HER2 Fluorescence In Situ Hybridization Testing Research Articles

Are There More HER2 FISH in the Sea? An Institution’s Experience in Identifying HER2 Positivity Using Fluorescent In Situ Hybridization in Patients with HER2 Negative Immunohistochemistry

BackgroundApproximately 20% of breast cancers express HER2-positive receptors in the USA. HER2 receptor immunohistochemistry (IHC) staining with equivocal (2+) results commonly undergoes fluorescence in-situ hybridization (FISH) for further classification. Current guidelines do not recommend routine FISH testing in IHC-negative (0 or 1+) cases. This study investigates an institution that performs both IHC and FISH testing on all cases to identify the true HER2-positive rate.Patients and MethodsA retrospective chart review from 2015 to 2021 was conducted at an institution where both HER2 IHC and FISH testing were performed at the time of diagnosis for all invasive breast cancers. The rate of true HER2-positive patients was determined, and patient and tumor characteristics were further explored.ResultsA total of 1835 invasive breast cancer cases were primarily treated at this institution. A total of 289 cases were HER2 positive on IHC and FISH testing (15.7%). An additional 38 cases were identified as HER2 negative on IHC, but reclassified as HER2 positive on reflex FISH testing. Total HER2 positive cases increased from 289 (15.7%) to 327 cases (17.8%) with reflex FISH testing.ConclusionsThe additional HER2-positive cases after completing FISH testing on IHC-negative tumors suggests there may be a role for routine FISH testing in addition to standard IHC staining to determine HER2 status for breast cancer. The ethical, prognostic and even benefits of a correct diagnosis outweigh the added expense of FISH testing.

HER2 amplification by next-generation sequencing to identify HER2-positive invasive breast cancer with negative HER2 immunohistochemistry

BackgroundHuman epidermal growth factor receptor 2 (HER2) positive breast carcinomas due to HER2 amplification are associated with aggressive behavior and a poor prognosis. Anti-HER2-targeted therapies are widely used to treat HER2-positive breast carcinomas with excellent outcomes. Accurate identification of HER2 amplification status in breast carcinomas is of important diagnostic and treatment value. Currently, HER2 amplification status is routinely determined by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) testing. This study will review our past HER2 data to determine and characterize discordant results between HER2 IHC and FISH. It will also determine a potential impact of HER2 amplification status by next-generation sequencing (NGS) on these patients.MethodsWe reviewed a total of 4884 breast carcinomas with coexisting HER2 IHC and HER2 FISH performed at our institution from 2010 to 2022. 57 cases also had a Next-Generation-Sequencing-based (NGS) gene panel performed. Given the advances in biostatic analysis pipelines, NGS methods were utilized to provide results on HER2 amplification status along with somatic mutations.ResultsWhile the majority (ranging from 98.5% with IHC score of 0 and 93.1% with IHC score of 1 +) of 4884 breast carcinomas had concordant results from HER2 IHC and HER2 FISH testing, a small percentage of patients (ranging from 1.5% in those with IHC score of 0, to 6.9% with IHC score of 1 +) had discordant results, with negative HER2 IHC and positive HER2 FISH results. These patients could be reported as HER2-negative breast carcinomas if only HER2 IHC testing has been performed according to a current cost-effective HER2 test strategy. 57 patients had HER2 amplification status determined by NGS, and all patients had concordant results between HER2 NGS and FISH tests. A HER2-amplified breast carcinoma by NGS had a negative IHC and a positive HER2 FISH result. This case was classified as a HER2-positive breast carcinoma, had anti-HER2-targeted therapy, and achieved a complete clinical response.ConclusionsA small percentage of HER2-positive breast carcinomas are unidentified because of a negative HER2 IHC based on our current cost-effective HER2 test strategy. It is not feasible and affordable in routine clinical practice to perform HER2 FISH for the cases with negative HER2 IHC (IHC score 0 and 1 +). Therefore, NGS assays capable of simultaneously detecting both somatic mutations and HER2 amplification could provide a more comprehensive genetic profiling for breast carcinomas in a clinical setting. Identification of HER2 amplification by NGS in HER2-positive breast carcinomas with negative HER2 IHC results is important since these cases are concealed by our current cost-effective HER2 test strategy with IHC first (for all cases) and FISH reflex (only for cases with IHC score of 2 +), and would offer the opportunity for potentially beneficial anti-HER2-targeted therapies for these patients.

RAI1 alternate probe identifies additional gastroesophageal adenocarcinoma cases as amplified following equivocal HER2 fluorescence in situ hybridization testing: experience from a national reference laboratory

The College of American Pathologists/American Society of Clinical Oncology recommends HER2 testing prior to initiation of targeted therapy for patients with advanced Gastroesophageal adenocarcinoma (GEA), using immunohistochemistry (IHC) followed by fluorescence in situ hybridization (FISH) in cases with an equivocal (score 2 + ) result on IHC. The FISH results are considered indeterminate if the HER2/CEP17 ratio is <2.0 with an average CEP17 copy number of ≥3.0 and a HER2 copy number ≥4.0 and ≤6.0 after counting additional tumor cells. Indeterminate results may be resolved by using an alternative chromosome 17 probe such as RAI1. The purpose of this study is to review our experience with RAI1 alternate probe in HER2 FISH testing of GEA in a large reference laboratory setting. Esophageal, gastroesophageal, and gastric adenocarcinomas received for HER2 FISH testing in our lab between 9/2018 and 1/2020 were included. HER2/CEP17 and HER2/ RAI1 ratios, and the average HER2, CEP17, RAI1 signals per cell were recorded. 328 GEA had HER2 testing performed in our lab during the study period. 101 (30.8%) were amplified, 169 (51.5%) were non-amplified and 58 (17.7%) were indeterminate. Following RAI1 testing, 42 (72.4%) of 58 indeterminate cases were reclassified as non-amplified and 16 (27.6%) were reclassified as amplified, increasing the total amplified cases to 117 (35.7%). The correlation between the average CEP17 and RAI1 copy number for all cases was weak (R2 = 0.095). In summary, using the alternate probe RAI1 reclassifies 27.6% of original HER2 FISH indeterminate gastroesophageal carcinomas as amplified, which makes them eligible for targeted therapies.

American Society of Clinical Oncology/College of American Pathologists 2018 Focused Update of Breast Cancer HER2 FISH Testing GuidelinesResults From a National Reference Laboratory

To review impact of the ASCO/CAP 2018 update on HER2 testing. HER2 fluorescence in situ hybridization (FISH) test requests from primary and metastatic breast cancers between August 2018 and January 2019 were included. FISH results requiring a changed algorithm under the new guidelines (groups 2, 3, and 4) were identified and HER2:CEN17 ratios, average HER2, CEN17 signals/cell, and HER2 immunohistochemistry (IHC) results were recorded. Of the HER2 FISH cases 176/812(21.7%) fell within groups 2, 3, or 4; 0/12, 1/12, and 2/152 cases were positive (3+) by IHC, and 1/12, 2/12, and 6/152 cases were positive after targeted scoring from groups 2, 3, and 4, respectively. Following 2018 updates, 8.3%, 25%, and 5.3% of the groups 2, 3, and 4 were positive, respectively. Groups 2, 3, and 4 constituted over 20% of HER2 FISH tests in a reference laboratory. The 2018 ASCO/CAP update significantly decreased the HER2 positivity rate.

Abstract ES1-1: What is a HER2-positive in breast cancer in 2018?

Abstract Remarkable progress has been made in the treatment of patients with HER2-positive breast cancer since trastuzumab was approved by the U.S. Food and Drug Administration 20 years ago. Among patients with HER2-positive disease, HER2-targeted therapy has been associated with substantial improvements in overall survival in the adjuvant setting, a high rate of pathologic complete response (pCR) after neoadjuvant therapy, and improvements in survival in patients with metastatic disease. While much of these improvements in outcome is due to more effective therapies, improvements in patient selection as a result of more accurate HER2 testing has also contributed to the better prognosis of patients with this disease. Data from clinical trials examining the benefit of trastuzumab in the adjuvant setting and published in the early 2000s (e.g., NSABP B31; NCCTG 9831) demonstrated that up to 25% of community-based assays reported as HER2-positive by immunohistochemistry (IHC) and up to 15% reported as HER2-positive by fluorescence in situ hybridization (FISH) could not be confirmed upon central laboratory testing. To address this high frequency of false-positive results and to improve the accuracy of HER2 testing, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2007 published guideline recommendations for HER2 testing. These guidelines were subsequently updated in 2013 and, more recently, in 2018. Since the implementation of these guidelines, the accuracy of HER2 testing has improved. In fact, in current clinical practice, when HER2 IHC and FISH testing are performed according to the ASCO-CAP guidelines, approximately 95% of breast cancers can ultimately be categorized as either unequivocally HER2-positive or HER2-negative. In the remainder of the cases, combined IHC and FISH assay results provide a less straightforward picture of the HER2 status and, in turn, create uncertainty regarding the likelihood of benefit from HER2-targeted therapy. These include cases which on dual-probe FISH testing show: 1) HER2/CEP17 ratio &amp;gt;2.0 and an average HER2 copy number &amp;lt;4.0 signals/cell (monosomy); 2) HER2/CEP17 ratio &amp;lt;2.0 and an average HER2 copy number &amp;gt;6.0 signals/cell (polysomy); and 3) HER2/CEP17 ratio &amp;lt;2.0 and an average HER2 copy number &amp;gt;4.0 and &amp;lt;6.0 signals/cell. However, the most recent (2018) update of the ASCO-CAP guidelines provides pragmatic recommendations to resolve the HER2 status of such cases, and this, in turn, should result in even fewer cases in which the HER2 status remains in doubt. It should be noted, however, that not all patients whose tumors are HER2-positive by IHC and/or FISH respond similarly to HER2-targeted therapy and that some patients who initially respond subsequently develop resistance. Active efforts are underway to understand the mechanisms of de novo and acquired resistance to HER2-targeted treatments and to identify biomarkers that can predict resistance to therapy. Thus, the clinically relevant question in 2018 is not “what is a HER2-positive breast cancer ”? Rather, the question that now needs to be addressed is “which HER2-positive breast cancers will respond to HER2 targeted-treatments and to which HER2-targeted treatments are they most likely to respond? ”. Citation Format: Schnitt S. What is a HER2-positive in breast cancer in 2018? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES1-1.

HER2 FISH concordance in breast cancer patients with both cytology and surgical pathology specimens

Fluorescence in Situ Hybridization (FISH) for Human Epidermal Growth Factor Receptor 2 (HER2) is traditionally performed on histologic specimens; there are no guidelines for validation of HER2 testing on cytology specimens. Our aim was to correlate HER2 FISH results on cytologic and histologic specimens. A retrospective search was conducted to identify breast carcinomas with HER2 FISH testing on both cytologic and histologic specimens. Results were interpreted using updated 2013 ASCO/CAP guidelines. 54 women with HER2 FISH results on both cytologic and histologic specimens were identified. HER2 FISH was performed on 26 formalin-fixed thrombin clot cell blocks, 6 Cellient (Hologic, Inc., Bedford, MA) cell blocks and 22 ThinPrep slides, and was negative in 43 (87%), positive in 8 (15%), and equivocal in 3 (5%) cases. Of 43 negative cytology cases, the histologic specimen was also negative in 40 (93%). Of 8 positive cytology cases, the histologic specimen was positive in 5 (63%). Overall, 48 of 54 cases (89%) had a concordant result. In patients with a discordant result, the average interval between HER2 FISH testing was 7 years. Three of 6 discordant cases showed HER2 genetic heterogeneity. Five patients received adjuvant chemotherapy, 2 received endocrine therapy, and 1 received trastuzumab. FISH is reliable for determining HER2 status in cytopathology specimens. Discordant results between cytologic and surgical specimens are uncommon; possible explanations include genetic heterogeneity or inherent molecular changes associated with disease progression and therapies that occur between testing of the primary carcinoma and the metastasis.

Impact of repeat HER2 testing after initial equivocal HER2 FISH results using 2013 ASCO/CAP guidelines.

The updated 2013 American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing have made some major changes in HER2 fluorescence in situ hybridization (FISH) interpretation criteria with additional FISH equivocal cases. Repeat HER2 testing is recommended after initial HER2 FISH equivocal results; however, little is known about its impact on final HER2 status. The aim of this study is to investigate whether reflex test clarifies HER2 status, and to characterize clinicopathological features of the newly defined HER2 equivocal group. A total of 886 consecutive cases of primary invasive breast cancer conducted with dual-probe HER2 FISH testing between November 2013 and December 2015 were reviewed. HER2 immunohistochemistry (IHC) and FISH testing were performed on a different tissue block or a new specimen after initial HER2 FISH equivocal results. Compared to 2007 guideline, 85 (9.6%) cases changed their category by using 2013 guideline. The major change of the 85 cases is that 57 (6.4%) cases in HER2 FISH-negative category changed to equivocal, and the equivocal category cases increased from 36 to 67. HER2 FISH equivocal was significantly associated with HER2 IHC equivocal (2+) and chromosome 17 polysomy (P<0.01). Repeat testing by IHC and FISH clarified HER2 status in 33 and 42% of HER2 equivocal cases, respectively. Overall 32 (48%) initial HER2 equivocal cases stayed HER2 equivocal after repeat FISH and or IHC testing. These tumors were ER/PR+, with high KI-67 index. New guidelines classify more HER2 FISH equivocal cases. Repeat HER2 testing clarifies HER2 status in about 50% of initial HER2 FISH equivocal cases. In addition, HER2 equivocal cases merit further study as there is limited information about prognosis and optimal treatment strategy for this population.

Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer

Fluorescence in situ hybridization (FISH) is one of the recommended techniques for human epidermal growth factor receptor 2 (HER2) status assessment on cancer tissues. Here we develop microfluidics-assisted FISH (MA-FISH), in which hybridization of the FISH probes with their target DNA strands is obtained by applying square-wave oscillatory flows of diluted probe solutions in a thin microfluidic chamber of 5 μl volume. By optimizing the experimental parameters, MA-FISH decreases the consumption of the expensive probe solution by a factor 5 with respect to the standard technique, and reduces the hybridization time to 4 h, which is four times faster than in the standard protocol. To validate the method, we blindly conducted HER2 MA-FISH on 51 formalin-fixed paraffin-embedded tissue slides of 17 breast cancer samples, and compared the results with standard HER2 FISH testing. HER2 status classification was determined according to published guidelines, based on average number of HER2 copies per cell and average HER2/CEP17 ratio. Excellent agreement was observed between the two methods, supporting the validity of MA-FISH and further promoting its short hybridization time and reduced reagent consumption.

Estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression in breast cancer FNA cell blocks and paired histologic specimens: A large retrospective study.

Molecular analysis represents an increasingly important component of the pathologic examination of tumor specimens. Notably, the characterization of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression in breast cancer specimens provides critical prognostic and predictive information. The objective of the current study was to compare the concordance of these markers as determined on fine-needle aspiration (FNA) cell blocks compared with tissue blocks prepared from surgical specimens. A total of 134 cases of breast carcinoma were identified from 2002 through 2014 with both FNA cell blocks (fixed in 10% formalin) and corresponding available tissue blocks and ER, PR, and HER2 were characterized in both specimens. Negative and positive concordances were determined for ER and PR in cell blocks compared with tissue blocks, and for HER2 immunohistochemistry on cell blocks and tissue blocks versus the corresponding reference method, fluorescence in situ hybridization (FISH). Concordance for ER expression evaluated on a cell block compared with the corresponding tissue block was 96.2%. Concordance for PR expression was 77.5%. Overall agreement of HER2 FISH testing between cell blocks and tissue blocks was 96.7%. For both cell blocks and tissue blocks, HER2 expression by immunohistochemistry demonstrated ≥98% positive and negative concordance with the FISH reference method. ER, PR, and HER2 determination on FNA-acquired cell block (fixed exclusively in 10% formalin) showed excellent agreement for ER and HER2 and moderate agreement for PR with the corresponding tissue block. These findings support the equivalency of ER and HER2 evaluation performed on FNA cell blocks compared with surgical tissue blocks. Cancer Cytopathol 2016;124:828-35. © 2016 American Cancer Society.

Abstract P6-08-53: Her2 FISH amplification in ER/PR/Her2 IHC negative breast cancer

Abstract Background: Triple negative (ER negative, PR negative, Her2 negative) breast cancer is an aggressive cancer that is not likely to respond to endocrine or anti-Her2 therapy. While Her2 positivity is a poor prognostic factor, patients with Her2 positive tumors are eligible for anti-Her2 therapy. Her2 positivity is based on either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) result. Her2 IHC scores of 0+ and 1+ are negative, 2+ is equivocal, and 3+ is positive. The Her2 gene is amplified when the Her2/CEP17 ratio is ≥2.0. While most tumors will show IHC/FISH concordance, some tumors may show discordant findings between IHC staining and FISH. Previous studies have shown benefit from anti-Her2 therapy in tumors with discordant IHC/FISH results. The purpose of this study was to determine the frequency of Her2 amplification in triple IHC negative breast cancer to determine whether routine Her2 FISH testing is necessary in this subset of patients. Methods: After Institutional Review Board approval, the pathology databases were searched for cases of triple IHC negative breast cancer from 2003-August 2013. All cases of triple IHC negative with corresponding Her2 FISH testing were included in this study. The results of Her2 FISH were correlated with Her2 IHC. The clinical history and IHC slides for all discordant Her2 cases were reviewed when available. Results: A total of 659 triple IHC negative breast cancer cases from 516 patients were found. The patients’ age at diagnosis ranged from 26-92. The cases were classified using the ratio of Her2/CEP17 per the 2013ASCO/CAP guidelines: non-amplified &amp;lt; 2.0, amplified ≥ 2.0. As the copy number for Her2 was not always available, the 2013 ASCO/CAP criterion for amplification based on the copy number was not used in reclassifying the cases. Based on the ratio, 20 tumors were amplified, 631 tumors were not amplified, and 8 tumors had insufficient tissue for Her2 FISH testing. The Her2/CEP17 ratio for amplified cases ranged from 2.0 to 11.1 and for non-amplified cases from 0.5 to 1.9. Of these 20 patients, 10 received some trastuzumab treatment, 6 did not receive any trastuzumab, and in 4 patients, there was no additional information available regarding treatment with trastuzumab. Of the 10 who received trastuzumab treatment, 2 died of disease (DOD), 26 months and 45 months after their diagnosis. The overall survival of the other patients in this group was 23-106 months. Of the 6 who did not receive trastuzumab, one DOD after 76 months. The other 5 patients have overall survival ranging from 15-93 months. Of the patients with unknown Her2 targeted therapy status, one is deceased, the status of 2 is unknown, and one patient is alive at 110 months. Discussion: Our study showed 3% of triple IHC negative breast cancers to be Her2 FISH amplified. Of the 20 tumors, 19 showed Her2/CEP17 between 2.0 to 4.2 with one case showing Her2/CEP17 of 11.1. Firm conclusions on the efficacy of trastuzumab treatment in this small subset of patients with Her2 amplification but no protein overexpression cannot be drawn. However, the overall survival of those treated and not treated is similar. Based on the small number of cases with Her2 IHC/FISH discordance, a compelling case for routine testing of all Her2 IHC negative tumors cannot be made. Citation Format: Sunati Sahoo, Helena Hwang. Her2 FISH amplification in ER/PR/Her2 IHC negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-53.

Barriers to implementing primary HER2 FISH testing.

e16642 Background: Two HER2/neu testing protocols are recommended by guidelines—primary Fluorescence In Situ Hybridization (FISH) and primary Immunohistochemistry (IHC) with reflex to FISH. FISH testing is recognized as more accurate at 4% error rate versus IHC at 20% (Wolf et al, J Clin Oncol. 2007). Transition to primary FISH should improve quality (Sauter et al, J Clin Oncol. 2009) but is hampered by factors of familiarity, cost, and convenience (Ross et al, Oncologist. 2009). Our goal is to further characterize the barriers to adopting primary HER2 FISH testing. Methods: 49 multi-disciplinary interviews with clinicians (n=13), executives (n=11) from 5 insurers, lab management (n=8) and pathologists (n=17) on barriers impacting a move to primary HER2 FISH testing protocol. We analyzed the interviews using the framework approach of qualitative research. Results: We identified themes impacting a move to primary HER2 FISH testing. Many care organizations lack a consistent comprehensive process to evaluate impact of a new testing protocol (Table). There are conflicting views on the impacts of primary FISH implementation: IHC pathologists and lab managers perceived it will cause ineffective workflow, clinician dissatisfaction from increased turn-around time and negative financial impact. However, FISH pathologists and lab managers reported effective workflow, clinician satisfaction, and positive/neutral financial impact (Table). Clinicians in ‘IHC primary’ centers did not express concerns regarding primary FISH, and reported preference for adopting it (Table). Most payers are concerned with HER2 quality issues but lack mechanisms to address them (82%). Conclusions: Organizational factors impact implementation of HER2 primary FISH testing. The dichotomy between expected and actual impact suggests lack of consistent framework and information available for decision making. Future studies assessing actual impacts of adopting primary FISH, and other testing protocols, are needed to inform diagnostic protocol evaluation and decision making. Factors Pathologists and lab managers – IHC Pathologists and lab managers – FISH Clinicians in IHC centers Payers Lack of process to evaluate 75% 78% 85% 64% Negative workflow, clinical, financial impact 88% 0% 8% 27%

Achieving 95% Cross-Methodological Concordance in HER2 Testing

We were interested in determining our concordance between fluorescence in situ hybridization (FISH) and a previously validated immunohistochemical HER2 assay to identify possible reasons for discordance and to determine if all reasons for discordance were addressed by the American Society of Clinical Oncology/College of American Pathologists guidelines. We reviewed 697 cases (2004-2007) in which HER2 immunohistochemical and FISH testing were concurrently done. Overall concordance between nonequivocal immunohistochemical and FISH results was 96%. Of the 19 discordant cases, 13 (68%) were interpreted as positive immunohistochemically but negative by FISH. The primary reason for this discordance was immunohistochemical interpretation. Weak stain intensity, granular staining, and interpretation in areas of crush artifact were identified as the most common issues. Of the 6 cases interpreted as immunohistochemically negative and FISH-positive, 2 were from patients known to be receiving trastuzumab at the time of biopsy, 1 was very close to the FISH equivocal category, and 4 cases had fewer than 1.5 CEP17 signals per cell (1 patient in this group was also receiving trastuzumab). Focusing on issues with HER2 immunohistochemical interpretation can improve concordance rates for immunohistochemically positive cases, but biologic reasons may explain some discordant immunohistochemically negative cases.

Rapid screening of tissue microarrays for Her‐2 fluorescence in situ hybridization testing is an accurate, efficient and economic method of providing an entirely in situ hybridization‐based Her‐2 testing service

Fluorescence in situ hybridization (FISH) testing is the 'gold standard' method for Her-2 status assessment in breast cancer patients, yet is only employed in about 30% of tests carried out because of cost and labour considerations. We have previously described tissue microarray (TMA)-based testing to eliminate cost constraints, and now describe a rapid screening approach to reduce time spent testing. We examined 88 cases of invasive breast cancer on TMAs comparing formal FISH scoring with a rapid screening technique. Each core was screened by two observers and results recorded as positive, equivocal or negative. Each approach was timed. Data were analysed by comparing the rapid screening results with formal counts. Using rapid screening, two-thirds of negative and half the positive cases could be eliminated with 100% accuracy. It took 2 min per observer per case to rapid screen six TMA cores at x100 magnification. The remaining cases required formal counting, which took no longer than with whole-section techniques. Rapid screening of TMAs for routine Her-2 FISH testing is safe, economical and time efficient. The technique ensures that all patients receive 'gold standard' testing.

Significance of HER2 Low-Level Copy Gain in Barrett's Cancer: Implications for Fluorescence In situ Hybridization Testing in Tissues

HER2 may be a relevant biomarker in Barrett's cancer. We compared three HER2 laboratory methods, standard fluorescence in situ hybridization (FISH), image-based three-dimensional FISH in thick (16 microm) sections, and immunohistochemistry, to predict patient outcome. Tissue microarray sections from 124 Barrett's cancer patients were analyzed by standard FISH on thin (4 microm) sections and by image-based three-dimensional FISH on thick (16 microm) sections for HER2 and chromosome-17, as well for p185(HER2) by immunohistochemistry. Correlations with clinical and follow-up data were examined. Only three-dimensional FISH on thick (16 microm) sections revealed HER2 gene copy gain to be associated with increased disease-specific mortality (relative risk, 2.1; 95% confidence interval, 1.06-4.26; P = 0.033). In contrast, standard FISH on thin (4 microm) sections and immunohistochemistry failed to predict clinical outcome. Low-level gain of HER2 occurred frequently in Barrett's cancer (>or=2.5-4.0 HER2 copies, 59.7%; HER2-to-chromosome-17 ratio, >or=1.1-2.0; 61.2%) and defined a subpopulation for patient outcome as unfavorable as HER2 gene amplification [disease-free survival, P = 0.017 (HER2 copies)]. This low-level group was neither definable by standard FISH nor immunohistochemistry. No prognostic significance was found for chromosome-17 aneusomy. Low-level copy gains of HER2 define a biologically distinct subpopulation of Barrett's cancer patients. Importantly, these subtle copy number changes are not reliably detected by standard FISH in thin (4 microm) tissue sections, highlighting a thus far unrecognized weakness in HER2 FISH testing. These results should be taken into account for accurate evaluation of biomarkers by FISH and for HER2 FISH testing in tissue sections.

The Use of TMA for Interlaboratory Validation of FISH Testing for Detection of HER2 Gene Amplification in Breast Cancer

HER2 fluorescence in situ hybridization (FISH) testing for breast cancer is largely limited to academic centers and commercial laboratories. As testing demands increase, methods for rapid and cost-effective technical validation and quality assessment will be required. Tissue microarray (TMA), a technique for high-throughput biomarker evaluation, could help facilitate these needs. Our objective was to assess the usefulness of TMA technology for validation of HER2 FISH testing. Two TMA blocks containing paired cores from 41 breast cancers were constructed. HER2 FISH was performed in parallel at two institutions and the results compared. One institution, with considerable HER2 FISH experience, served as the reference laboratory. HER2 chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) were compared to the FISH results. For positive and negative results, the concordance rate between laboratories was 100%. Using kappa statistical analysis to determine interobserver agreement, HER2 to chromosome 17 gene copy ratios showed strong agreement between laboratories with kappa = 0.85 (perfect agreement = 1.0). Four cases displaying low-level amplification by CISH contained chromosome 17 polysomy and gene copy ratios of <2.0 by FISH. Good concordance was observed between HER2 IHC and in situ hybridization testing. TMA is a robust and effective method for the technical validation of HER2 FISH testing and should be considered for use by quality assessment programs.