Fluorescence In Situ Hybridization Amplification Research Articles

Evaluation of HER2 immunohistochemistry-positive and immunohistochemistry-negative FISH amplification breast cancers using next-generation sequencing

Abstract Objectives In this study, we aimed to assess the concordance between HER2 positivity, defined as scores of 2+ or 3+ by immunohistochemistry (IHC), and HER2 gene amplification results obtained by fluorescence in situ hybridization (FISH) and gene panel testing (GPT). Methods A total of 45 breast cancer patients with sufficient biopsy, breast-conserving surgery, or mastectomy specimens collected between 2018 and 2024 at Hokuto Hospital were included in the study. HER2 status was defined as HER2+ in cases with scores of 3+ on IHC staining, HER2-FISHamp in cases with scores of 2+ on IHC staining plus FISH amplification. FISH amplification was defined as a ratio of HER2 to the amplification of the chromosome 17 centromere ≥2.0. Next-generation sequencing (NGS) for these genes was conducted using MiSeq. The criterion for ERBB2 amplification requires gene amplification of more than 2 standard deviations (SD) above the average of the panel genes and CN ≥4. Results HER2+ correlated with the presence of NGS amplification of ERBB2 (p<0.001). NGS amplification was significantly more detectable in HER2+ than in HER2-FISHamp. HER2-FISHamp correlated with larger tumor size (p=0.009) and the presence of lymph node metastases (p<0.001). No correlations were observed between the HER2 status and other parameters such as age, histological grade, stage histological type, recurrence, treatment response evaluation, and breast cancer mortality. Conclusions To achieve a comprehensive diagnosis, it is crucial to integrate GPT findings with other diagnostic approaches and to have a though understanding of the characteristics and limitations of each method.

Clinical application of the HM-1000 image processing for HER2 fluorescence in situ hybridization signal quantification in breast cancer

BackgroundAccurate quantification of human epidermal growth factor receptor 2 (HER2) gene amplification is important for predicting treatment response and prognosis in patients with breast cancer. Fluorescence in situ hybridization (FISH) is the gold standard for the diagnosis of HER2 status, particularly in cases with equivocal status on immunohistochemistry (IHC) staining, but has some limitations of non-classical amplifications and such cases are diagnosed basing on additional IHC and FISH. This study investigated the clinical utility of a novel super-resolution fluorescence microscopy technique for the better FISH signal visualization and HER2 FISH classification.MethodsFourteen breast cancer tissue samples were retrospectively collected between September 2018 and February 2022, and FISH HER2 signal quantification was evaluated by determining the HER2/chromosome 17 centromere (CEP17) ratio and the number of HER2 signals per nucleus in super- versus conventional-resolution images.ResultsSuper-resolution images maintained the same overall HER2 diagnosis from routine, but HER2 FISH amplification changed negative to monosomy in two cases. Two Letrozole non-response relapses coincided to monosomy samples. The median number of HER2 signals per nucleus was 7.5 in super-resolution images and 4.0 in conventional-resolution images in HER2-positive samples and 2.8 and 2.1 signals per nucleus, respectively, in HER2-negative samples.ConclusionsSuper-resolution images improved signal visualization, including a significant difference in the number of countable HER2 and CEP17 signals in a single nucleus compared with conventional-resolution images. Increased accuracy of signal quantification by super-resolution microscopy may provide clinicians with more detailed information regarding HER2 FISH status that allows to better FISH classification such as HER2-low samples.

MDM2 FISH testing criteria in adipose tissue tumors with mature adipocytic morphology-A resection case-based study

The current criteria for utilizing MDM2 Fluorescence In Situ Hybridization (FISH) in adipose-derived tumors were first introduced in 2015 and have been widely adopted. However, these criteria may fail to identify some atypical lipomatous tumors / well-differentiated liposarcoma (ALT/WDL) with mature adipocytic morphology in clinical practice, possibly due to the fact that the existing criteria are primarily based on biopsy cases. Hence, a criterion based on resection cases is needed. In this study, we included 87 adipose tissue tumors with mature adipocytic morphology which were first resected, as well as 9 consultation cases and 25 recurrent resection cases. The final diagnosis was based on MDM2 amplification status. Among the 87 first-time resection cohort, MDM2 FISH amplification was observed in only 2 (5%) of the 39 superficial cases. Marginal infiltration was significantly different in both the MDM2 FISH negative and positive groups (p<0.05). Of the 37 intramuscular tumors, 17 (46%) showed MDM2 FISH amplification. The MDM2 amplification positive group had a larger tumor size than MDM2 amplification negative group (p=0.042). Tumors of larger size (≥11cm) were highly correlated with MDM2 amplification (p=0.003), but still, 35.3% of the MDM2 amplification-positive cases had tumor sizes less than 11cm. Eight (66.7%) out of twelve retroperitoneal/ pelvic cases were MDM2 FISH positive. Among the 25 recurrent cases, twenty (80%) of them had MDM2 FISH amplification. In conclusion, we recommend MDM2 FISH for: 1. superficial cases with marginal infiltration based on adequate margin sampling; 2. all intramuscular tumors, retroperitoneal/pelvic tumors and recurrent tumors, both in resection cases and biopsy cases.

Changes of HER2 low expression status in primary and recurrent/metastatic breast cancer

Objective: To investigate the evolution and clinical significance of HER2 low expression status in HER2 negative patients in primary and recurrent/metastatic breast cancers. Methods: The data and archived sections of 259 breast cancer patients with recurrence/metastasis and HER2-negative primary foci were collected from January 2015 to January 2022 at the Fourth Hospital of Hebei Medical University, and the HER2 status of primary and recurrence/metastasis foci was determined by immunohistochemistry (IHC), among which IHC 2+patients were subject to fluorescence in situ hybridization (FISH). The HER2 status was classified as HER2-0 group; patients with IHC 1+, IHC 2+and no FISH amplification were classified as HER2 low expression group; and patients with IHC 3+, IHC 2+and FISH amplified were classified as HER2-positive group. The changes of HER2 status in patients with HER2 low expression in primary versus recurrent/metastatic breast cancer foci were compared, and their clinicopathologic characteristics and prognosis were analyzed. Results: The overall concordance rate between primary and recurrent/metastatic HER2 status in breast cancer was 60.6% (157/259, κ=0.178). A total of 102 patients (102/259, 39.4%) had inconsistent primary and recurrent/metastatic HER2 status; 37 patients (37/259, 14.3%) had HER2-0 at the primary foci and HER2-low expression at the recurrent/metastatic; and 56 patients (56/259, 21.6%) had HER2-low expression in the primary foci and HER2-0 in the recurrent/metastatic. The recurrent/metastatic foci became low-expressing compared with the recurrent/metastatic foci which remained HER2-0 patients, with longer overall survival time, higher ER and PR positivity, lower Ki-67 positivity index, and lower tumor histological grade; all with statistically significant differences (all P<0.05). In the primary HER2-low group, patients with recurrent/metastatic foci became HER2-0 while those with recurrent/metastatic foci remained low expression; there were no statistically significant differences in clinicopathological features and overall survival time (all P>0.05). Conclusions: Unstable HER2 status in patients with HER2-0 and low expression in primary versus recurrent/metastatic breast cancer foci, and HER2-0 in the primary foci but low HER2 expression status in recurrence/metastasis is associated with favourable prognosis, and testing HER2 status in recurrence/metastasis can provide more treatment options for such patients.

Potential impact of HercepTest™ mAb PharmDx (Dako Omnis) (ge001) in breast cancer diagnosis.

TheHER2 gene is abiomarker for breast cancer prognosis and treatment. Overexpression ofHER2 protein determined by immunohistochemistry (IHC) or amplification oftheHER2 gene determined by fluorescence in situ hybridization (FISH) is acondition for qualifying patients for anti-HER2 therapy. Due to thehigh toxicity ofanti-HER2 treatment, proper patient selection is essential. In our study we compared 40 cases with IHC staining ofHER2 antibody determined by Ventana PATHWAY anti-HER2/neu antibody (4B5) as HER2 2+ with thenew antibody (HercepTest™ mAb PharmDx [Dako Omnis] [GE001]). Then using adouble-blind study we compared the(IHC) evaluation with FISH results. In 65% ofcases (26/40) theIHC 2+ score remained unchanged, in 32.5% ofcases (13/40) expression ofHER2 protein after IHC with new antibody was indicated as 3+ score, and in one case we observed adecrease ofHER2 protein expression to 1+. In all cases but one, in which we found IHC HER 3+ with new antibody, there was FISH amplification. We have reason to believe that thenew antibody will reduce thediagnostic time and avoid unnecessary costs. Due to thesmall study group, further investigation is needed.

Abstract PS10-33: Analysis of factors associated with pathological complete response (pCR) in patients with HER2+ breast cancer receiving neoadjuvant chemotherapy

Abstract Background: Pathologic complete response (pCR) following neoadjuvant chemotherapy (NAC) is associated with improved survival outcomes among women with HER2 positive (HER2+) breast cancer. We aimed to analyze the clinical, pathological and molecular factors associated with pCR in our series of HER2+ breast cancer patients. Methods: Between 2009 and 2019, breast cancer patients receiving NAC at our centre were enrolled in a prospective database. For this study we selected women with HER2+ unilateral breast cancer stages II or III who received anthracycline-taxane NAC regimens (with trastuzumab given concurrently with the taxane) and subsequently underwent breast and axillary surgery with curative intent. Medical charts of these 161 women were retrospectively reviewed to determine what clinical, pathological and molecular characteristics were associated with pCR using bivariate and multivariate analysis. Results: Of the 161 HER2+ women median age at diagnosis was 49 years (range 25-78) with 90 (56%) women under age 50. Total of 104 (65%) tumors were stage II, 108 (67%) expressed estrogen and/or progesterone receptors, 139 (86%) had HER2 3+ expression by immunohistochemistry (IHC) and 22 (14%) had HER2 IHC 2+ expression with amplified HER2 on fluorescence in situ hybridization (FISH) test. The NAC regimens were: Doxorubicin-Cyclophosphamide-Paclitaxel for 83 (52%) and Fluorouracil-Epirubicin-Cyclophosphamide-Docetaxel for 78 (48%) women. A total of 73 patients [45%; 44/108 HR+ (41%) and 29/53 HR- (55%)] achieved pCR (p=0.94). pCR rate among HER2 IHC 3+ tumors was 69/139 (50%), while for HER2 IHC 2+ tumors was 4/22 (18%). On bivariate analysis HER2 IHC 3+, absence of estrogen receptors and absence of progesterone receptors were predictive of pCR, while age, menopausal status, histologic subtype, tumor grade and NAC regimen were not. In the multivariate model only HER2 IHC 3+ expression remained a significant predictor of pCR (OR =4.443, 95% CI 1.376 - 14.344). Conclusion: Our analysis suggests that for HER2+ breast cancers treated with NAC with anthracycline-taxane and trastuzumab, HER2 IHC 3+ expression is associated with a higher rate of pCR compared to HER2 IHC 2+ expression with FISH amplification. Further analysis in a larger cohort is warranted. Citation Format: Neda Stjepanovic, Katarzyna Jerzak, Maureen Trudeau, Andrea Eisen, Sonal Gandhi, Ellen Warner, Danilo Giffoni MM Mata, Anthony Lott, Maria Romero, Sharon Lemon-Wong, Althea VanMassop, Xingshan Cao, William Tran, Rossanna Pezo. Analysis of factors associated with pathological complete response (pCR) in patients with HER2+ breast cancer receiving neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-33.

HER-2/neu over expression in gall bladder adenocarcinoma: A quest for potential therapeutic target.

Gall bladder carcinoma (GBC) is an aggressive malignancy with high mortality and aggressive course, with palliation as the only available option. To evaluate frequency of HER-2/neu overexpression in GBC and to seek its correlation, if any with conventional clinicopathological parameters and survival. Immunohistochemistry (IHC) was performed on 200 cases of GBC, 32 cases of dysplasia, and 100 cases of chronic cholecystitis. Fluorescent in situ hybridization (FISH) was performed on 30 randomly selected cases of GBC to validate IHC. HER-2/neu overexpression (IHC 3+/FISH amplification ≥2.2) was correlated with clinicopathological parameters by Chi-square test.P < 0.05 was considered significant. Survival analysis was done by log-rank test and Kaplan-Meier analysis. HER-2/neu overexpression was seen in 14% (28/200) GBC cases but was not found in dysplasia and chronic cholecystitis. Majority of these cases were ≤grade 2 and in advanced stage, however this was not statistically significant. A lower mean survival in HER-2/neu positive group as compared to HER-2/neu negative group (17.1 ± 2.3 month versus 67.6 ± 8.5 month, respectively) was observed. Concordance between IHC and FISH was seen in 18/19 cases. This study delineates a subset of GBC patients with HER-2/neu overexpression, in whom targeted therapy can offer a survival benefit.

Granular cell tumors overexpress TFE3 without gene rearrangement: Evaluation of immunohistochemistry and break-apart FISH in 45 cases.

Transcription factor E3 (TFE3) is a useful marker for tumors with Xp11.2 translocation, including alveolar soft part sarcoma and renal cell carcinoma. Recently, TFE3 overexpression was also found in granular cell tumors (GrCTs). However, the case cohorts of these two studies were limited to only 11 and 6 cases. Whether aberrant TFE3 expression is a common feature of Asian patients with GrCT requires further investigation. In the present study, immunohistochemical staining and TFE3 break-apart fluorescence in situ hybridization (FISH) assay were performed in 45 samples of GrCTs obtained from Chinese patients recruited from three medical centers in northeast China. Diffusive and marked nuclear staining for TFE3 was identified in 11/45 (24%) cases, which was lower than previously reported. Focal or weak TFE3 staining was identified in 13/45 (29%) cases. The remaining 21 cases were negative stained. In addition, GrCTs in subcutaneous tissue exhibited a relatively higher ratio (8/45, 18%) for TFE3 expression, compared with those in other sites. Furthermore, according to FISH data, no rearrangement or amplification of TFE3 was identified in these cases, whether they were positively or negatively stained for TFE3. The results from the present study demonstrated that part of patients GrCTs exhibited TFE3 overexpression, which suggested that this may not be derived from gene rearrangement.

High Fidelity of Breast Biomarker Metrics: A 10-Year Experience in a Single, Large Academic Institution.

Recommendations for standardization of breast biomarkers including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) led to the creation of American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines to provide continuous guidance. Included in these recommendations is the "ongoing assay assessment procedures." We report these biomarker metrics as there is a dearth of published information on this topic. ER, PR, and HER2 positivity rates of all newly diagnosed, recurrent, and metastatic invasive breast cancers on core biopsies, and repeated testing on resection specimen by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) were collected from April 1, 2008 to December 31, 2017. The positivity rates of ER, PR, and HER2 over almost 10 years of monitoring showed high fidelity. Total ER-positive rate was 83.6% (81.4% to 86.8%), ER+/PR+ was 71.7% (68.6% to 75.5%), ER+/PR- was 17.6% (11.0% to 15.0%), ER-/PR- was 16.0% (13.5% to 18.2%), and ER-/PR+ was 0.6% (0.2% to 1.0%). The HER2-positive rate was 13.7% (10.2% to 17.4%) including 9.9% (7.3% to 11.9%) by IHC and 3.8% (1.9% to 5.9%) by FISH reflexed from IHC 2+ results. FISH amplification rate of HER2 IHC 2+ cases was 11.0% (5.8% to 19.2%). Annual quality-assurance check for HER2 IHC/FISH percent positive and percent negative agreement (as defined by Food and Drug Administration) was 96% to 100%. This longitudinal active assessment of 9564 breast biomarker cases shows the achievement of high fidelity of breast biomarker results when following the ASCO/CAP guidelines. Continuous monitoring of breast biomarkers may minimize assay analytical drift and assure quality clinically relevant results.

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.

MET exon 14 splice site mutation, protein expression, and amplification in pulmonary sarcomatoid carcinoma: A multi-center study from China.

e20595 Background: MET exon 14 splice site mutation (MET exon 14Δ) was identified as a potential driver mutation in non-small cell lung cancer (NSCLC). Previously, several small sample size studies have revealed a higher frequency in pulmonary sarcomatoid carcinoma (PSC) than other types of NSCLC. In order to reveal the accurate frequency of MET gene alterations in Chinese PSCs, we comprehensively studied the frequency and the relationship among MET exon 14Δ, protein expression and amplification in a large cohort of PSCs. Methods: MET exon 14Δ, protein expression and amplification were detected in 148 surgically resected PSC samples from multiple medical centers by using Sanger sequencing, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. Results: MET exon 14Δ was identified in 9 (6.1%) of 148 cases. Six were pleomorphic carcinoma, two were spindle cell carcinoma and one was carcinosarcoma. MET exon 14Δ was more common in pleomorphic carcinoma compared with other subtypes of PSCs (p = 0.01, X2 test) and have an obvious tendency to occur in elder patients. MET protein overexpression was detected in 16.2% (24/148) of PSCs, and was found significantly associated with MET exon 14Δ (p = 0.045). MET FISH positive was detected in 15.5% (23/148), and 6 (4.1%) of them were confirmed as gene amplifications. MET FISH positivity (P &lt; 0.001, X2 test) and amplification (P &lt; 0.001, X2 test) were significantly associated with the IHC positive. No significant relationship between MET exon 14Δ and amplification was found. Conclusions: Chinese PSC has high incidence of METexon 14Δ and amplication. MET inhibition may benefit this specific subgroup of patients.

Change in Pattern of HER2 Fluorescent in Situ Hybridization (FISH) Results in Breast Cancers Submitted for FISH Testing: Experience of a Reference Laboratory Using US Food and Drug Administration Criteria and American Society of Clinical Oncology and College of American Pathologists Guidelines.

Purpose In 1998, the US Food and Drug Administration (FDA) approved human epidermal growth factor receptor 2 (HER2) testing guidelines to determine eligibility for HER2-directed therapy (HDT) in breast cancer. ASCO and the College of American Pathologists published immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) HER2 testing guidelines in 2007 (AC2007) and updated these guidelines in 2013 (AC2013). We compared the HER2 FISH amplification frequency using these three guidelines. Methods Patient samples that were sent to the Mayo Clinic cytogenetics laboratory for FISH testing (n = 2,851; from November 2013 to October 2014) were analyzed. Frequency of HER2 FISH amplification was examined and impact of AC2013 assessed. Results IHC results were available for 1,922 patient samples (67.4%), 137 of which were from Mayo Clinic. Distribution was 2.4% IHC 0, 7.9% IHC 1+, 84.8% IHC 2+, and 2.5% IHC 3+. Among IHC 2+ patients, HER2 FISH positivity was 11.8% (FDA), 9.4% (AC2007), and 24.1% (AC2013). Overall, 11.8% (n = 339) were positive with a FISH ratio ≥ 2.0, 1.3% (n = 35) with a FISH ratio ≥ 2.0 despite a HER2 signal < 4.0, and 3.0% (n = 86) with HER2 signal ≥ 6.0 despite FISH ratio < 2.0. Among 405 patients (14.2%) who were initially considered FISH-equivocal (ratio < 2.0 with HER2 signal ≥ 4.0, but < 6.0; AC2013), use of an alternative chromosome 17 probe reassigned 212 (7.4% overall) patients to FISH-positive and 36 (1.3% overall) patients to FISH-negative, whereas 157 (5.5% overall) patients remained equivocal. Final HER2 positivity with AC2013 (23.6%) was increased (P < .001) compared with FDA (13.1%) and AC2007 (11%) guidelines. Conclusion In a reference laboratory cohort that was highly enriched for IHC 2+ patient samples, AC2013 guidelines led to a larger number of FISH-equivocal patients. Approximately one half of these FISH-equivocal patients (7.4% overall) became HER2-positive upon alternative FISH probe testing. However, these patients would not have participated in the pivotal HDT trials. Clinical utility data on HDT benefit in these patients and other special subsets are needed.

Validity of immunohistochemistry method in predicting HER-2 gene status and association of clinicopathological variables with it in invasive breast cancer patients.

Human epidermal growth factor receptor-2 is an important and prognostic factors and one of the most targeted proteins in breast cancer's therapy. There is no globally accepted method for determining its status. Here, we aimed to evaluate the immunohistochemistry method validity in predicting HER-2 status by Fluorescence in situ hybridization method and investigate some clinicopathological variables association with HER-2 amplification. A total of 190 HER-2 2+ and 3+ by immunohistochemistry (IHC) invasive breast cancer cases were enrolled in this study. Fluorescence in situ hybridization (FISH) was performed for these cases using FDA criteria and the association between clinicopathological variables and HER-2 status evaluated. Study consisted of 190 invasive breast cancer patients (160 HER-2 2+ and 30 HER-2 3+). HER-2 FISH amplification according to FDA criteria was found 27.5% (44/160 patients) in HER-2 2+ patients and 83.3% (25/30 patients) in HER-2 3+ patients. Tumors with HER-2 amplification were more likely to be ER-negative (51.0% vs 31.2%, p = 0.013) and PR-negative (52.9% vs 27.0%, p < 0.001). This study showed that immunohistochemistry is not a good method for evaluating HER-2 status and decision-making about trastuzumab therapy even with 3+ score patients. However, this result may not be too strong for IHC 3+ cases due to the limited number of these patients in this study.

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.

HER2 as a target in invasive urothelial carcinoma.

We evaluated primary tumors from two cohorts, Spain (N = 111) and Greece (N = 102), for patients who were treated with platinum-based chemotherapy. Patients were tested for HER2 status (IHC score of 3+ or FISH ratio of ≥2.2) by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), DNA copy number, mRNA expression, and mutation status in patients with metastatic urothelial carcinoma (UC), and its impact on survival. ERBB2 mutation was determined by hotspot sequencing. mRNA expression was assessed using NanoString counting. Association of overall survival (OS) and HER2 status was assessed by a Cox regression model. NIH-3T3 cells containing HER2 V777L were assessed for growth, invasion, and HER2 kinase activation. In all, 22% of Spanish and 4% of Greek cohorts had 3+ HER2 staining by IHC. FISH amplification was identified in 20% of Spanish and 4% of Greek cohorts. Kappa coefficient between FISH and IHC was 0.47. HER2 status was not associated with OS in univariate (Spanish P = 0.34; Greek P = 0.11) or multivariate (Spanish P = 0.49; Greek P = 0.12) analysis. HER2-positive tumors expressed higher levels of HER2 mRNA than HER2-negative tumors (P < 0.001). HER2 mutations (V777L and L755S) were identified in two (2%) patients. In vitro analysis of V777L results in transformation of NIH-3T3 cells, leading to increased growth, invasion on soft agar, and HER2 kinase constitutive activation. In summary, HER2 overexpression or amplification in the primary tumor did not predict OS in patients with metastatic UC. HER2 positivity rates can differ between different populations. Further trials in genomically screened patients are needed to assess HER2-targeted therapies in UC.

Abstract 2840: Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology

Abstract With the advent of multiple HER-2 targeting agents, the need for a diagnostic test that accurately predicts the levels of HER2 in newly diagnosed breast cancer patients has never been greater. The goal of this study was to develop an immunofluorescence based assay to objectively and reproducibly quantify HER-2 protein level in Formalin-Fixed Paraffin-Embedded (FFPE) specimens using AQUA Technology and to provide a binary readout, thus eliminating reflex testing and enabling quick treatment decisions. Methods: Three well known HER-2 antibody clones, A0485, CB11, and SP3 were evaluated across a three-log dilution series under four different antigen retrieval conditions on a tissue microarray (TMA) containing 80 breast cancer cases with known central HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) amplification status. The top six immunofluorescence assay combinations out of 108 conditions were quantified by AQUA scoring based on the their ability to accurately separate negative and positive cases using receiver operating characteristic (ROC) analysis. These top six assay conditions were then assessed on an independent TMA containing 94 breast cancer cases and their AQUA scores were compared to HER2 protein levels determined by reverse phase protein microarray (RPMA®), HER2 mRNA levels assessed by RNAscope® as well as to the current “gold standard” IHC and FISH amplification. Results: Top three conditions clearly segregating HER2 positive and HER2 negative breast cancer cases were selected and analyzed on an independent TMA. Conditions associated with both SP3 and A0485 antibodies demonstrated high correlations with both HER2 protein levels determined by RPMA analysis and HER2 mRNA levels assessed by RNAscope (spearman's rho &amp;gt;0.85). Conclusion: Assessment of HER2 status using AQUA Technology was confirmed using two expression platforms, including both protein (RPMA) and RNA (RNAscope) supporting the utilization of the AQUA methodology for diagnostic test development. Citation Format: Jennifer Bordeaux, Krupa Chandrasekaran, Sue Beruti, Mike Nerenberg, Corinne Ramos, David Rimm, Jelveh Lameh, Naveen Dakappagari. Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2014-2840

Clinicopathological variables predicting HER-2 gene status in immunohistochemistry-equivocal (2+) invasive breast cancer.

Human epidermal growth factor receptor-2 (HER-2) gene status is crucial to guide treatment decisions regarding the use of HER-2-targeted therapies in breast cancer. An invasive breast cancer with HER-2 2+ score is regarded as HER-2 status equivocal and should further determine by fluorescent in situ hybridization (FISH), which is considered the standard test for HER-2 status. Here, we aimed to establish a risk score to allow for prediction of the presence of HER-2 gene status. A total of 182 HER-2 2+ by immunohistochemistry (IHC) invasive breast cancer cases were enrolled in this study. The association between clinicopathological variables like age, sex, tumor grade, hormone receptor (HR) status, P53 and proliferation index (Ki67), and FISH result using US Food and Drug Administration (FDA) criteria was evaluated. Also, we compared the HER-2 FISH results using FDA criteria and 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline. The study population had a median age of 48 years (range, 29-78 years). Estrogen receptor (ER) was expressed in 131 (72.0%) patients. 73.1% of patients (133/182) were progesterone receptor (PR) positive. The median Ki67 value was 20% (range, 3-90%). There was good agreement between the FDA and 2013 ASCO/CAP guideline. Sixty-three of all patients were HER-2 FISH amplified (positive) based on FDA criteria. Tumors with HER-2 amplified were more likely to harbor ER negative (58.8% vs. 25.2%, P<0.001) or PR negative (57.1% vs. 26.3%, P<0.001) or P53 negative (44.8% vs. 29.8%, P=0.048). A significant high level of Ki67 was detected in HER-2 amplified groups (P=0.006). We created a risk score that comprised HR, P53 and Ki67. A significant association between risk score and HER-2 FISH amplification was observed (χ(2)=30.41, P<0.001). This novel immunohistochemical risk score could be highly useful to predict the presence of HER-2 gene status in invasive breast cancer.

Abstract P3-05-01: Accurate assessment of HER2 status in “triple-negative” breast cancer requires both IHC and FISH testing

Abstract Introduction: Triple-negative invasive breast carcinoma (TNBC) does not over-express estrogen, progesterone receptors and HER2/neu. Approximately 15% breast cancers are “triple negative” and have poor overall survival with no targeted therapeutic options. HER2 positive patients also have a poor prognosis which is markedly improved with anti-HER2 therapy. ASCO/CAP guidelines have made substantial improvement in biomarker assessment by standardizing pre-analytic, analytic and post-analytic variables. Since 2009, we have performed HER2 fluorescence in situ hybridization (FISH) analysis in addition to immunohistochemistry (IHC) on all triple negative patients in an effort to accurately assess HER2 status. Materials and Methods: Pathology database at our institute was queried for triple negative breast cancer cases 2009-2012. 175 cases were identified with ER(SP1; Ventana)&amp;lt; = 1%, PR(1E2; Ventana)&amp;lt; = 1% and HER2 IHC (HercepTest® DAKO 2009-2011; 4B5-Ventana- 2012)&amp;lt; = 1+. FISH for Her2 was also performed on these cases. HER2/CEP17&amp;gt; = 2 was classified as HER2 positive. FISH for HER2 amplification was performed using the FDA-approved HER2 DNA Probe Kit (Vysis, Inc.). The hybridization results were recorded and analyzed by BioView Duet system (Allegro Plus Automated Scanner) with HER2 application software (BV-HER2-AF). Fisher's exact test was performed to correlate HER2 amplification with clinico-pathological parameters. Results: 175 breast cancers were classified as “triple-negative” by IHC between 2009-2012. The patients’ age ranged from 25 to 93 years old (mean = 58); 109 were Caucasian (62%), 33 African American (19%), 9 Asian (5%) and 4 Hispanic (2%). The majority of the cases (152, 86%) were invasive ductal carcinoma. The rest include 9 cases of medullary carcinoma, 3 cases of metaplastic carcinoma, 3 cases of apocrine carcinoma, 2 cases of papillary carcinoma and 2 cases of invasive lobular carcinoma. 34 had regional lymph node (axillary) metastasis (19%) and 9 had distant metastasis (5%). 23 of the 175 “triple-negative” cases (13%) showed HER2 amplification by HER2 FISH analysis (HER2/CEP17&amp;gt; = 2). 14.9% (20/134) TNBC cases were FISH positive 2009-11 (HercepTest®, DAKO) and 7.3% (3/41) cases were FISH positive in 2012 (4B5, Ventana). 14 of the 23 patients were Caucasian (61%), 4 African American (17%), 2 Asian and 1 Hispanic. 20 of the 23 cases (87%) had invasive ductal carcinoma, of which 18 (78%) were poorly differentiated. 8 of the 23 cases (35%) had lymph node metastasis and 1 case had distant metastasis. No statistically significant correlation was observed between the HER2 gene amplification and clinicopathological factors analyzed, including race (P = 1), age (P = 1), tumor size (P = 0.6), pathologic stage (P = 0.6), lymph node status (P = 0.08), and Ki-67 index (P = 0.8 with 20% cutoff; P = 0.1 with 70% cutoff). Conclusion: In this cohort, 13% TNBC were re-classified as HER2 positive by performing HER2 FISH test in addition to immunohistochemistry analysis. Rate of FISH positivity in TNBC declined over the four year study period. No clinicopathological variables are correlated with HER2 FISH amplification. These data suggest that dual testing of HER2 should be considered routinely in TNBC for accurate classification to guide therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-01.

Evaluation of serum HER2 extracellular domain in in metastatic gastric or gastroesophageal junction cancer: Correlation with HER2 status by immunohistochemistry and fluorescence in situ hybridization and clinicopathologic parameters.

4107 Background: To explore the association between serum human epidermal growth factor receptor-2 (HER2) extracellular domain (ECD) and tissue HER2 status, their relationship with clinicopathological parameters and impact on overall survival (OS) in metastatic gastric or gastro-oesophageal junction (GEJ) adenocarcinoma. Methods: A total of 219 histologically confirmed inoperable locally advanced, recurrent, or metastatic gastric or GEJ adenocarcinoma were included. Serum HER2 ECD was measured by chemiluminescent assay. Tissue HER2 status was accessed by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) assay. Results: The median serum ECD level was 9.3 ng/ml (range 3.0 to &gt;350). Tissue HER2 status was positive (IHC3+ or 2+ with FISH amplification) in 37 patients (16.9%) and negative in 182 patients (83.1%). Statistically significant associations were found between serum HER2 ECD level and HER2 status assessed by IHC and FISH. The ROC-analysis suggested the cutoff of 16.35 ng/ml (24 of 219 patients had HER2 ECD &gt;16.35 ng/ml ) could produce a sensitivity of 51.4% and a specificity of 97.3% to predict tissue HER2 status. If the cutoff value was increaseed to 22 ng/ml, then all 12 patients with serum HER2 ECD &gt;22 ng/ml were HER 2 positive in primary tumor, corresponding to a specificity of 100% and a sensitivity of 32.4%. High serum HER2 ECD levels were strongly associated with liver metastasis (P &lt; 0.001), the intestinal histologic type (Lauren's classification) (P =0.003), large number of metastais (&gt;2) (P=0.012) and increased LDH level (P &lt; 0.001) . High serum HER2 ECD levels were more common in GEJ adenocarcinoma although the difference had no stastical significance. HER2 ECD levels did not show a significant impact on OS. Conclusions: A significant association was observed between serum HER2 ECD levels and tissue HER2 status in metastasis gastric or GEJ adenocarcinoma. The high specificity of serum HER2 ECD assay to predict tissue HER2 status suggests its potential use as a surrogate marker of the HER2 status in gastric cancer.

Multidimensional investigation of HER2 in advanced urothelial carcinoma (UC).

4580 Background: Incidence of Her2 positivity and association with overall survival (OS) are controversial in advanced UC. Activating Her2 mutations have been identified in other cancers, but they have not been previously reported in UC. We determined Her2 status by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and copy number gain (CNG) via array CGH of primary UC tumors from patients (pts) with metastatic disease. Targeted Her2 sequencing was performed at known mutation hotspots, and mutation effect was investigated in vitro. Methods: Tissue microarrays of formalin fixed paraffin-embedded tumor from 98 UC pts treated with platinum-based combination chemotherapy for metastatic disease were evaluated for Her2 protein and for Her2 gene amplification by using standard clinical protocols. Positive staining was defined as an IHC score of 3+ or a FISH ratio of ≥2 using scoring criteria established for evaluation of breast cancer. Her2 CNG was evaluated by aCGH with cutoff log base 2 ratio &gt; 0.9. Mutation status was validated by hME sequencing. OS was measured from start of treatment for metastatic disease. Association of OS and Her2 status was assessed by a Cox regression model. NIH-3T3 cells with Her2 V777L were assessed for growth, invasion, and Her2 kinase activation. Results: 22% of pts had 3+ Her2 staining by IHC. 21% of pts had FISH amplification. These were concordant in 78% of pts. CNG was identified in 16% and was concordant with FISH and IHC 85% and 88% of the time, respectively. Her2 status by any modality showed no significant association with OS in either univariate [HR=0.94, 95% CI: (0.52, 1.70), p=0.83] or multivariate [HR=1.12, 95% CI: (0.61, 2.06), p=0.72] analysis. Her2 mutations (V777L and L755S) were identified in 2 pts (2%). In vitro analysis of V777L results in transformation of NIH-3T3 cells, leading to increased growth, invasion on soft agar, and Her2 kinase constitutive activation. Conclusions: Her2 overexpression or amplification in the primary tumor does not predict OS in pts with metastatic UC. Other research has suggested that V777L sensitizes cells to lapatinib, while L755S leads to lapatinib resistance. These rare oncogenic Her2 mutations occur and may be therapeutic targets in selected pts.