Am J Clin Pathol. 2018 Oct 18.
Gibbons-Fideler IS, Nitta H, Murillo A, Tozbikian G, Banks P, Parwani AV, Li Z.
PMID: 30339245 | DOI: 10.1093/ajcp/aqy136
OBJECTIVES:
Either immunohistochemistry (IHC) or in situ hybridization (ISH) can be used to determine human epidermal growth factor receptor 2 (HER2) status. Breast cancers (BCs) with HER2 IHC-negative (IHC-) and ISH-amplified (ISH+) results have been rarely reported but not well studied. We investigated the frequency of HER2 IHC-/ISH+ BCs and their response to anti-HER2 neoadjuvant chemotherapy (NAC).
METHODS:
Seventeen BCs with HER2 IHC-/ISH+ results were identified from 1,107 consecutive invasive BCs (1.5%, 17/1,107).
RESULTS:
Gene protein assay confirmed the original HER2 IHC and ISH results. Increased HER2 RNA level was detected in HER2 IHC-/ISH+ cases compared with HER2 IHC-/ISH- cases. Eight patients had anti-HER2 NAC; three had pathologic complete response, and five had residual tumors.
CONCLUSIONS:
A small percentage of patients (1.5%) showed discordant HER2 IHC and ISH results (IHC-/ISH+) and would have lost the opportunity for potentially beneficial anti-HER2-targeted therapy if only HER2 IHC testing had been used."
PLoS One. 2014 May 30;9(5):e98528.
Seo AN, Kwak Y, Kim DW, Kang SB, Choe G, Kim WH, Lee HS.
PMID: 24879338 | DOI: 10.1371/journal.pone.0098528
This study aimed at determining the incidence and clinical implications of HER2 status in primary colorectal cancer (CRC). HER2 status was investigated in two retrospective cohorts of 365 consecutive CRC patients (cohort 1) and 174 advanced CRC patients with synchronous or metachronous distant metastasis (cohort 2). HER2 status was determined by performing dual-color silver in-situ hybridization (SISH), mRNA in-situ hybridization (ISH), and immunohistochemistry (IHC). The incidence of HER2 protein overexpression (IHC 2+/3+) was approximately 6% (22 of 365 in cohort 1; 10 of 174 in cohort 2). HER2 gene amplification was observed in 5.8% of the patients from cohort 1 and 6.3% of the patients from cohort 2. HER2 gene amplification was more frequently observed in CRCs located in the rectum than in the right and left colon (P = 0.013 in cohort 1; P = 0.009 in cohort 2). HER2 status, determined by IHC, ISH, and dual-color SISH, was not significantly associated with aggressive CRC behaviour or patients' prognosis in both the cohorts. Of the combined cohort with a total of 539 cases, the concordance rate was 95.5% between dual-color SISH and IHC detection methods. On excluding equivocally immunostained cases (IHC 2+), the concordance rate was 97.7%. HER2 mRNA overtranscription, detected by ISH, significantly correlated with protein overexpression and gene amplification (P<0.001). HER2 gene amplification was identified in a minority of CRC patients with high concordance rates between dual-color SISH and IHC detection methods. Although HER2 status did not predict patients' prognosis, our findings may serve as a basis for future studies on patient selection for HER2 targeted therapy.
PLoS One. 2014 Jun 26;9(6):e99131.
Vassilakopoulou M, Togun T, Dafni U, Cheng H1, Bordeaux J, Neumeister VM, Bobos M, Pentheroudakis G, Skarlos DV, Pectasides D, Kotoula V, Fountzilas G, Rimm DL, Psyrri A.
PMID: 24968015 | DOI: 10.1371/journal.pone.0099131.eCollection2014.
BACKGROUND: We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients. METHODS: A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan - Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates. RESULTS: HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson's Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman's Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31-0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22-0.70, p = 0.002). CONCLUSIONS: The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.
Zhonghua Bing Li Xue Za Zhi.
Wu SF, Liu YY, Liu XD, Jiang Y, Luo YF, Cui QC, Liang ZY, Zeng X.
PMID: 29996317 | DOI: 10.3760/cma.j.issn.0529-5807.2018.07.008.
Objective: To investigate human epidermal growth factor 2 (HER2) gene status and in situ mRNA expression in breast cancers with immunohistochemistry(IHC) 1+ , and to reveal HER2 positive rate in these patients to provide reference data for obtaining precise HER2 results and modifying relevant clinical strategy to breast cancer. Methods: Sixty-five IHC 1+ formalin-fixed and paraffin-embedded samples of invasive breast carcinoma of no special type (IBC-NST) were collected by surgical operation at Peking Union Medical College Hospital during 2011 to 2013. HER2 status and in situ mRNA expression were tested by fluorescence in situ hybridization (FISH) and RNAscope, respectively, by using tissue microarray. Metastatic lymph node was re-tested by FISH if HER2 status was equivocal or negative and with high expression of mRNA in the primary lesion. Results: Four of 65 samples (6.2%) were FISH positive, which included 2 cases of HER2/CEP17>2 and average HER2 copy number>4 and 2 cases of HER2/CEP17<2 and average HER2 copy number>6. In the 4 samples of HER2 positive, 2 patients showed high in situ mRNA expression (3 scores by RNAscope), 2 patients showed moderate in situ mRNA expression (2 scores by RNAscope). In addition, 3 specimens with HER2/CEP17>2 and average HER2 copy number<4 were found in all patients, which included 2 cases of high in situ mRNA expression (3 and 4 scores by RNAscope) and 1 cases of moderate in situ mRNA expression (2 scores by RNAscope). There was no significant association between HER2 status or mRNA expression and clinicopathological characteristics, including tumor size, histopathological differentiation, lymph node metastasis and lymphovascular invasion (P>0.05). Conclusions: A small number of HER2 IHC 1+ patients exist mRNA expression by using FISH method, which suggested that these patients might benefit from anti-HER2 therapy potentially. Since the importance for patients with breast cancers to develop diagnostic and therapeutic strategies from accurate molecular typing, further studies based on a larger cohort are needed to validate our findings.
Seung BJ, Cho SH, Kim SH, Lim HY, Sur JH
PMID: 32059046 | DOI: 10.1371/journal.pone.0229031
Spontaneously occurring canine mammary gland tumors share many features with human breast cancer, including biological behavior and histologic features. Compared to transgenic murine model, canine models have advantages including naturally occurring models of human diseases and cancer. In humans, breast cancer is divided into molecular subtypes based on ER, PR, and HER2 expression. In contrast with humans, few studies have evaluated these subtypes in canine mammary gland tumors, including expression of HER2. HER2 expression in canine mammary tissues has been further complicated by controversy regarding the antibody's specificity. This study aimed to investigate c-erbB2 mRNA expression in retrospective formalin-fixed paraffin embedded samples, using RNA in situ hybridization with a novel quantitative assay and to compare this method with immunohistochemistry. Using 48 canine mammary tumor samples and 14 non-neoplastic canine mammary tissues, RNA in situ hybridization was performed with RNAscopeﾮ using a canine-specific target gene probe (ERBB2), and quantitative measurement was performed using the housekeeping gene (POLR2A) to calculate the target gene/housekeeping gene ratio. The ratio of ERBB2/POLR2A was quantified using open-source image analysis programs and compared with the immunohistochemistry results. A significant correlation was observed between the HER2 immunohistochemistry score and ERBB2/POLR2A RNA in situ hybridization (P < 0.001). When the HER2 immunohistochemistry score was 3+, significantly higher expression of HER2 mRNA was observed by RNA in situ hybridization. Interestingly, HER2 mRNA was also observed in non-neoplastic mammary tissues by RNA in situ hybridization. This assay potentially facilitates the reliable quantification of mRNA expression levels in retrospective formalin-fixed paraffin-embedded samples. Further studies are required to elucidate the role of HER2 in canine mammary gland tumors and to implement clinical trials in dogs
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Wang, S;Zhou, X;Niu, S;Chen, L;Zhang, H;Chen, H;Zhou, F;
PMID: 36841435 | DOI: 10.1016/j.modpat.2023.100148
As the most common type of HPV-independent (HPVI) endocervical adenocarcinomas (ECAs), gastric-type endocervical adenocarcinomas (GEAs) account for approximately 10% of all ECAs Although anti-HER2 therapy has been proven effective in many cancers, it has not been utilized in ECAs including GEAs, which is at least partly due to the lack of a well-defined guideline. Limited available data regarding HER2 in GEAs and ECAs have considerable variations likely caused by variations in tumor types selection, testing methods, and scoring criteria. Here, we selected 58 GEA cases to examine the HER2 status using IHC and FISH and to investigate the prognostic value and their association with other known or potential prognostic factors. When strong complete or lateral/basolateral membranous reactivity in ≥10% tumor cells was used to define HER2 positivity, relatively high prevalence of HER2 overexpression (17.2%, 10/58) and amplification (15.5%, 9/58), as well as high IHC-FISH concordance rate (90%, 9/10) was found in GEAs. A lateral/basolateral staining pattern ('U-shaped') was observed, at least focally, in the majority of HER2-positive (3+) and equivocal (2+) tumors. Notably, considerable heterogeneity of HER2 expression was observed in HER2 positive and equivocal cases (80.0% and 83.3%, respectively). HER2 overexpression and amplification were associated with worse progression-free survival (PFS) (p=0.047 and p=0.032, respectively). PD-L1 expression was associated with worse PFS (p=0.032), while mutant type p53 demonstrated no prognostic significance. Our work laid a solid foundation for the eventual development of a future standard HER testing guideline for GEAs.
Wilding, B;Scharn, D;Böse, D;Baum, A;Santoro, V;Chetta, P;Schnitzer, R;Botesteanu, DA;Reiser, C;Kornigg, S;Knesl, P;Hörmann, A;Köferle, A;Corcokovic, M;Lieb, S;Scholz, G;Bruchhaus, J;Spina, M;Balla, J;Peric-Simov, B;Zimmer, J;Mitzner, S;Fett, TN;Beran, A;Lamarre, L;Gerstberger, T;Gerlach, D;Bauer, M;Bergner, A;Schlattl, A;Bader, G;Treu, M;Engelhardt, H;Zahn, S;Fuchs, JE;Zuber, J;Ettmayer, P;Pearson, M;Petronczki, M;Kraut, N;McConnell, DB;Solca, F;Neumüller, RA;
PMID: 35883003 | DOI: 10.1038/s43018-022-00412-y
Oncogenic alterations in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of patients with non-small cell lung cancer and predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinical-grade tyrosine kinase inhibitors are limited by either insufficient selectivity against wild-type (WT) epidermal growth factor receptor (EGFR), which is a major cause of dose-limiting toxicity or by potency against HER2 exon 20 mutant variants. Here we report the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing WT EGFR, which reduce tumor cell survival and proliferation in vitro and result in regressions in preclinical xenograft models of HER2 exon 20 mutant non-small cell lung cancer, concomitant with inhibition of downstream HER2 signaling. Our results suggest that HER2 exon 20 insertion-driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing WT EGFR, paving the way for clinical translation.
Chinese Journal of Pathology
Shafei W, Yuanyuan L, Ying J, Yufeng L, Quancai C, Zhiyong L, Xuan Z.
PMID: - | DOI: -
Objective:
To investigate in situ mRNA expression of HER 2 oncogene in breast cancers with equivocal immunohistochemical results , and to explore the potential feasibility of RNAscope technique in evaluating HER2 status in breast cancers .Methods Sixty-nine FFPE samples of invasive ductal breast cancer with equivocal HER 2 immunohistochemistry results ( IHC 2+) were collected from surgical excisions from Peking Union Medical College Hospital between June 2010 and June 2013.HER2 status and in situ mRNA expression were tested by fluorescence in situ hybridization ( FISH) and RNAscope respectively using tissue microarray constructed from tumor paraffin blocks .The results of HER2 mRNA expression were scored 0 to 4 ( from low to high levels ) according to mRNA expression in 100 cancer cells .HER2 mRNA expression was evaluated in two groups of patients , with positive and negative FISH results .Results Twenty-three of the 69 samples were FISH positive, including 16 samples that were scored 4 by RNAscope (70%,16/23), 6 samples were scored 3 ( 26%,6/23 ) and one sample was scored 2 ( 4%,1/23 ) .High in situ mRNA expression (score 4 or 3) were observed in 96%of HER2 FISH positive samples.All of samples that were scored 4 by RNAscope were FISH positive .Forty-six samples were FISH negative , including 17 samples that were scored 3 by RNAscope (37%,17/46), 25 samples were scored 2 (54%,25/46), and 4 samples were scored 1 (9%,4/46).Conclusions Breast cancer with HER2 IHC 2 +could be further classified according to in situ mRNA expression status .Among them, RNAscope score of 4 could be one of the interpretation criteria for re-testing IHC 2+samples.In situ detection of HER2 mRNA may be an additional candidate method of confirmation for HER 2 gene amplification or protein overexpression , and has potential clinical utility.
Personalized therapeutic strategies in HER2-driven gastric cancer
Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association
Ughetto, S;Migliore, C;Pietrantonio, F;Apicella, M;Petrelli, A;D'Errico, L;Durando, S;Moya-Rull, D;Bellomo, SE;Rizzolio, S;Capelôa, T;Ribisi, S;Degiuli, M;Reddavid, R;Rapa, I;Fumagalli, U;De Pascale, S;Ribero, D;Baronchelli, C;Sgroi, G;Rausa, E;Baiocchi, GL;Molfino, S;Manenti, S;Bencivenga, M;Sacco, M;Castelli, C;Siena, S;Sartore-Bianchi, A;Tosi, F;Morano, F;Raimondi, A;Prisciandaro, M;Gloghini, A;Marsoni, S;Sottile, A;Sarotto, I;Sapino, A;Marchiò, C;Cassoni, P;Guarrera, S;Corso, S;Giordano, S;
PMID: 33755862 | DOI: 10.1007/s10120-021-01165-w
Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 "hyper"-amplified (≥ 8 copies) tumors. We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody-drug conjugates, in a selected subgroup of HER2 "hyper"-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody-drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-"hyper"-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.
Human pathology, 44(4):487–94.
Kim MA, Jung JE, Lee HE, Yang HK, Kim WH (2013)
PMID: 23084583 | DOI: 10.1016/j.humpath.2012.06.022.
The importance of anti-HER2 therapy has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. In the present study, we evaluated HER2 mRNA expression using a new mRNA in situ detection technique called RNAscope in 211 cases of formalin-fixed, paraffin-embedded gastric carcinoma. In addition, we compared the results with the conventional methods of immunohistochemistry, fluorescence in situ hybridization, and dual-color silver in situ hybridization. RNA in situ hybridization (in situ hybridization) showed that 162 cases (76.8%) were score 0, 5 cases (2.4%) were score 1, 10 cases (4.7%) were score 2, 13 cases (6.2%) were score 3, and 21 cases (10.0%) were score 4. HER2 transcription levels were found to be significantly related to pT class, pN class, and tumor recurrence. mRNA expression was well correlated with protein overexpression and gene amplification; 20 cases out of 23 with DNA amplification showed a score of 4 in RNA in situ hybridization (P < .001). Three cases showed false negative and one case showed false positive results by in situ hybridization. More studies are needed to determine whether the in situ hybridization method can identify additional patients that may benefit from anti-HER2 therapy or exclude those who may be resistant to anti-HER2 therapy.
The Journal of Molecular Diagnostics, 15(2), 210–219.
Wang Z, Portier BP, Gruver AM, Bui S, Wang H, Su N, Vo HT, Ma XJ, Luo Y, Budd GT, Tubbs RR (2013).
PMID: 23305906 | DOI: 10.1016/j.jmoldx.2012.10.003.
Patient management based on HER2 status in breast carcinoma is an archetypical example of personalized medicine but remains hampered by equivocal testing and intratumoral heterogeneity. We developed a fully automated, quantitative, bright-field in situ hybridization technique (RNAscope), applied it to quantify single-cell HER2 mRNA levels in 132 invasive breast carcinomas, and compared the results with those by real-time quantitative PCR (qPCR) and Food and Drug Administration-approved methods, including fluorescence in situ hybridization (FISH), IHC, chromogenic in situ hybridization, and dual in situ hybridization. Both RNAscope and qPCR were 97.3% concordant with FISH in cases in which FISH results were unequivocal. RNAscope was superior to qPCR in cases with intratumoral heterogeneity or equivocal FISH results. This novel assay may enable ultimate HER2 status resolution as a reflex test for current testing algorithms. Quantitative in situ RNA measurement at the single-cell level may be broadly applicable in companion diagnostic applications.
Predictive significance of HER2 intratumoral heterogeneity, determined by simultaneous gene and protein analysis, for resistance to trastuzumab-based treatments for HER2-positive breast cancer
Virchows Archiv : an international journal of pathology
Horii, R;Nitta, H;Nojima, M;Maruyama, R;Ueno, T;Ito, Y;Ohno, S;Banks, P;Kanda, H;Akiyama, F;
PMID: 33496805 | DOI: 10.1007/s00428-021-03036-2
Gene-protein assay (GPA), a combination of immunohistochemistry and dual in situ hybridization, allows simultaneous visualization of HER2 protein and gene on a single slide. We aimed to clarify the clinical significance of HER2 intratumoral heterogeneity (ITH) using GPA. We investigated the relationships between various HER2 ITH indicators and clinical course in 102 patients with HER2-positive breast cancer, treated with neoadjuvant trastuzumab and chemotherapy. Five representative microscopic images were captured from each GPA slide of pre-therapeutic biopsy materials. All evaluable cancer cells in the images were individually assessed for HER2 gene copy number and protein expression. Mean and coefficient of variation (CV) of both gene copy number and protein category were calculated, and each was divided into negative, equivocal, and positive. Based on their combined status, cancer cells were classified into nine types. Pathological complete response (pCR) to neoadjuvant treatments showed positive relationships to mean gene copy number (P < 0.001), mean protein category (P < 0.001), and proportion of gene- and protein-positive tumor cells (P < 0.001) and showed negative relationships to the CV of protein category (P < 0.001) and the proportion of gene-amplified but protein-negative tumor cells (P = 0.002). Two diagnostic models, created by combining clinicopathological factors and ITH indicators, showed excellent potential diagnostic ability for pCR (mean gene copy number and protein category CV; AUC = 0.837, proportion of gene- and protein-positive tumor cells; AUC = 0.831). HER2 ITH quantified by GPA is a potential predictive indicator for efficacy of HER2-targeted treatment.
