HER2 Testing In Breast Cancer Research Articles

Navigating HER2-Low Testing in Invasive Breast Cancer: Update Recommendations for Pathologists.

The article discusses the importance of accurately distinguishing HER2-low from HER2-negative breast cancer, as novel ADCs have demonstrated activity in a large population of patients with HER2-low-expressing BC. While current guidelines recommend a dichotomous classification of HER2 as either positive or negative, the emergence of the HER2-low concept calls for standardization of HER2 testing in breast cancer, using currently available assays to better discriminate HER2 levels. This review covers the evolution and latest updates of the ASCO/CAP guidelines relevant to this important biomarker in breast cancer, including still-evolving concepts such as HER2 low, HER2 heterogeneity, and HER2 evolution. Our group presents the latest Mexican recommendations for HER2 status evaluation in breast cancer, considering the ASCO/CAP guidelines and introducing the HER2-low concept. In the era of personalized medicine, accurate HER2 status assessment remains one of the most important biomarkers in breast cancer, and the commitment of Mexican pathologists to theragnostic biomarker quality is crucial for providing the most efficient care in oncology.

HER2 amplification in cases with low HER2 expression by immunohistochemistry (IHC): A Single Institutional Experience

Abstract Introduction/Objective HER2 testing in invasive breast carcinoma (IBC) is the standard of care to determine patient eligibility for HER2 directed therapy with trasuzumab. Initial testing of tissue by HER2 immunohistochemistry (IHC) can be positive, negative, or equivocal. In equivocal cases as well as negative cases in our institution HER2 FISH is also performed. According to ASCO/CAP guidelines a subset of equivocal and negative IHC cases are considered HER2 positive if they meet criteria for increased HER2/CEP17 ratio and HER2 copy number by FISH, allowing more patients to be treated with HER2 directed therapies. HER2 grouping per ASCO is as follows; Group 1: HER2 copy number ≥4 signals/cell, Group 2: <4.0 signals/cell, Group 3: ≥6 signals/cell, Group 4: HER2 copy number ≥4 and <6 signals/cell, Group 5: HER2 copy number <4 signals/cell Methods/Case Report All cases of IBC resection with HER2 IHC and FISH results from Jan 2018-Dec 2021 were included in this study. All cases positive for HER2 by IHC alone (3+ staining) were excluded. The ASCO/CAP 2018 guidelines for HER2 testing in breast cancer were used to evaluate HER2 positivity by FISH and the relation to IHC scores Results (if a Case Study enter NA) A total of 2630 IBC cases with both HER2 IHC and FISH testing were included. Of these, 131 (5.0%) were in FISH group 1, 17 (0.7%) in group 2, 11 (0.4%) in group 3, 161 (6.1%) in group 4, and 2310 (87.8%) in group 5. A total of 142 (5.4%) cases that were HER2 equivocal or negative by IHC were HER2 positive by FISH. Specifically, 0.3% of cases with an IHC score of 0, 1.4% with a score of 1+, and 14.6% of cases with a score of 2+ were positive by FISH. Of the 14.6% IHC 2+ cases that were FISH positive 13.3% were in FISH group 1 (115 cases) and 1.3% (11 cases) in FISH group 3. Of FISH positive cases 1.4% had an IHC score of 0, 9.9% had an IHC score of 1 and 88.7% had a score of 2 Conclusion At our institution 14.6% of IBC with equivocal HER2 staining on IHC, 1.4% of cases with negative (1+) staining and 0.3% of cases with negative (0) were HER2 FISH positive. Since HER2 FISH is not reflexively performed on cases HER2 negative by IHC, this additional 1.7% will qualify for HER2 targeted therapy Interpretation of low HER2 expression by IHC by traditional IHC assay may miss a small percentage of cases eligible for HER2 directed therapy. Accurate interpretation of HER2 low by IHC is critical as recent clinical trials have shown that low HER2 expression with IHC scores of 1+ or 2+ without gene amplification by FISH could benefit from Trastuzumab Deruxtecan

Assessment of the Impact of Alternative Fixatives on HER2 Detection in Breast Cancer and Gastric Cancer Tumor Specimens.

The type of fixative used for preserving tumor specimens can significantly impact the performance of the immunohistochemistry and in situ hybridization assays used for assessing human epidermal growth factor receptor 2 (HER2) status. This study reports the prevalence of the use of alternative fixatives other than the guideline-recommended 10% neutral buffered formalin (NBF) during HER2 testing in a real-world setting. The effects of alternative fixatives [20% NBF and 10% unbuffered formalin (UBF) fixatives] on HER2 testing of breast cancer (BC) and gastric cancer (GC) cell lines and tissues are also assessed. Overall, 117,636 tumor samples received at a central laboratory from >8000 clinical trial sites across 60 countries were reviewed to determine the prevalence of alternative fixative usage. To investigate the impact of alternative fixatives, 27 cell lines (21 BC and 6 GC) and 76 tumor tissue samples (50 BC and 26 GC) were fixed in 10% NBF, 20% NBF, or 10% UBF, and evaluated for HER2 status by immunohistochemistry and in situ hybridization. Real-world data showed that 9195 (7.8%) tumor samples were preserved using an alternative fixative. In cell lines, overall percentage agreement, negative percentage agreement, and positive percentage agreement among the 3 fixatives were 100%. In tumor tissues, the agreement among 10% NBF, 20% NBF, and 10% UBF ranged between 94.7% and 96.6% for negative percentage agreement and 90.9% for overall percentage agreement compared with a range of 58.3% to 66.7% for positive percentage agreement. These results suggest that alternative fixatives may have the potential to convert HER2 status in tissues from positive to negative.

Abstract 5457: Prevalence of “low” HER2 expression is frequent in breast cancer but also in cancers of other origin: A tissue microarray study on 131 tumor types

Abstract HER2 is a receptor tyrosine-protein kinase coded by the ERBB2 gene. The HER2 status is routinely assessed to select patients eligible for targeted therapy with anti-HER2 drugs. HER2 positivity was restricted to cancers with a 3+ score at immunohistochemistry (IHC) and/or gene amplification. The successful use of HER2 antibody-drug conjugates in tumors with IHC scores of 1+ (HER2 low) or 0 with incomplete and faint staining in ≤10% of tumor cells (HER2 ultralow) is currently changing the traditional dichotomy of HER2 testing in breast cancer. To determine HER2 expression in neoplastic tissues, a tissue microarray containing one 0.6mm tissue spot each from 7,505 tumor samples from 131 different tumor types and subtypes was analyzed by immunohistochemistry. IHC scoring included 3+, 2+, 1+, and a category “+” (faintly positive in less than 10% of tumor cells; ultralow). The cohort included 310 breast cancers for which the HER2 amplification was also assessed by fluorescence in situ hybridization. Among 555 evaluable breast cancers, HER2 IHC was 3+ in 7.6%, 2+ in 3.2%, 1+ in 14.6%, “+” in 15.9% and 0 in 58.7%. The HER2 amplification rate was in 95.8% of 3+, 61.5% of 2+, 18.2% of 1+, but 0% in “+” and HER2 0 breast cancers. Among 4,912 non-breast cancers, the HER2 status was 3 in 0.6%, 2 in 0.7%, 1 in 3.3%, “+” in 2.3%, and 0 in 93.2%. In these tumors, 3+ positivity was largely restricted to gastric/esophageal adenocarcinoma, urothelial carcinoma, ovarian cancer as well as adenocarcinomas of the lung, colon, and pancreas. A 2+ positivity was mostly seen in tumor entities that had also 3+ cases. A 1+ or “+” HER2 positivity was found in 50 of 147 analyzed non-breast cancer categories. HER2 low and ultralow (1+ or “+”) was most commonly seen in basal cell carcinoma of the skin (52.2%), clear cell carcinoma of the ovary (33.3%), urothelial carcinoma of the renal pelvis (33.3%), gallbladder adenocarcinoma (26.7%), muscle-invasive urothelial carcinoma of the bladder (24.5%), prostatic adenocarcinoma, Gleason 4+4 (23.3%), endometrial serous carcinoma (22.7%), prostatic adenocarcinoma, Gleason 5+5 (19.2%), carcinosarcoma of the ovary (18.8%), renal oncocytoma (17.9%), serous carcinoma of the ovary (17.8%), gastric adenocarcinoma, intestinal type (13.2%), neuroendocrine tumor (NET) of the lung (11.1%), carcinosarcoma of the uterus (11.1%), endometrioid carcinoma of the ovary (11.1%), squamous cell carcinoma of the larynx (10.7%), parathyroid gland adenoma (10%), endometrioid endometrial carcinoma (9.7%), adenocarcinoma of the esophagus (9.5%), cholangiocarcinoma of the liver (9.3%), and pancreatic/ampullary adenocarcinoma (8.6%). While 3+ IHC is limited to only few cancer types, there is a much broader range of tumor entities that can show HER2 low and ultralow expression. HER2 antibody-drug conjugates may therefore be successful drugs in a large variety of different tumor entities. Citation Format: Maximilian Lennartz, Florian Viehweger, David Dum, Ria Uhlig, Andrea Hinsch, Doris Hoeflmayer, Christoph Fraune, Christian Bernreuther, Patrick Lebok, Soeren Weidemann, Guido Sauter, Till Sebastian Clauditz, Frank Jacobsen, Till Krech, Andreas H Marx, Sarah Minner, Ronald Simon, Natalia Gorbokon, Stefan Steurer, Eike Burandt. Prevalence of “low” HER2 expression is frequent in breast cancer but also in cancers of other origin: A tissue microarray study on 131 tumor types. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5457.

Quantitative Imaging Analysis Fluorescence In Situ Hybridization Validation for Clinical HER2 Testing in Breast Cancer.

Quantitative imaging is a promising tool that is gaining wide use across several areas of pathology. Although there has been increasing adoption of morphologic and immunohistochemical analysis, the adoption of evaluation of fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue has been limited because of complexity and lack of practice guidelines. To perform human epidermal growth factor receptor 2 (HER2) FISH validation in breast carcinoma in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guideline. Clinical validation of HER2 FISH was performed using the US Food and Drug Administration-approved dual-probe HER2 IQFISH (Dako, Carpinteria, California) with digital scanning performed on a PathFusion (Applied Spectral Imaging, Carlsbad, California) system. Validation parameters evaluated included z-stacking, classifier, accuracy, precision, software, and hardware settings. Finally, we evaluated the performance of digital enumeration on clinical samples in a real-world setting. The accuracy samples showed a final concordance of 95.3% to 100% across HER2 groups 1 to 5. During clinical implementation for HER2 groups 2, 3, and 4, we achieved a final concordance of 76% (95 of 125). Of these cases, only 8% (10 of 125) had discordances with clinical impact that could be identified algorithmically and triaged for manual review. Digital FISH enumeration is a useful tool to improve the efficacy of HER2 FISH enumeration and capture genetic heterogeneity across HER2 signals. Excluding cases with high background or poor image quality and manual review of cases with ASCO/CAP group discordances can further improve the efficiency of digital HER2 FISH enumeration.

Abstract P6-04-19: Engaging pathologists in a social peer-to-peer learning collaborative to discuss the emergence of HER2-low breast cancer

Abstract Introduction: Recent advances in research have shown clinical effectiveness when targeting the lower range of HER2 expression (ie, HER2-low) in patients with metastatic breast cancer. American Society for Clinical Pathology worked in collaboration with Q Synthesis to develop a peer-to-peer learning collaborative to proactively prepare pathologists for HER2-low and to discuss the clinical implications around this emerging classification. This CME project was supported by an educational grant from AstraZeneca Pharmaceuticals LP and Daiichi Sankyo Inc. Methods: ASCP launched a peer-to-peer (P2P) learning collaborative (HER2 Breast Trailblazers) where small groups of pathologists met to discuss some of the practical implications associated with HER2-low. 38 pathologists from a mix of academic and community settings participated in this CME program. For foundational knowledge, learners completed online modules covering scientific updates on HER2-low. Through small-group, case-based discussions, learners reviewed operational challenges and opportunities to prepare for HER2-low. They applied this knowledge to lead projects at their own institutions focusing on the anticipated changes around HER2-low. ASCP also launched a series of peer-led Twitter Chats that were designed to reach a broad audience and foster open dialogue about the emerging science of HER2-low breast cancer. This approach engaged Twitter users who were eager to share and disseminate the education to their colleagues. Twitter Chats provided peer-to-peer feedback regarding ways to navigate obstacles, barriers, and other challenges affecting HER2 testing in breast cancer. Results: The learners identified the following challenges and opportunities: Defining HER2-low: Several learners had heard misconceptions around the definition of HER2-low. Recent studies have defined HER2-low as IHC 1+ or IHC 2+ with ISH-negative. Interobserver concordance with IHC 0 vs 1+: Several learners discussed the challenges around interpreting IHC 0 vs 1+. They felt that some pathologists may need guided feedback to improve their diagnostic skills. Use of IHC vs. ISH: Several learners only performed ISH for HER2 testing on all breast cancer samples. If HER2-low emerges as a third category, they would need to return to IHC. Implications for non-metastatic breast cancer: Recent HER2-low studies have focused on patients with metastatic breast cancer. If HER2-low emerges as a third category, it is unclear whether this designation will also be used in patients who have early-stage breast cancer. Leadership: As pathologists prepare for HER2-low, they have opportunities to lead projects to assess and improve IHC interobserver concordance, coach others on IHC interpretation, increase operational efficiency, strength communication skills, and build up the team by proactively anticipating challenges around HER2-low. Conclusions: HER2-low breast cancer appears to be emerging as a new classification and pathologists need to be prepared to ensure accurate testing and interpretation. Through a peer-to-peer learning collaborative, pathologists identified ways to proactively prepare and demonstrate leadership so that cancer centers and laboratories may be ready to embrace a new paradigm of HER2 classification in breast cancer. A series of public Twitter Chats broadened this discussion and increased awareness among pathologists. Citation Format: Joseph Kim, Kellie Beumer, Melissa Kelly. Engaging pathologists in a social peer-to-peer learning collaborative to discuss the emergence of HER2-low breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-19.

The concordance between IHC and ISH for HER-2 testing in breast cancer in Nakhon Pathom Hospital, Thailand, based on the ASCO/CAP 2018 guidelines: a retrospective study.

Human epidermal growth factor receptor 2 (HER-2) is the prognostic and predictive biomarker for breast cancer found in a quarter of all breast cancer cases. Precise testing of HER-2 will impact treatment response.Based on the updated American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER-2 testing guidelines, we report 141 cases of positive concordance rate between HER-2 IHC stain and dual-probe ISH for HER-2 testing classified in equivocal (2+) and positive (3+) IHC groups in Nakhon Pathom Hospital, Thailand. Our study showed statistical significance in the relationship between positive (3+) IHC and dual-probe ISH for HER-2 testing with a correlation rate of 91.76% (rs = 0.38031, p < 0.0001) and 37.50% in equivocal (2+) IHC groups.This is the first report of a positive concordance rate between equivocal and positive HER-2 IHC and dual-probe ISH for HER-2 based on the ASCO/CAP 2018 guidelines in Thailand. Our study confirmed a statistically significant relationship in the positive IHC (3+) group. In addition, we suggested reflexing ISH testing only in equivocal IHC (2+) cases to decrease the waiting time and economical approach.

Retrospective observational study of HER2 immunohistochemistry in borderline breast cancer patients\xa0undergoing neoadjuvant therapy, with an emphasis on Group 2 (HER2/CEP17 ratio \u22652.0, HER2 copy number

BackgroundThe ASCO/CAP guidance on HER2 testing in breast cancer (BC) has recently changed. Group 2 tumours with immunohistochemistry score 2+ and HER2/CEP17 ratio ≥2.0 and HER2 copy number <4.0 signals/cell were re-classified as HER2 negative. This study aims to examine the response of Group 2 tumours to neoadjuvant chemotherapy (NACT).Methods749 BC cases were identified from 11 institutions. The association between HER2 groups and pathological complete response (pCR) was assessed.Results54% of immunohistochemistry HER2 positive (score 3+) BCs showed pCR, compared to 19% of immunohistochemistry 2+ FISH amplified cases. 27% of Group 2 treated with HER2 targeted therapy achieved pCR, compared to 19 and 11% in the combined Groups 1 + 3 and Groups 4 + 5, respectively. No difference in pCR rates was identified between Group 2 and Group 1 or combined Groups 1 + 3. However, Group 2 response rate was higher than Groups 4 + 5 (p = 0.017).ConclusionNo difference in pCR was detected in tumours with a HER2/CEP17 ratio ≥2.0 and a HER2 score 2+ by IHC when stratified by HER2 gene copy number. Our data suggest that ASCO/CAP HER2 Group 2 carcinomas should be evaluated further with respect to eligibility for HER2 targeted therapy.

Rate of reclassification of HER2-equivocal breast cancer cases to HER2-negative per the 2018 ASCO/CAP guidelines and response of HER2-equivocal cases to anti-HER2 therapy.

The 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline on HER2 testing in breast cancer permits reclassification of cases with HER2-equivocal results by FISH. The impact of such reclassification is unclear. We sought to determine the proportion of HER2-equivocal cases that are reclassified as HER2-negative and the impact of anti-HER2 therapy on survival in HER2-equivocal cases. We reviewed medical records of breast cancer patients who had HER2 testing by fluorescence in stitu hybridization (FISH) and immunohistochemistry (IHC) performed or verified at The University of Texas MD Anderson Cancer Center during April 2014 through March 2018 and had equivocal results according to the 2013 ASCO/CAP guideline. The population was divided into 2 cohorts according to whether the biopsy specimen analyzed came from primary or from recurrent or metastatic disease. HER2 status was reclassified according to the 2018 ASCO/CAP guideline. Overall survival (OS) and event-free survival (EFS) were calculated using the Kaplan-Meier method, and the relationship between anti-HER2 therapy and clinical outcomes was assessed. We identified 139 cases with HER2-equivocal results according to the 2013 ASCO/CAP guideline: 90 cases of primary disease and 49 cases of recurrent/metastatic disease. Per the 2018 ASCO/CAP guideline, these cases were classified as follows: overall, HER2-negative 112 cases (80%), HER2-positive 1 (1%), and unknown 26 (19%); primary cohort, HER2-negative 85 (94%), HER2-positive 1 (1%), unknown 4 (4%); and recurrent/metastatic, HER2-negative 27 (55%) and unknown 22 (45%). Five patients in the primary-disease cohort and 1 patient in the recurrent/metastatic-disease cohort received anti-HER2 therapy. There was no significant association between anti-HER2 therapy and OS or EFS in either cohort (primary disease: OS, p = 0.67; EFS, p = 0.49; recurrent/metastatic-disease, OS, p = 0.61; EFS, p = 0.78. The majority of HER2-equivocal breast cancer cases were reclassified as HER2-negative per the 2018 ASCO/CAP guideline. No association between anti-HER2 therapy and OS or EFS was observed. HER2-equivocal cases seem to have clinical behavior similar to that of HER2-negative breast cancers.

HER2 Testing for Breast Cancer in the Genomics Laboratory: A Sea Change for Fluorescence In Situ Hybridization.

Guidelines for HER2 testing in breast cancer have changed over time, from the US Food and Drug Administration guideline to the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines published in 2007, 2013, and 2018. To investigate the change in assignment of HER2 status in breast cancers with equivocal (2+) immunohistochemistry (IHC) results by fluorescence in situ hybridization (FISH) following implementation of the ASCO/CAP 2018 guideline. The study included 3556 invasive breast cancers that were HER2 equivocal (2+) by IHC and were submitted to our FISH laboratory after July 2018. Reflex testing (with repeat IHC staining) was performed on certain categories of FISH results known as groups 2, 3, and 4. Concomitant review of IHC and FISH was performed on these reflex cases per 2018 guideline recommendations. The FISH data were analyzed to compare US Food and Drug Administration and ASCO/CAP 2007, 2013, and 2018 interpretations. Of 3548 invasive breast cancers with complete data available, the percentage agreement for FISH according to different guidelines was highest for ASCO/CAP 2018 versus US Food and Drug Administration (96.5%), followed by ASCO/CAP 2018 versus 2007 (93.8%), and lowest with ASCO/CAP 2018 versus 2013 (83.7%). Per the 2018 guideline, reflex IHC testing was performed on 633 breast cancers (17.8%); the majority of reflex testing results were negative (541 of 633; 85.5%). The overall distribution of HER2 FISH results (per the 2018 guideline) was 88.5% negative and 11.5% positive. By eliminating the equivocal FISH category, the 2018 ASCO/CAP guideline significantly reduced the HER2 FISH-positive rate in tumors with equivocal (2+) IHC results.

Biomarker heterogeneity between primary breast cancer and synchronous axillary lymph node metastases.

Whether the expression status of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2) receptor and Ki-67 show concordance between the primary tumors and the synchronous axillary lymph node (ALN) metastases has been discussed in numerous studies. However, to date, the results of these studies remain controversial. Therefore, the present study aimed to investigate whether the expression of ER, PR, HER-2 and Ki-67 was in concordance between the primary tumors and synchronous ALN metastases in patients with operable breast cancer (BC). A total of 60 tissue samples were collected from patients with primary operable BC diagnosed with primary tumors and synchronous ALN metastases. The expression levels of the four biomarkers, ER, PR, HER-2 and Ki-67, were assessed in primary lesions and synchronous ALN metastases samples using immunohistochemistry. The cut-off values were set to 10% for ER and PR, while the labeling index of Ki-67 was set to 14%. The immunostaining intensity of ER and PR was scored as negative (−), 1+, 2+ and 3+. The criteria for HER-2 testing in BC were implemented according to the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) guidelines. The concordance rates for ER, PR and HER-2 were 96.7 (58/60), 96.7 (58/60) and 90% (54/60), respectively. In addition, the kappa values of consistency in the primary lesions and the synchronous ALN metastases were 0.773 for ER, 0.654 for PR and 0.785 for HER-2. Furthermore, the P-values of ER, PR and Ki-67 numerical variables between the two groups were 0.393, 0.400 and 0.331, respectively, as demonstrated using a non-parametric Wilcoxon signed rank test. The findings of the present study demonstrated a high degree of concordance between the expression of ER, PR, HER-2 and Ki-67 in the primary tumors and that in the synchronous ALN metastases, suggesting that the BC primary tumor biomarkers may be used for the prognosis of synchronous ALN metastases in patients with operable BC.

Applying the New Guidelines of HER2 Testing in Breast Cancer.

The human epidermal growth factor receptor 2 (HER2) is an important prognostic and predictive biomarker in the breast cancer. The American Society of Clinical Oncology/College of American Pathology (ASCO/CAP) has published HER2 testing guidelines in breast cancer. We herein reviewed the HER2 testing guidelines in breast cancer with a focus on the application of the current guidelines. The continual investigation of HER2 testing in breast cancer has resulted in updates in the HER2 testing guidelines. The current guidelines focus on the uncommon clinical scenarios and emphasize the coordination between immunohistochemistry and in situ hybridization results, in an effort to improve clarity and accuracy. The ASCO/CAP guidelines provide valuable recommendations to ensure the accurate evaluation of HER2 status in breast cancer patients through standardization. Additional studies, particularly those with long-term outcome data are still needed to validate the guideline recommendations, especially the uncommon cases.

HER2 Dual In Situ Hybridization: Correlations and Cautions

Accurate HER2 testing in breast cancer is crucial for appropriate precision therapy. HER2 testing is most commonly accomplished by a combination of immunohistochemistry and in situ hybridization techniques, as gene amplification is closely tied to protein overexpression. During the last 5+ years, brightfield dual in situ hybridization (DISH) has replaced fluorescence methods (fluorescence in situ hybridization [FISH]) in some laboratories. To analyze routine HER2 DISH performance in the field. We reviewed our experience with HER2 DISH performed at outside laboratories and referred for patient care. Of 273 identified retrospective DISH results, 55 had repeated FISH testing at our institution; 7 (13%) were discordant. Additional cases had technical flaws hampering appropriate scoring. In 23 cases (42%), HER2 DISH was performed without immunohistochemistry. Slide review of a prospective cohort of 42 consecutive DISH cases revealed 14 (33%) with technical or interpretative limitations potentially jeopardizing results. Commonly identified problems include lack of or weak signals in most tumor cells, and silver precipitate or red signals outside of nuclei, resulting in false-negative or false-positive interpretations, respectively. Further, 44% (24 of 55) of DISH reports lacked complete data, specifically average HER2 signals/cell. While HER2 DISH can be an efficient and effective alternative to FISH, we illustrate pitfalls and reinforce that careful attention to slide quality and technical parameters are critically important. HER2 DISH cotesting with immunohistochemistry could help minimize false-negative or false-positive HER2 results.

Reassignment of HER2 status for subgroups of breast cancer according to the 2018 updated American Society of Clinical Oncology and College of American Pathologists guidelines: The impact of combined immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) reflex testing in a large national reference laboratory.

3144 Background: Updated ASCO/CAP Guidelines for HER2 testing in breast cancer have been most impactful on the resolution of certain challenging groups of FISH results. We review the change in assignment of HER2 status in a large series of breast cancers referred to a large national reference laboratory for FISH testing since the introduction of the 2018 updated guidelines. Methods: Patient samples submitted to the Mayo Clinic Cytogenetics Laboratory (N = 2208) were analyzed by FISH. Samples with Group 2, Group 3, or Group 4 FISH results were reflexed to immunohistochemistry (IHC) in our central laboratory; FISH slides for those cases with equivocal 2+ IHC results were re-scored in the regions of invasive cancer showing more intense membranous staining. A subset of 202 samples with Group 4 FISH results were also reflexed to the previously employed reflex FISH assay (HER2/D17S122), and these were also re-analyzed according to the new reflex IHC/FISH process. Results: 382 of 2208 breast cancer samples tested (17.3%) had FISH results categorized as Group 2 (N = 17, 0.8%), Group 3 (N = 34, 1.5%), or Group 4 (N = 331, 15%) and required reflex IHC testing, and of those, 75% were 2+ equivocal and required targeted re-analysis of the FISH slide according to the 2018 updated guidelines. Re-analysis of the FISH slide resulted in switching between Groups 1-5 in 19.4% of cases, but HER2 status was changed by FISH re-scoring in only 7.7% of cases re-scored (1.0% of all samples), generally due to only minor shifts in HER2 copy number and HER2/control ratios between the initial and IHC-guided reflex FISH scores. In the subset of 202 cases tested by both reflex methods, the previously employed HER2/D17S122 reflex probe set was positive in 123 cases (60.9%), whereas reflex IHC/FISH was positive in only 10 cases (7.9%). Including positive reflex IHC (0.4%) and positive reflex FISH results (2.1%), the overall assignment of positive HER2 status on our series of 2208 cases was 11.5%. Conclusions: Overall rates of HER2 positive FISH results have declined under the most recent ASCO/CAP guideline update as a consequence of new recommendations for reflex testing for Groups 2-4. This change is largely due to reassignment of Group 2 and Group 4 results as negative in the absence of positive IHC.

Abstract P4-02-08: Statistical modeling of influential variables affecting HER2-positivity in breast cancer: Final analyses from two large, multicenter, noninterventional studies in Germany

Abstract Background: While HER2 testing in breast cancer (BC) has been routine for over a decade, testing quality remains a challenge. Currently, HER2-positivity rate is the only recommended quality indicator. However, the large, observational, prospective NIU HER2 study in Germany quantified the impact of patient- and tumor-related characteristics such as histologic grade, hormone receptor (HR) status, histologic subtype, age, and nodal status on HER2-positivity, indicating that these factors need to be considered when evaluating HER2-positivity as a measure of testing quality (Rüschoff et al. Mod Pathol 2017). We now report the final analyses from the multicenter EPI HER2 BC study (ML29763, NCT02666261) in Germany where we compared NIU and EPI study data, and aimed to validate the NIU study model. Methods: Data from eligible patients with invasive BC were collected (HER2 test result; patient- and tumor-related factors) and variables influencing HER2-positivity identified and compared between studies. The NIU study model was validated and its predictive power determined using newly collected data from the EPI study, with cutoff and variable coefficients from the previous NIU analysis. Additional promising variables were explored, and their relative influence investigated, using multiple stepwise logistic regression. Results: In total, 14,729 (EPI) and 15,281 (NIU) BC samples were analyzed. Distributions of the main variables were comparable; overall HER2-positivity rates were 13.47% (EPI) and 14.24% (NIU). Fitting the NIU study model to EPI study data demonstrated that all five covariates from the NIU study analyses significantly affected HER2-positivity (p &amp;lt; 0.01); the influence for each covariate differed only slightly between studies (in EPI, histologic grade had most influence followed by histologic subtype, HR status, nodal status, and age). Prediction profiles were used to visualize the relationship between the model-predicted probability of HER2-positivity and the five identified covariates, which showed good comparability between studies. The receiver operating characteristics area under the curve (ROC AUC) of the NIU model used to predict HER2-positivity in the EPI study data was close to that of the model fitted to the NIU data, thus successfully validating the NIU model. To further improve the model, the categorical HR status was replaced by estrogen receptor (ER) and progesterone receptor (PgR) expression. Inclusion of ER and PgR as continuous variables improved the predictive strength of the model (ROC AUC = 0.74; sensitivity = 0.76; specificity = 0.63). Based on this improved model, PgR status had the highest influence on HER2-positivity, followed by histologic grade, histologic subtype, nodal status, ER status, and age. Conclusions: Results from our analyses confirm the statistically and clinically significant influence of patient- and tumor-related factors on HER2-positivity, and highlight the necessity to integrate these factors into the quality control assessment of HER2 testing. Implementation of this model in routine practice may assist in addressing issues with interlaboratory variation, and help to identify centers with HER2 testing problems more accurately. Citation Format: Rüschoff J, Lebeau A, Kreipe H, Sinn P, Schildhaus H-U, Decker T, Ammann J, Künzel C, Koch W, Untch M. Statistical modeling of influential variables affecting HER2-positivity in breast cancer: Final analyses from two large, multicenter, noninterventional studies in Germany [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 P4-02-08.

HER2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update Summary.

HER2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update Summary.

Abstract P2-03-06: Draft recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer will decrease HER2 positivity rates

Abstract Introduction: A focused update of American Society of Clinical Oncology (ASCO) and College of American Pathologist's (CAP) guideline recommendations for HER2 testing in breast cancer (BrCa) was published in May, 2017 for public comment (http://www.cap.org/ShowProperty?nodePath=/UCMCon/Contribution Folders/WebContent/pdf/her2-breast-summary-draft-recommendations-2017.pdf). The focused update for in situ hybridization (ISH) addresses following uncommon scenarios: 1) HER2/CEP17 ratio ≥ 2.0, and HER2 /cell &amp;lt;4.0; 2) HER2/CEP17 ratio &amp;lt;2.0 and HER2 /cell ≥6.0; 3) HER2/CEP17 &amp;lt;2.0 and HER2 /cell ≥4.0 and &amp;lt;6.0. The first two groups are currently reported as positive, the third as equivocal. It is recommended that immunohistochemistry (IHC) performed by the same lab for all these groups. If the IHC testing is 3+ or 0/1+ the final diagnosis will be reported as positive or negative respectively. If the reflex IHC is 2+, a recount of the original ISH area of carcinoma with IHC 2+ staining is recommended. The purpose of this study is to review the results of targeted FISH testing following IHC to predict the effects of proposed guidelines in a high volume national reference lab. Materials and methods: HER2 FISH tests performed on BrCa between 4/2015-5/2017 are included. Our lab offers HER2 testing by IHC (HercepTest™ Dako) and dual probe FISH (Dako IQ). Equivocal (2+) cases showing ≥10% weak or moderate circumferential membrane staining or intense but &amp;lt;10% circumferential staining are circled by a pathologist and reflexed to HER2 FISH with preferential counting performed in the circled areas. Equivocal FISH cases are re-tested with the alternate RAI1 probe (Agilent Technologies). FISH scoring is done manually by 2 people following 2013 ASCO/CAP guidelines with at least 20 cells counted in amplified and non-amplified cases and 40 cells counted in equivocal cases. Results: 2460 HER2 FISH test requests were received during the study period. 7 cases failed at initial testing and 13 cases failed at repeat testing of equivocal cases with reflex probe. 389 (16.2%) cases were amplified, 1686 (68.7%) non amplified, and 369 (15.0%) were equivocal with FDA approved probe set. 116 (32.6%) of equivocal cases re-tested with alternate probe were amplified increasing overall amplification rate to 21%. The table below shows cases that fall under 3 uncommon categories that are proposed for changes in draft guidelines. HER2/CEP17 RatioAverage HER2/CellNumber of Cases with IHC (Total Number)IHC n(result)Results per 2013 ASCO/CAP before Reflex ProbeResults per 2013 ASCO/CAP after Reflex ProbeProposed Guidelines≥ 2&amp;lt; 48 (28)7 (2+)POSITIVE-NEGATIVE 1 (1+) &amp;lt; 2≥ 69 (31)9 (2+)POSITIVE-NEGATIVE &amp;lt; 2≥ 4-698 (372)96 (2+)EQUIVOCAL32/98 (32.7%) POSITIVE1/98 (1%) POSITIVE 1 (3+) 1 (0) Conclusions: In our lab where all FISH tests with prior IHC are counted on targeted areas, all of the cases within the first two groups will be reclassified as negative. The use of reflex probe in equivocal cases is not recommended in the draft recommendations further decreasing positivity rates. Approximately 5% decrease is expected in HER2 FISH positivity rates following proposed recommendations in reference laboratory setting. Citation Format: Johnson E, Gulbahce E. Draft recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer will decrease HER2 positivity rates [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-06.

Abstract P6-03-01: Variables influencing HER2-positivity in breast cancer: Assessment and validation of a statistical model based on two multicenter noninterventional studies in Germany

Abstract Background: HER2 testing in breast cancer (BC), routine for &amp;gt;10 years, allows selection of patients (pts) for HER2-targeted therapy; however, testing quality remains a concern. While guidelines recommend assessment of HER2-positivity rates as a quality indicator, the influence of patient- or tumor-related factors on variability was unknown until we identified the effect (in order of influence) of histologic grade, hormone receptor (HR) status, histologic subtype, age, and nodal status in a large, multicenter, observational study in Germany (NIU HER2 study; Rüschoff et al., Mod Pathol 2017). Based on these variables and the statistical model developed, potential issues with HER2 testing quality in local practice may be identified. We now report interim analyses from a multicenter study in Germany (EPI HER2 BC study; NCT02666261), where data from the NIU and EPI studies were compared and the validity of the NIU study model assessed. Methods: Routine HER2 test results and patient- and tumor-related data were collected from eligible pts with BC. Factors influencing HER2-positivity rates in the EPI study were compared with those identified in the NIU study. The predictive power of the NIU study model, fitted to EPI data, was determined and assessments performed using the variable coefficients and cutoff resulting from the NIU study analysis. Attempts were also made to improve the model. Results: Analyses included 15281 (NIU) and 6019 (EPI) invasive BC samples. The distribution of relevant variables, including HER2-positivity rate (NIU: 14.4%; EPI: 13.5%), was comparable. When the NIU study model was fitted to EPI study data, all five covariates identified in the NIU analyses had a significant effect on HER2-positivity (p&amp;lt;0.001); the order of influence for covariates differed between studies (EPI [in order of influence]: histologic grading, histologic subtype, HR status, nodal status, and age). The relationship between HER2-positivity rate and the combined influence of covariates, visualized with the NIU study prediction profiler, was reproduced with EPI study data. The NIU study statistical model, with variable coefficients and cut-point determined in the NIU study, was used to predict the HER2-positivity of samples in EPI; if their NIU model-estimated probability of positivity was &amp;gt;0.1407, the resulting sensitivity, specificity, and receiver operating characteristic (ROC) area under the curve (AUC) were 0.7032, 0.6622, and 0.7259, respectively. Thus, initial validation of the NIU study model with EPI data was successful. Semiquantitative estrogen and progesterone receptor expression data were available from EPI only; their inclusion as independent continuous, rather than categorical, variables improved the model (ROC AUC = 0.7533). Conclusions: The statistical modeling approach used to analyze data from the NIU study showed that patient- or tumor-related characteristics should be considered when assessing HER2 testing quality. Our present analysis validates and improves upon this statistical model and further highlights the need to assess HER2 testing quality in BC. Comparison of calculated vs actual positivity rates may help identify centers with potential HER2 testing quality issues. Citation Format: Rüschoff J, Lebeau A, Kreipe H, Gerharz CD, Sinn P, Schildhaus H-U, Tennstedt-Schenk C, Ammann JU, Künzel C, Koch W, Untch M. Variables influencing HER2-positivity in breast cancer: Assessment and validation of a statistical model based on two multicenter noninterventional studies in Germany [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-03-01.

Comparative analysis of the EGFR, HER2, c-MYC, and MET variations in colorectal cancer determined by three different measures: gene copy number gain, amplification status and the 2013 ASCO/CAP guideline criterion for HER2 testing of breast cancer

BackgroundThe purpose of this study was to explore gene copy number (GCN) variation of EGFR, HER2, c-MYC, and MET in patients with primary colorectal cancer (CRC).MethodsDual-colour silver-enhanced in situ hybridization was performed in tissue samples of 334 primary CRC patients. The amplification status (GCN ratio ≥2) and GCN gain (average GCN ≥4) data for the EGFR, HER2, c-MYC and MET genes were obtained. GCN variation was also assessed by the criterion of the 2013 ASCO/CAP guidelines for HER2 testing.ResultsAmplification of EGFR, HER2, c-MYC and MET was detected in 8 (2.4%), 20 (6.0%), 29 (8.7%), and 14 (4.2%) patients, respectively. Of 66 patients with at least one amplified gene, five exhibited co-amplification of genes studied (HER2-MET co-amplification: two patients; HER2-c-MYC co-amplification: two patients; EGFR-c-MYC co-amplification: one patient). There were 109 patients with GCN gains of one or more genes (EGFR: 11/334, HER2: 29/334, c-MYC; 60/334, MET: 48/334) and 32.1% (35/109) had multiple GCN gains. When each GCN was assessed by the criterion of the ASCO/CAP 2013 guideline for HER2 testing, 116 people showed positive or equivocal results for one or more genes. The cumulative amplification status had no association with patients’ outcome. However, the cumulative results of the GCN gain and GCN status determined according to the ASCO/CAP guideline had a significant prognostic correlation in the univariate analysis (P values of 0.006 and 0.022, respectively). In the multivariate analysis, GCN gain and GCN status were independent prognostic factors (P values of 0.010 and 0.017, respectively).ConclusionsIn this study, we evaluated GCN variation of four genes in a large sample of Korean CRC patients. The amplification status was not related to patient outcome. However, the GCN gain and GCN status according to the ASCO/CAP 2013 guideline were independent prognostic factors.

Analysis of molecular subtypes for the increased HER2 equivocal cases caused by application of the updated 2013 ASCO/CAP HER2 testing guidelines in breast cancer.

Accurate testing of the status of human epidermal growth factor receptor type 2 (HER2) is a prerequisite for HER2-directed therapy. The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) published joint guideline recommendations for HER2 testing in breast cancer in 2007 and it was updated in 2013. We compared the HER2 gene amplification status based on these two guidelines and analyzed the molecular characteristics of the equivocal cases. A total of 1894 patient samples were analyzed for both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). HER2 FISH amplification was examined and re-assessed using 2013 guidelines. According to the 2013 ASCO/CAP recommendations, 763 (40.3%) cases were classified as HER2 positive compared with 729 (38.5%) cases defined by 2007 guidelines. There was a significant increase of 6.1% in the proportion of HER2 FISH equivocal cases that were interpreted using ASCO/CAP 2013 (7.3%) compared with 2007 (1.2%) guidelines (P<0.001). Of 138 FISH equivocal cases defined by 2013 guidelines, 125 cases were IHC2+ and 13 cases were IHC1+. These 125 cases included 4 double equivocal cases which were defined as equivocal by both 2007 and 2013 guidelines and 121 cases whose status was changed from negative defined by 2007 guidelines to equivocal defined by 2013 guidelines. Compared with luminal A type and luminal B type respectively, these 121 equivocal cases demonstrated no significant difference with luminal B type in T stage and N stage (P=0.192, P=0.421). When we divided the luminal B type into two parts that included HER2 negative cases and HER2 positive cases, the equivocal cases also showed no significant difference with these two subtypes in T stage and N stage. Our study suggested that implementation of the revised ASCO/CAP 2013 guidelines resulted in an increase of 1.7% in overall HER2 positivity rate and of 6.1% in equivocal cases. Pathological analysis revealed that these equivocal cases exhibit similar biological behavior with luminal B type tumors. Clinical utility data on targeted therapy in equivocal patients should be further investigated.