). Consistent with other studies, Her-2/neu overexpression in this cohort was 20.26%. CONCLUSION: Her-2/neu positivity may activate ER expression through signaling kinases, and the combined target of mitogenic estrogen plus the monoclonal antibody therapy against Her-2/neu-overexpressing tumors expand chances of survival for patients in developing countries such as Kenya. The cost factor for these tests, selection for appropriate combined therapies and lack of awareness were noted as limiting factors for access to basic health care service and resulted in advanced tumor grade at time of patient presentation.     

Comparison of chromogenic <Emphasis Type="Italic">in situ</Emphasis> hybridization with other methodologies for HER2 status assesment in breast cancer

The high incidence of human epidermal growth factor receptor (HER)2 overexpression on breast and various other cancer cells and the prognostic and potentially predictive value of HER2 render this growth receptor a novel and important therapeutic target. Out of a wide range of assays that have been used in research for the detection of HER2 status, only two techniques are now predominant and readily applicable in the routine clinical pathology laboratory: determination of HER2 overexpression by immunohistochemistry (IHC) and HER2 gene amplification by fluorescence in situ hybridisation (FISH). In a retrospective study on a cohort of 173 archival invasive breast carcinomas a chromogenic in situ hybridisation (CISH) assay for the detection of HER2 amplification was established. Results were compared to HercepTest, which is the most frequently used method for detecting HER2 alteration. Additionally, HER2 gene copy number was investigated using differential PCR (dPCR) as a testing system. Discrepant cases between CISH and HercepTest and all IHC positive cases (2+ and 3+), a total of 42 cases, were analysed with FISH Path Vysion (Vysis) assay. HER2 overexpression was found by IHC in 24.3%, HER2 amplification by CISH in 19.1% and by dPCR in 9.2% of the tumours. The overall concordance rate between CISH and IHC was 95.9%, between dPCR and IHC 85% and between CISH and FISH 100%, respectively. Among 25 HercepTest positive cases (score 3+) two showed no gene amplification and four out of 13 tumours with score 2+ were negative with CISH and FISH. The current study showed that CISH offers an ideal approach that allows detection of HER2 amplification in the context of morphology, whereas the major drawback of dPCR is the impracticability of tissue differentiation of invasive and non-invasive carcinoma.     

胃癌HER2蛋白表达及基因拷贝分析

目的：探讨胃癌组织HER2蛋白表达和基因拷贝数增加频率,以及蛋白表达与基因拷贝数之间的相关性。方法：分别采用免疫组织化学（1HC）和显色原位杂交（ClSH）方法,检测80例青岛人胃癌组织中HER2蛋白表达和基因扩增情况。结果：HER2免疫组织化学0者51例、1＋者12例、2＋者12例、3＋者5例。通过CISH分析：这组患者中HER2基因拷贝数扩增者共7例（8．8％）,其中包括基因临界扩增3例（3．8％）。HER2的蛋白表达与基因拷贝数增加相关（P〈0．05）,这两个指标与肿瘤分化程度相关。HER2免疫组织化学3＋和基因扩增结果一致,与乳腺癌相似。结论：这组患者中,具有较高的HER2蛋白表达和基因拷贝数增加的比率,基因扩增可能是其蛋白过表达的主要分子机制。     

HER-2/neu oncogene expression, DNA ploidy and proliferation index in breast cancer.

HER-2/neu gene expression, DNA ploidy and proliferation index were studied in 250 cases of breast cancer. Expression of HER-2/neu was determined by using an antibody to the HER-2/neu receptor. Ki-67 antibody was used to determine the proliferation index of the breast cancers, and the Feulgen method was used to assess DNA amounts in the tumor cells. Histochemical staining was quantitated by image analysis. Of the cancers studied, 72 were positive for overexpression of HER-2/neu protein; of these, 62 (86%) possessed near-tetraploid DNA content, and 47 (65%) had more than one G0G1 stem line (polyploid) of DNA distribution. Cells from the cases negative for HER/2-neu overexpression contained DNA amounts that ranged from diploid to varying degrees of aneuploid. A significant difference in the amounts of cellular proliferation in HER-2/neu overexpressing cancers was found between those that expressed the HER-2/neu receptor on their membranes and those that exhibited mainly cytoplasmic receptors.     

Fluorescence in situ hybridization for HER-2/neu amplification of breast carcinoma in archival fine needle aspiration biopsy specimens

OBJECTIVE: To assess the usefulness of fluorescence in situ hybridization (FISH) for HER-2/neu amplification of breast carcinoma in archival fine needle aspiration biopsy (FNAB) specimens. STUDY DESIGN: All FISH performed on formalin-fixed, paraffin-embedded surgical specimens during January 2003-August 2003 at the University of California Irvine Medical Center were selected. Prior FNABs were retrieved. One cytologic slide was destained in each case. The results were compared with those obtained on histologic specimens using the paired t test. RESULTS: FISH was performed on 41 surgical specimens of breast carcinoma. Thirteen patients had prior FNABs that were positive for adenocarcinoma. After hybridization on destained fine needle aspiration slides, no cells were found in 2 cases, and the results were not readable in 2 cases. In the remaining 9 cases, the results, expressed as the ratio of copies of the HER-2/neu gene to copies of the chromosome 17 centromere, were 5.10, 1.14, 1.21, 1.12, 0.74, 1.11, 1.21, 9.87 and 2.4. Results on the corresponding histologic specimens were 5.25, 1.05, 1.13, 1.22, 1.13, 1.12, 1.21, 9.35 and 2.61, respectively. No significant difference was found (p = 0.23). CONCLUSION: HER-2/neu amplification status by FISH can be accurately and reliably evaluated in existing archival cytologic slides.     

Evaluation of HER-2/neu in serum and tissue of primary and metastatic breast cancer patients using an automated enzyme immunoassay

Serum HER-2/neu concentrations were evaluated in 172 healthy subjects, 176 primary and 55 metastatic breast cancer patients, employing a new automated assay (Bayer Immuno 1 serum HER-2/neu). Using 13 ng/mL as the cutoff, abnormal HER-2/neu serum levels were found in 8% (14/176) of primary and 50.9% (28/55) of metastatic breast cancer patients. Both in primary and metastatic breast cancer a significant relationship was found with the stage of the disease when serum HER-2/neu was considered as a categorized variable (p=0.0003 and p=0.02, respectively), but not when it was taken as a continuous variable (p=0.247 and p=0.146, respectively). Moreover, we evaluated the correlation between Immuno 1 HER-2/neu and Oncogene Research Products ELISA assay in 53 normal subjects, 46 primary and 34 metastatic breast cancer patients. The correlation was relatively good (p<0.0001), although substantial differences could be found in single cases. The Immuno 1 assay was also evaluated for the first time in breast cancer tissue. The method, which showed good performance both in terms of imprecision and linearity, was used to measure HER-2/neu protein in 140 cytosol samples from primary breast cancer tissue and in homogenates from 40 matched cases. The correlation between the two matrixes was very close (p<0.0001). By contrast, no correlation was found between serum and matched cytosol (p=0.101) or homogenate samples (p=0.511).     

HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.     

Predicting the efficacy of trastuzumab-based therapy in breast cancer: current standards and future strategies

Breast cancer is the most common female malignancy in many industrialized countries. Approximately one fourth of all women diagnosed with early breast cancer present with tumors that are characterized by erbB2 amplification. While the associated Her-2/neu receptor overexpression results in a high risk of relapse and poor prognosis, these tumors also represent a target for a selective monoclonal antibody therapy with trastuzumab (Herceptin). The combination of trastuzumab with chemotherapy has led to a considerable reduction of recurrences and to a significant reduction in breast cancer mortality both in the adjuvant and metastatic setting. Unfortunately, despite Her-2/neu overexpression, not all patients equally benefit from trastuzumab treatment, and almost all women with metastatic breast cancer eventually progress during antibody therapy. Moreover, trastuzumab is burdened with cardiotoxicity, thus increasing the risk of symptomatic congestive heart failure. In addition, the marginal costs for a 1 year therapy of trastuzumab-based therapy, which is currently considered to be the most effective treatment regimen in the adjuvant setting, may amount for up to US$ 40.000. Testing for erbB2 oncogene amplification by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH), respectively, and staining for Her-2/neu receptor overexpression by immunohistochemistry (IHC) represent the current standard for determining patient eligibility for trastuzumab-based therapy. However, while the negative predictive value of these assays for predicting the absence of benefit from trastuzumab-based therapy is sufficiently high, their positive predictive value remains insufficient, i.e. only a proportion of patients selected by these tests substantially benefit from trastuzumab-containing regimen. Accordingly, over the last years a number of biomarkers have been evaluated in their potential to predict response to trastuzumab-based therapies. These include markers auf activation of Her-2/neu (e.g., tyrosine phosphorylated Her-2/neu in tissue and cleaved Her-2/neu extracellular domain in serum) and its dimerization partners (e.g., EGFR), respectively, but also components of Her-2/neu-induced downstream signaling pathways that are crucial for the growth inhibitory effects of trastuzumab (e.g., PTEN and PI3K). Other parameters, such as topoisomerase-II alpha and c-myc co-amplifications, have also been identified as potentially useful predictors of response to trastuzumab-based chemotherapy regimen. While the benefit of these predictive biomarkers in the metastatic setting is currently explored, their usefulness in the adjuvant setting is still largely unknown. It is, however, undisputable that, within the group of Her-2/neu overexpressing tumors, further response predictors are needed in order to minimize trastuzumab-associated side effects, and to reduce the considerable societal costs that are associated with trastuzumab-based treatment regimen.