Immunohistochemistry Equivocal Cases Research Articles

HER2 Status Assessment in Endometrial Serous Carcinoma: Comparative Analysis of Two Proposed Testing and Interpretation Algorithms.

HER2 status is now routinely assessed in endometrial serous carcinoma (ESC) due to the reported predictive value of HER2 protein overexpression and/or gene amplification. Herein the authors compare 2 proposed testing and interpretation guidelines for HER2 in ESC. Forty-three consecutive cases of ESC that had been dually tested by both HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were interpreted using 2 sets of guidelines. Guideline set 1 (GS1) is the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines for breast cancer. Guideline set 2 (GS2) is a recent proposal that is a slight modification of the enrollment criteria for the clinical trial (NCT01367002) that demonstrated a survival benefit for anti-HER2 therapy in ESC. By IHC, GS1 and GS2, respectively classified 39.5% (17/43) and 28% (12/43) of ESC as HER2-negative, 37.2% (16/43) and 53.4% (23/43) as HER2 equivocal, and 23.2% (10/43) and 18.6% (8/43) as HER2-positive (P > 0.05 for all). IHC and FISH were highly concordant at the extremes using either set of guidelines, as no cases were found to be IHC3+/FISH-negative or IHC 0-1+/FISH-positive. GS1 and GS2 were comparable regarding the proportion of IHC equivocal cases that were HER2 amplified by FISH (19% vs 23% respectively; [P = 0.71]). GS1 and GS2 displayed 98% (42/43) concordance regarding the final (IHC and/or FISH-based) classification of tumors as being HER2-positive or negative, and the same 13 cases were ultimately classified as HER2 amplified using either GS1 or GS2. One "discordant" case was classified as HER2-positive using GS2 but HER2-negative using GS1 (HER2 IHC score 2+ using both guidelines, HER2:CEP17 signal ratio of 3, HER2 signal number of 3.4). Six (14%) of the 43 cases (FISH Groups: 2, 3, and 4) would require IHC results to interpret the FISH findings using GS1. Because GS1 requires that the HER2 IHC staining be observed within a homogeneous and contiguous invasive cell population, and this is not a requirement in GS2, GS2 may be better suited for ESC given its frequently heterogeneous staining pattern. Additional studies may be required on the optimal interpretation of problematic dual-probe FISH scenarios in GS2 and the necessity for IHC correlation in such scenarios. Using either set of guidelines, our findings support a reflex testing strategy of restricting FISH testing to cases that are IHC equivocal.

Comparison of breast cancer HER-2 receptor testing with immunohistochemistry and in situ hybridization.

Human epidermal growth factor receptor-2 (HER2) status can be tested with immunohistochemistry (IHC) and in situ hybridization (ISH). The 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 testing guidelines suggest initial HER2 testing using IHC and further testing IHC equivocal cases with ISH. However, many institutions perform both IHC and ISH on the same specimen. This study aims to analyze the concordance between HER2 IHC and ISH in order to evaluate the benefit of repeating HER2 testing on the same breast cancer specimens. Patients diagnosed with invasive breast cancer through BreastScreen NSW Sydney West program between January 2018 and December 2020 were identified and their HER2 IHC and HER2 ISH results on core needle biopsy (CNB) and surgical excisions (SE) were retrospectively collected. Specimens with both IHC and ISH results were then analyzed for agreement and concordance using unweighted kappa values. Equivocal IHC (2+) cases were excluded from concordance analysis. Overall, there were 240 invasive breast cancer specimens (CNB and SE) with both IHC and ISH recorded. Concordance between HER2 IHC and ISH was 100% (95% CI: 96.2-100%; κ=1.00 (P<0.001)). Of the IHC equivocal cases (n=146), 94.5% were ISH negative. There was perfect positive concordance and agreement between non-equivocal IHC and ISH results. This reinforces that IHC alone can be utilized reliably for testing HER2 status of non-equivocal cases consistent with the 2018 ASCO/CAP guidelines.

Reckoning the Status of HER2/neu Protein and Oncogene in Breast Cancer Specimens: Comparison of IHC Assay with FISH Assay at a Tertiary Care Centre, West India

Introduction: Breast carcinoma is the most prevalent malignant neoplasm among women with more than one million new cases per year. The benefit of humanised anti-Human Epidermal growth factor Receptor 2 (HER2)/neu monoclonal antibody trastuzumab (Herceptin) in HER2/neu-positive breast cancers has been well documented. Immunohistochemistry (IHC) for protein overexpression and Fluorescence In Situ Hybridisation (FISH) for detecting HER2/neu gene amplification is widely used. Some laboratories use IHC as primary test with FISH for subset of cases while using FISH as primary investigation followed by IHC if needed is done by others. Aim: To evaluate IHC assay in breast carcinoma cases for HER2/neu as screening test before FISH. Materials and Methods: A cross-sectional descriptive type of observational study was conducted in the Department of Pathology and Multi-Disciplinary Research Unit, SMS Medical College and attached hospitals Jaipur, India, between April 2020 and December 2021. A total 122 samples of invasive breast carcinoma were included in study for IHC and FISH analysis. On paraffin embedded breast tumour tissue sections, IHC was performed using mouse monoclonal antibody targeting the intracellular domain of HER2/neu protein and FISH was implemented by dual colour probes targeting the HER2/neu gene on chromosome 17. Cases were classified according to HER2/neu status on IHC interpretation and FISH interpretation as per American Society of Clinical Oncology/ the College of American Pathologists (ASCO/CAP) HER2/neu-2018 testing guidelines. Chi-square test, kappa coefficient and Z-test were applied for statistical analysis. The p-value&lt;0.05 was considered significant. Results: Of the 122 patients of Invasive Duct Carcinoma (IDC), the mean age was 50.3±12.7 years with a age range of 25-70 years. HER2/neu IHC score 3+ (over expression) was seen in 19 (15.6%) cases while 2+ (equivocal) result was obtained in 84 (68.8%) cases and 19 (15.6%) cases showed negative (1+/0) HER2/neu expression. Out of 19 IHC positive cases, all cases were amplified by FISH and all 19 IHC negative cases, were non amplified by FISH. There were 84 IHC equivocal cases of which 37 (44%) cases were amplified, 45 (53.6%) non amplified and 2 (2.4%) cases were equivocal when analysed by FISH. Also, 11 (9%) cases were found Centromere Enumeration Probe (CEP) amplified in the study, they were negative or equivocal on IHC and none was IHC positive. Conclusion: The study concluded that combined FISH and IHC methodologies could optimise information on HER2/neu status in breast cancer patients. Also, testing algorithm is emphasised where laboratories may use IHC as a screening method and FISH can be used as accurate and specific method in IHC (2+) equivocal cases. Thus, patients with HER2/neu status positive of IHC (3+) or FISH (gene amplified) can be proposed to be treated with herceptin (trastuzumab).

Breast Carcinoma - A Comparative Study of Immunohistochemistry and Fluorescence in Situ Hybridization for Her-2 Assessment and Association of ER, PR, HER-2 and Ki-67 Expression with Clinico-Pathological Parameters.

Breast cancer is the most common cancer in developed and developing countries. This study mainly addresses the issue of an equivocal result in IHC, which then needs further assessment if the patient has to receive targeted therapy. The study aimed to detect the expression of Her2/neu protein in breast cancer by immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH) and evaluate concordance and discordance between the two methods. Also, the clinicopathological parameters in these patients were studied in association with ER, PR, HER-2, and Ki-67. This study was conducted on 34 female carcinoma breast specimens, including core biopsies and mastectomies. Each case underwent histopathological and immunohistochemical studies for (Estrogen Receptor) ER, (Progesterone Receptor) PR, (Human Epidermal growth factor Receptor 2) HER-2, and Ki-67. In addition, FISH was done on all the samples to detect Her2 gene amplification. The overall concordance between the two tests was 79.41% while the concordance between the two tests in equivocal cases, was 14.3%. ER/PR expression and HER-2 amplification were inversely associated. Also, Ki-67 expression was not associated with the side size of the lesion, lymphovascular invasion, and lymph node metastasis. Age less than 50 at presentation and infiltrating ductal carcinoma histological type showed increased proliferation index. The highest concordance between FISH and IHC was noted in IHC positive and negative cases, whereas IHC equivocal cases showed low concordance. FISH accurately determines the assessment of HER2 expressions in equivocal cases.

Indian Data on HER2 Fluorescence In Situ Hybridization in Invasive Breast Cancer with Immunohistochemically Equivocal Results As Per 2018 ASCO/CAP Guidelines.

Biren ParikhIntroduction Hormonal status and HER2 expression are valuable biomarkers and dictate the management of the patients diagnosed with invasive breast cancer (IBC). It is crucial to identify the patients who truly respond to anti-HER2 targeted therapy. Updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines has recommended certain modifications in HER2 interpretation by fluorescence in situ hybridization (FISH) with concomitant immunohistochemistry (IHC). Objectives We aimed to evaluate HER2 FISH interpretation in IBC with equivocal IHC results as per 2018 ASCO/CAP recommendations and compare FISH results with hormonal receptor status. Materials and Methods FISH results of 502 cases of IBC with equivocal IHC report between January 2016 to January 2022 were reviewed retrospectively. FISH results were categorized according to ASCO/CAP guidelines 2018 into five respective groups. Results FISH testing in IHC equivocal cases showed 219 (43.6%) cases were classic amplified (positive) belonged to group 1, 217(43.2%) cases were classic nonamplified (negative) fell into group 5, 39 (7.8%) and 02 (0.4%) patients were in group 2 (negative) and group 3 (positive), and 25 (5.0%) cases were in group 4 (negative). About 52.1 and 49.3% of cases with estrogen receptor and progesterone receptor positivity were reported as HER2 positive. Among 502 cases, 25 equivocal cases according to the 2013 guidelines were redefined as HER2 negative and 02 (0.4%) cases reported positive were classified negative as per updated 2018 guidelines. Conclusion Revised 2018 guidelines is helpful in accurate identification of HER2 status and in avoiding targeted therapy in unwarranted cases. Updated 2018 guidelines has removed equivocal HER2-FISH category that has eliminated management dilemma in these cases. Only long-term clinical follow-up will establish the validity of the updated guidelines.

P59.27 Complementary Utility of Combined ALK/ROS1 FISH with Immunohistochemistry for ALK/ROS1 Rearrangement Testing in Lung Cancer

Testing for EGFR mutations, ALK and ROS1 rearrangements and BRAF mutations are now recommended as the essential minimum predictive biomarkers to be tested in primary non-squamous NSCLC patients presenting with advanced disease. While the ALK D5F3 Ventana immunohistochemistry (IHC) Assay is approved for detection of ALK rearrangements and for targeted therapy, ROS1 IHC only serves as a screening tool for ROS1 rearrangements with demonstration of gene fusions by molecular methods required for confirmation and targeted treatment. Nevertheless, equivocal IHC results, although uncommon, still occur with the D5F3 ALK assay necessitating complementary methods for confirmation. The recent availability of combined ALK/ROS1 break-apart fluorescence in-situ hybridisation (FISH) probes offer the advantage of tissue conservation with ability to analyse both gene alterations in a single tissue section. The aim of the present study was to study the utility of the combined ALK/ROS1 FISH assay as a complement to ALK and ROS1 IHC in routine predictive biomarker testing algorithms. Over a 6-year study period, all EGFR wild type NSCLC samples were subject to automated ALK D5F3 Ventana IHC assay and ROS1 IHC using the D4D6 (Cell signalling technology) clone as a manual lab developed test. Diffuse strong granular staining in tumor cells for ALK and ROS1 protein was interpreted as positive. All cases of equivocal ALK staining and positive ROS1 staining was subject to combined ALK/ROS1 FISH using the FlexISH ALK/ROS1 DistinguISH Probe (Vysis) performed as per manufacturers’ instructions. Presence of ALK/ROS1 break apart signals and/or isolated ALK/ROS1 3’ signals in >15% of interpretable tumour nuclei was considered as positive for ALK or ROS1 rearrangement. The ALK Ventana Assay was performed in 711 formalin fixed paraffin embedded EGFR wild type NSCLC samples including biopsies, lymph node excisions and cell blocks. Of these, 12% (85/711) were positive, 2% (13/711) were equivocal, 75% (539/711) were negative, while in the remaining 11% (76/711), result could not be evaluated due to exhaustion of tumor tissue. FISH analysis in four of the ALK IHC equivocal cases show absence of ALK translocations while no tissue remained for FISH analysis in the remaining equivocal cases. ROS1 IHC was performed in 434 cases of the above cohort of which 7% (30/434) were positive. ROS1 immunopositivity was enriched among ALK IHC positive cases with 43% (19/44) of ALK IHC positive cases also showing protein expression of ROS1. FISH analysis revealed ROS1 translocation in two cases (both showing diffuse strong ROS1 protein expression and negative ALK IHC) while all dual ALK/ROS IHC positive cases tested by FISH showed presence of only ALK rearrangements with non-rearranged ROS1 signals. Tissue adequacy remains the major limiting factor for predictive biomarker testing in lung cancer. The use of combined ALK-ROS FISH allows for excellent demonstration of ALK and ROS1 rearrangements in the same section using the same criteria validated in single gene break apart FISH probes. ROS1 protein is commonly overexpressed in ALK rearranged NSCLC and may represent cross reactivity with ALK or a related protein.

Effect of the 2013 ASCO-CAP HER2 Testing Guideline on the Management of IHC/HER2 2+ Invasive Breast Cancer.

With advances in anti-HER2 treatment and improved prognoses of HER2-positive breast cancer, the American Society of Clinical Oncology and the American Society of Pathologists (ASCO/CAP) have revised the HER2 diagnostic guidelines several times. We examined how to respond clinically to the revisions of the interpretation of the immunohistochemistry (IHC) method. We re-evaluated 254 patients diagnosed as HER2 IHC equivocal, who underwent fluorescence in situ hybridization (FISH) before and after the IHC diagnostic criteria update in 2013. Twenty of 131 (15.3%) IHC equivocal cases by the ASCO/CAP 2007 guideline were IHC score 3+ and one of 20 (0.76%) was negative for FISH. Five of 123 (4.1%) IHC equivocal cases by the ASCO/CAP 2013 guideline were negative for IHC as per the 2007 guideline and four were positive for FISH. After revision of the ASCO/CAP 2013 guideline, 3.3% of HER2-negative cases before the revision should have received anti-HER2 treatment.

An analysis of HER2 amplification in cervical adenocarcinoma: correlation with clinical outcomes and the International Endocervical Adenocarcinoma Criteria and Classification.

Few studies have explored HER2 status in cervical adenocarcinoma, particularly in the gastric‐type adenocarcinoma (GAC), a nonhuman‐papillomavirus‐related subtype with poor clinical outcomes. In this study, we investigated HER2 expression and amplification by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 209 well annotated cervical adenocarcinomas diagnosed using the International Endocervical Adenocarcinoma Criteria and Classification. IHC identified HER2 protein expression in 57.4% (123/209) of adenocarcinomas, of which 62 were IHC 1+ (negative), 38 2+ (equivocal) and 23 3+ (positive). HER2 amplification was found in 13 cases (6.2%) including 10 with IHC 3+ and 3 with IHC 2+. Among all the major histotypes of cervical adenocarcinoma, HER2 amplification was most common in GAC cases with a frequency of 14.7% (5/34). Moreover, HER2 amplification was more frequently associated with 2018 International Federation of Gynecology & Obstetrics (FIGO) stage III/IV, perineural involvement and ovarian spread (p < 0.05) while IHC 3+ was more common in patients with lymphovascular invasion and ovarian involvement (p < 0.05). Survival analysis indicated that FIGO stage III/IV, GAC, and p53 overexpression were associated with poor disease‐specific survival and tumor recurrence (p < 0.05). In conclusion, HER2 amplification was present in a subset of adenocarcinomas, and more common in GAC, pointing to a potential benefit from trastuzumab treatment. HER2 overexpression does not identify gene amplification status in cervical adenocarcinoma; therefore, FISH is suggested for both IHC positive and equivocal cases. Further investigation on more cases with longer follow‐up times is required to consolidate these findings.

HER2 status in breast cancer: changes in guidelines and complicating factors for interpretation.

Human epidermal growth factor receptor 2 (HER2) protein overexpression and/or HER2 gene amplification is found in about 20% of invasive breast cancers. It is a sole predictive marker for treatment benefits from HER2 targeted therapy and thus, HER2 testing is a routine practice for newly diagnosed breast cancer in pathology. Currently, HER2 immunohistochemistry (IHC) is used for a screening test, and in situ hybridization is used as a confirmation test for HER2 IHC equivocal cases. Since the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines on HER2 testing was first released in 2007, it has been updated to provide clear instructions for HER2 testing and accurate determination of HER2 status in breast cancer. During HER2 interpretation, some pitfalls such as intratumoral HER2 heterogeneity and increase in chromosome enumeration probe 17 signals may lead to inaccurate assessment of HER2 status. Moreover, HER2 status can be altered after neoadjuvant chemotherapy or during metastatic progression, due to biologic or methodologic issues. This review addresses recent updates of ASCO/CAP guidelines and factors complicating in the interpretation of HER2 status in breast cancers.

Correlation of Immunohistochemistry and Fluorescence in Situ Hybridization for HER-2 Assessment in Breast Cancer Patients: Single Centre Experience

BACKGROUND:Accurate assessment of HER-2 is imperative in selecting patients for targeted therapy. Most commonly used test methods for HER-2 are immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). We evaluated the concordance between FISH and IHC for HER-2 in breast cancer samples using Food and Drug Administration approved tests.MATERIAL AND METHODS:Archived paraffin tissue blocks from 73 breast cancer patients were used. HER-2 immunostaining was performed using Ventana anti–HER-2 monoclonal antibody. The FISH assay was performed using PathVysion™ HER-2 DNA Probe Kit.RESULTS:Of the 73 cases 68.5% were IHC 0/1+, 15.07% were IHC 2+ and 16.44% were IHC 3+. Successful hybridisation was achieved in 72 cases. HER-2 FISH amplification was determined in 16.67% cases. Ten IHC 3+ and two IHC 2+ cases were FISH positive. Two of the IHC 3+ cases were FISH negative. Concordance rate was 100%, 18.18% and 83.33% for IHC 0/1+, 2+ and 3+ group, respectively. Total concordance was 84.72%, kappa 0.598 (p < 0.0001). The sensitivity of IHC in detecting IHC 2+ and IHC 3+ cases was 16.7% and 83.3%, and the specificity was 85% and 96.67%, respectively.CONCLUSION:The consistency between the methods was highest for IHC negative and lowest for IHC equivocal cases. The immunohistochemistry showed high sensitivity for IHC 2+/3+ cases and high specificity for IHC 3+ cases. Our results support the view that false-positive rather than false-negative IHC results are a problem with HER-2/IHC testing, and that IHC should be used as an initial screening test, but IHC 2+/ 3+ results should be confirmed by FISH.

Digital image analysis of HER2 immunohistochemistry in gastric- and oesophageal adenocarcinoma: a validation study on biopsies and surgical specimens.

To test the validity of diagnostics incorporating digital image analysis (DIA) for human epidermal growth factor 2 (HER2) immunohistochemistry (IHC) in gastro-oesophageal adenocarcinomas, as an alternative to current standard diagnostics using manual scoring. We included 319 consecutive gastro-oesophageal adenocarcinomas (232 biopsies and 87 surgical specimens). DIA was applied to determine HER2 IHC classification, using both standard breast cancer (BC) and modified gastro-oesophageal cancer (GEC) cut-offs. Consensus manual scores were established by four independent observers. Chromogenic in-situ hybridization (CISH) was performed on all 2+ cases by manual scoring, DIA or both. HER2 status was considered positive in 3+ and CISH-positive 2+ cases. Overall agreement between DIA and consensus manual scores was 76.5% (weighted κ = 0.66, BC cut-offs) and 85.6% (weighted κ = 0.80, GEC cut-offs). Agreement was similar for biopsies and surgical specimens. All disagreement occurred in the manual IHC equivocal cases. DIA resulted in a reduction of 2+ cases: 75.8% with BC cut-offs and 46.5% with GEC cut-offs. HER2 status was positive in 48 cases (15%) with standard diagnostics and DIA using GEC cut-offs, and 46 cases (14.4%) using BC cut-offs (all with CISH in 2+ cases). Considering standard diagnostics as a reference, DIA showed 93.8% sensitivity and 99.6% specificity (BC cut-offs) or 97.9% sensitivity and 99.6% specificity (GEC cut-offs). DIA is a reliable and feasible alternative to manual HER2 IHC scoring in gastro-oesophageal adenocarcinoma, both in biopsies and surgical specimens, leading to a reduction of 2+ cases for which subsequent ISH testing is required.

Abstract P1-09-18: A clinically validated DNA microarray for high-resolution HER2 testing defines a new genomic subtype in high-risk breast cancer with equivocal results by IHC and FISH

Abstract Background: In all stages of breast cancer, the HER2 status of a patient's tumor is critically important as both a prognostic indicator, and for predicting response to targeted anti-HER2 therapies. CAP/ASCO 2013 guidelines recommend that newly diagnosed, recurrent, and metastatic breast tumors be evaluated for HER2 positivity by protein-based immunohistochemistry (IHC) and/or chromosome-based fluorescence in situ hybridization (FISH). In the majority of cases, these testing modalities provide a clearly actionable “positive” or “negative” answer. However, in an estimated 10- to 20% of breast cancers, both tests are reported as “equivocal” leaving the clinician with a treatment decision dilemma and no definitive alternative testing method. Here we report validation of an IHC-targeted DNA microarray comparative genomic hybridization (array CGH) assay for HER2 equivocal breast cancer, and definition of a new genomic subtype of HER2 status in high-risk breast cancer with equivocal IHC and FISH results. Methods: IHC-targeted HER2 receptor “hot spot” DNA samples extracted from 25 formalin fixed paraffin embedded (FFPE) breast tumor tissue samples previously characterized by IHC and FISH, were analyzed by array CGH. Eight tumors were known to be highly HER2 positive, seven tumors had IHC scores of 0 with negative FISH, and ten tumors had HER2 receptor staining by IHC (1-2+) and equivocal results by FISH (4-6 HER2 gene copies.) Tumor DNA (test) and human genomic DNA (reference) were fluorescently labeled, and competitively hybridized to a custom-designed genomic DNA microarray with high-density probe coverage of the HER2 amplicon on chromosome 17 (Agilent Technologies, Santa Clara CA). The array design includes over 4,600 chromosome 17 probes representing the p arm, q arm, telomeric and centromeric regions with 66 tiling probes over the HER2 (ERBB2) gene. Following hybridization, average HER2 gene copy number was calculated for each tumor sample by converting mean log2 signal intensity ratio value into genomic region copy number adjusted for % clonal fraction and experimentally established log2 ratio compression of the assay. Results: 25/25 (100%) of samples yielded adequate DNA for analysis and all highly HER2 positive and HER2 negative results were confirmed by array CGH. In 10/10 IHC equivocal cases with HER2 gene copy number 4-6 by FISH, CGH results confirmed HER2-Low gene copy number. Results for 25 Validation SamplesNumber of CasesIHC ScoreFISHCGH Copy NumberResult83+Positive&amp;gt; 6HER2-Positive101-2+Equivocal4-6HER2-Low70Negative&amp;lt; 6HER2-Negative Conclusions: High-resolution HER2 testing by IHC-targeted DNA microarray analysis accurately classifies HER2 status in breast cancer and better defines the HER2-Low genomic subtype most often called “equivocal” by standard IHC and FISH testing. This subcategory is characterized at the protein level by IHC evidence of anti-HER2 therapy target receptor expression on the surface of the cells, and at the genomic level by HER2 gene copy number &amp;lt; 6. Results of the NSABP-B47 clinical trial and further studies with larger numbers of HER2-Low patients are needed to determine if these patients benefit from anti-HER2 therapy. Citation Format: Gunn S, Yaziji H, Sims C, Govender S, Moore M, Cotter P, Jones S. A clinically validated DNA microarray for high-resolution HER2 testing defines a new genomic subtype in high-risk breast cancer with equivocal results by IHC and FISH [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-18.

ALK immunohistochemistry for ALK gene rearrangement screening in non-small cell lung cancer: a systematic review and meta-analysis.

The aim of this study was to investigate the diagnostic accuracy of anaplastic lymphoma kinase (ALK) immunohistochemistry (IHC) for ALK gene rearrangement in non-small cell lung cancer (NSCLC) through systematic review, meta-analysis and diagnostic test accuracy review. The current study included 11,806 NSCLC cases in 42 eligible studies. We performed concordance analyses between ALK IHC and fluorescence in situ hybridization (FISH). The diagnostic accuracy of ALK IHC was analyzed based on ALK IHC criteria and antibodies. The overall ALK IHC results were positive in 13.2%. The overall concordance rate between ALK IHC and FISH was 0.950 (95% confidence interval [CI], 0.927-0.966). In the ALK IHC-positive and negative groups, the concordance rates were 0.805 (95% CI 0.733-0.861) and 0.985 (95% CI 0.978-0.990), respectively. The ALK FISH-positive rates were 0.009 (95% CI 0.004-0.023), 0.378 (95% CI 0.217-0.572), 0.628 (95% CI 0.420-0.796) and 0.900 (95% CI 0.840-0.939) in the ALK IHC 0, 1+, 2+ and 3+ groups, respectively. In diagnostic test accuracy review for ALK IHC, the pooled sensitivity and specificity were 0.92 (95% CI 0.89-0.94) and 0.91 (95% CI 0.90-0.91), respectively. The diagnostic odds ratio and the area under the curve on the summary receiver operating characteristic curve were 266.56 (95% CI 110.83-641.14) and 0.983, respectively. Our results suggested that ALK IHC equivocal (score 1+ and 2+) cases should not be considered as IHC-negative in screening for ALK gene rearrangement. Additional detailed criteria for ALK IHC equivocal cases are necessary to determine how to best apply this approach in daily practice.

A nCounter CNV Assay to Detect HER2 Amplification: A Correlation Study with Immunohistochemistry and In Situ Hybridization in Advanced Gastric Cancer.

Screening amplified genes for targeted therapy with high-throughput technology is very important. The NanoString nCounter system allows multiplexed digital quantification of target molecules through the use of color-coded barcodes with the great advantage that formalin-fixed, paraffin-embedded (FFPE) tissue can be utilized. We tested nCounter custom copy number variation (CNV) panels in 220 gastric cancer samples and evaluated the utility of this method as a screening tool for the detection of CNV using HER2. For the validation of results, we compared the nCounter results with immunohistochemistry (IHC), and we further performed in situ hybridization (ISH) in discrepant cases. The average HER2 gene copy numbers (CNs) by nCounter were 17.25, 2.0 and 2.61 for the HER2 IHC positive (3+), equivocal (2+), and negative cases, respectively. Out of the 16 IHC 3+ cases, 13 (81.3%) were reported as HER2 CN gain (≥4). Gastric cancers with homogeneous HER2 overexpression or high tumor purity showed HER2 CN ≥10. Among the 192 cases with HER2 IHC negative and without HER2 gene amplification, 29 showed a HER2 CN ≥4 with the nCounter assay. The nCounter assay had a concordance rate of 83.4% (kappa value, 0.35), a sensitivity of 66.7%, a specificity of 85.2%, a negative predictive value of 96%, and a positive predictive value of 32.6% compared with HER2 IHC/ISH results. Fresh frozen (FF) samples revealed a higher concordance rate (91.5%, kappa value, 0.59) than FFPE samples (78.5%, kappa value 0.27) and showed a high specificity (97.2%). The nCounter CNV assay is a reliable and practical method to detect high CN variations. Given the intra-tumoral HER2 heterogeneity and normal cell contamination, additional IHC and/or FISH is necessary and needs caution in interpretation, especially in FFPE tissue samples.

HER2 FISH classification of equivocal HER2 IHC breast cancers with use of the 2013 ASCO/CAP practice guideline.

The status of human epidermal growth factor receptor 2 (HER2, ERBB2) determines the eligibility of breast cancer patients to receive HER2-targeted therapy. The majority of HER2 testing in the U.S. is performed using a combination of immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH) for IHC equivocal cases. In 2013, the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) updated the guideline for HER2 testing. This study evaluates the impact of the 2013 ASCO/CAP updated guideline on final HER2 FISH classification of breast cancers with an equivocal IHC result. For each case, we reported a FISH result according to the 2013 updated guideline and recorded a separated result using the 2007 guideline for investigational purpose. McNemar's test and Bowker's symmetry test were used to compare the classifications by the two guidelines. Among 172 HER2 IHC 2+ equivocal cases, use of the 2103 guideline changed classifications in 36 cases (21%) when compared with the results expected by use of the 2007 guideline, and yielded a higher proportion of positive (28.5 vs. 23.3%) and equivocal (16.3 vs. 4.1%), and a lower proportion of negative (55.2 vs. 72.7%) cases (p<0.001). The major classification change with use of the updated guideline is from the HER2 FISH negative to equivocal in 26 cases (15%). Our study has shown that implementation of the 2013 ASCO/CAP updated guideline has significant impact on HER2 classification for breast cancers with an equivocal HER2 IHC result and therefore increased the use of HER2-targeted therapy. Our data have also shown that reflex FISH is effective for final classification of the IHC equivocal cases and that polysomy 17 (CEP17 copy number ≥3/cell) is present in a significantly higher proportion of cases with an equivocal HER2 FISH classification.

Digital Image Analysis of HER2 Immunostained Gastric and Gastroesophageal Junction Adenocarcinomas.

Manual assessment of human epidermal growth factor receptor 2 (HER2) protein expression by immunohistochemistry (IHC) in gastric and gastroesophageal junction (GGEJ) adenocarcinomas is prone to interobserver variability and hampered by tumor heterogeneity and different scoring criteria. Equivocal cases are frequent, requiring additional in situ hybridization analysis. This study aimed to evaluate the accuracy of digital image analysis for the assessment of HER2 protein expression. In total, 110 GGEJ adenocarcinomas were included in tissue microarrays with 3 tissue cores per case. Two immunoassays, PATHWAY and HercepTest, and fluorescent in situ hybridization analysis were performed. The Visiopharm HER2-CONNECT Analysis Protocol Package was applied through the ONCOtopix digital image analysis software platform. HER2 membrane connectivity, calculated by the Analysis Protocol Package, was converted to standard IHC scores applying predetermined cutoff values for breast carcinoma as well as novel cutoff values. Cases with excessive cytoplasmic staining as well as HER2 amplified IHC negative cases were excluded from analysis. Applying HER2-CONNECT with connectivity cutoff values established for breast carcinoma resulted in 72.7% sensitivity and 100% specificity for the identification of HER2 positive gene amplified cases. By application of new cutoff values, the sensitivity increased to 100% without decreased specificity. With the new cutoff values, a 36% to 50% reduction of IHC equivocal cases was obtained. In conclusion, HER2-CONNECT with adjusted cutoff values seem to be an effective tool for standardized assessment of HER2 protein expression in GGEJ adenocarcinomas, decreasing the need for in situ hybridization analyzes.

Abstract P5-02-07: Effect of the new 2013 ASCO / CAP guidelines on HER2 reporting

Abstract Introduction: In 2013 ASCO/CAP published new guidelines for HER2 testing to decrease false negatives. We retrospectively classified cases submitted to a national reference laboratory for HER2 testing using the new guidelines in order to 1) see the overall effect of the 2013 vs 2007 guidelines and 2) predict shifts in interpretation of different HER2 testing methods in the future. M&amp;M: Our laboratory offers HER2 immunohistochemistry (IHC) by HercepTest (Dako) or 4B5 (Ventana) and HER2 dual probe FISH (Abbott Molecular). HER2 IHC and/or FISH tests performed between 1/2010-8/2013, originally scored with the 2007 guidelines, were reclassified with the 2013 guidelines. For IHC, the guideline scores of 0, 1+, 2+ and 3+ were recorded along with intensity (weak, moderate, strong) and % circumferential staining. For FISH, the HER2 and CEP 17 copy number per cell, and HER2:CEP 17 ratio were recorded. Results: 2358 samples submitted for HER2 FISH were available for review. Using the 2007 guidelines, 246 (10.4%) were amplified (Amp), 62 (2.7%) equivocal (Eq), and 2050 (86.9%) were non-amplified (NA). 29/62 (46.8%) FISH Eq cases had HER2:CEP17 ratio between 2.0-2.2, 1 (1.6%) had HER2 copy number &amp;gt;6 per cell. Therefore 30/2358 (1.3%) of all FISH tests previously classified as Eq would be reclassified as Amp following new guidelines increasing the overall FISH Amp rate by 12.5% to 11.7%. Over the same time period, 4043 HER2 IHC tests were performed using old guidelines 1097/4043 (27.1%) of which were Eq (2+). 731/1097 (66.6%) had reflex FISH requested on 2+ IHC cases. 24/731 (3.3%) did not have an iterpretable FISH result. Using the 2007 guidelines, 49 (6.9%) of the 2+ cases were FISH Amp, 23 (3.3%) remained Eq, 635 (89.8%) were NA. Using the new guidelines, an additional 8 (1.1%) of these 707 reflex FISH would be classified as Amp (7 cases due to HER2:CEP17 ratio 2.0 to 2.2 and one case for HER2 copy number &amp;gt;6). Also 8 NA cases would be reclassified as Eq due to a HER2 copy number between 4 and 5.9. With the new guidelines, the overall FISH Amp rate identified from reflex 2+ IHC is 8.1%. 507/707 (71.7%) of HER2 Eq (2+) cases reflexed to FISH had 10-30% circumferential staining (8 strong; 136 moderate; 363 weak). HER2 Amp rates for these are shown in Table 1. HER2 Amplification Status of IHC Equivocal (2+) Cases by Percent Membrane StainingIHC 2+FISH Amplified (2013 Guidelines) (%)2+ Cases (All)57/707 (8.1%)2+ Cases, 10-30% Membrane Staining37/507 (7.3%)-------Strong-------1/8 (12.5%)-------Moderate-------16/136 (11.8%)-------Weak-------20/363 (5.5%) Conclusion: 2013 guidelines will identify a higher % (1.1% for FISH) of eligible patients for targeted therapy. In this series, 2+ IHC cases with reflex FISH testing also had higher Amp rates by 2013 guidelines (8.1% vs 6.9%). Evaluating these rates over time will help assess the effect of the change in the scoring criteria. Amp rates for Eq (2+) IHC cases with 10-30% strong and moderate membrane staining are similar, although only the former group is classified as positive (3+) following new guidelines and would not be retested by FISH. Cases with less than 30% membrane staining may represent a biological spectrum that is not well represented by current IHC testing guidelines, and FISH confirmation on these cases may be justified. Citation Format: H Evin Gulbahce, Rachel E Factor, Erinn Downs-Kelly, Margaret Coppin, Katherine B Geiersbach. Effect of the new 2013 ASCO / CAP guidelines on HER2 reporting [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 P5-02-07.

Re-evaluation of HER2 status in 1 501 invasive breast cancers according to the 2013 American Society of Clinical Oncology/College of American Pathology guidelines

To retrospectively evaluate the HER2 status of 1 501 invasive breast cancer (IBC) by immunohistochemistry (IHC) and fluorescent in situ hybridizaion (FISH), and to compare and analyze the changes and their effects, using the 2007 and 2013 American Society of Clinical Oncology/College of American Pathologist(ASCO/CAP) HER2 testing guidelines. Tissue handling and HER2 testing were performed according the 2007 ASCO/CAP guideline recommendations. The HER2 status of all newly diagnosed IBC were routinely assessed by IHC, and reflex FISH assay was done on all the IHC equivocal (IHC 2+) cases. The HER2 status of 1 501 cases of IBC was re-evaluated according to these two criteria. Using the 2007 and 2013 ASCO/CAP criteria, the overall positive, equivocal and negative rates of HER2 over-expression and/or amplification in the 1 501 IBCs were 23.05% and 23.52%, 11.59% and 12.52%, and 65.36% and 63.96%, respectively. The positive and equivocal rates increased by 0.47% and 0.93% respectively, but the negative rate decreased by 1.40% when using the new criteria. For HER2 IHC staining using the 2007 and 2013 guidelines, the positive, equivocal and negative rates were 17.99% and 18.13% (+0.13%), 32.51% and 32.91% (+0.40%) and 49.50% and 48, 97% (-0.53%), respectively. FISH for HER2 amplification was done in 348 of the 1 501 IBCs, and using the 2007 and 2013 guidelines, the positive, equivocal and negative rates were 27.59% and 29.02% (+1.43%), 1.15% and 3.74% (+2.59%) and 71.26% and 67.24% (-4.02%), respectively. The application of 2013 ASCO/CAP guideline could lead to an increase in positive and equivocal rates, and a decrease in negative rate. The influence could be more prominent for the evaluation of FISH result, and would raise the positive and equivocal rates since a mean HER2 copy number is used in the new criteria. Our re-estimation of IHC result was concordant with the prediction of the guideline.

Bright-Field Microscopy for HER2 Gene Assessment

The human epidermal growth factor receptor gene ERBB2 (HER2) is located on chromosome 17 and amplified in approximately 20% of invasive breast carcinomas.1,2 Due to the availability of effective anti-HER2 therapies, the prognostic significance of HER2 gene amplification, and the predictive value of HER2 status with regard to response to chemotherapy, laboratory testing for protein overexpression and/or amplification is critical for clinical decision making in individual patients. Testing of HER2 on all newly diagnosed invasive breast carcinoma is standard of care. Although there is no gold standard for assessing HER2 status in breast cancer, immunohistochemistry (IHC) for protein expression and fluorescence in situ hybridization (FISH) for HER2 gene copy number are the preferred assays.3 Although no test is perfect, clinical utilization depends on a number of factors, including test accuracy but also consistency of results, as well as ease of performance and interpretation. Due to early standardization, consistency in reporting, and only a small number of equivocal results, the FISH assay became the preferred method for assessing HER2 gene status.4,5 FISH is also used as the reflex test in IHC equivocal (2+ score) cases. FISH on IHC equivocal cases can identify a small percentage of amplified cases but also aneuploidy and other difficult to assess abnormalities of chromosome 17. Despite its widespread use, the FISH assay does have some drawbacks. FISH is considered a morphology-based assay, but the fluorescent nature of the assay results in less than optimal histologic details (compared with bright-field microscopy), so the area of invasive tumor generally needs to be marked on a parallel H&E-stained section. The procedure …

Détermination de l’amplification de l’oncogène HER-2 dans le cancer du sein invasif par hybridation chromogénique in situ double couleur (dc-CISH) : étude comparative avec l’hybridation fluorescente in situ (FISH)

The amplification of the gene encoding for the human epidermal growth factor receptor 2 (HER-2 oncogene), located on chromosome 17 (17q21-q22), or the overexpression of this receptor have prognostic and therapeutic implications in invasive breast cancer. An evaluation of the HER-2 status by immunohistochemistry (IHC) is performed on all invasive breast cancer cases. Fluorescent in situ hybridization (FISH) is considered as the gold standard for the detection of HER-2 gene amplification for IHC equivocal cases (score 2+). A more recent in situ hybridization technique, the dual-color chromogenic in situ hybridization (dc-CISH), has been proposed as an alternative to FISH. The aim of this study was to measure the correlation between dc-CISH and FISH for HER-2 oncogene amplification assessment in invasive breast cancer. We built four tissue micro-array (TMA) blocs with 100 breast invasive cancer cases that had been previously tested by IHC for HER-2 detection: 10 score 0 cases, 10 score 3+cases, 39 score 1+and 41 score 2+cases. Both FISH and dc-CISH techniques were applied on all TMA cases as well as on two additional slides serving as controls. Interpretation of dc-CISH was carried out by a pathologist using an optical microscope. For FISH, the interpretation was done by a professional from the medical genetics department using a fluorescent microscope linked to a computer system for image capturing and analysis. The interpretation of the HER-2/CEN-17 ratio for both tests was in accordance with the values of the updated recommendations from the Canadian National Consensus Meeting on HER-2/neu testing in breast cancer and from the ASCO/CAP. Among the 100 cases initially included in the study, eight were excluded from the analysis due to sampling or technical flaws. From the 92 remaining cases, we obtained a concordance of 97.8% (90/92 cases) between the two techniques (Kappa coefficient 0.97, 95% confidence interval). The correlation coefficient (rho) between ratios was estimated at 0.57. This study shows a strong concordance between FISH and dc-CISH techniques and indicates that dc-CISH is a good alternative method for HER-2 gene amplification assessment in breast cancer.