HER2 mRNA Expression Research Articles

Abstract 44: U3-1402, a novel HER3-targeting ADC, and a novel DNA topoisomerase I inhibitor inhibit the growth of non-small cell lung cancer with EGFR mutation

Abstract Background HER3, a member of the HER family, is overexpressed in non-small cell lung cancer (NSCLC), especially in those with EGFR mutation. Anti-HER3 antibody therapies, including patritumab, are effective, but limited in their efficacy, for patients with NSCLC. U3-1402 is a novel ADC composed of an anti-HER3 antibody (patritumab) and a novel potent topoisomerase I inhibitor DX-8951. U3-1402 achieved a high drug-to-antibody ratio (DAR: 7-8), because it is homogeneously conjugated with the payload. Here, we aimed to preclinically evaluate U3-1402’s efficacy in NSCLC, especially in those with EGFR mutation. Materials and methods An in vitro growth inhibition assay was used to evaluate the sensitivity of 14 NSCLC cell lines to U3-1402. Cells were treated with U3-1402 at different concentrations (0-10 μg/ml) over 7 days; 50% growth inhibitory concentration relative to control (IC50) was calculated. PC9, HCC827, HCC827GR5, Ma70, Ma70GR, 11-18, H1650, HCC4006, and H1975 cells had the EGFR mutation. HCC827GR and Ma70GR were EGFR-TKI resistant clones, with MET genomic amplification and an unknown resistance mechanism, established from HCC827 and Ma70 cells, respectively. HER3 mRNA expression levels were measured by quantitative reverse transcription-PCR (qRT-PCR) and the ratio was calculated against house-keeping genes in each cell line. Results The in vitro growth inhibition assay indicated that HCC827GR5, Ma70GR, and 11-18 cells were sensitive to U3-1402 (IC50 values 1.0, 5.2, 3.2 μg/ml, respectively). However, other cells were relatively resistant to U3-1402, having IC50 values greater than 10 μg/ml. Notably, HCC827GR5 cells were more sensitive to U3-1402 than parental HCC827 cells were. Specifically, 1.0 μg/ml U3-1402 reduced the viable cell proportion to 50% of control in HCC827GR5 cells, but the effect was limited to 95% of control in HCC827 parental cells. Furthermore, EGFR-TKI erlotinib increased sensitivity to U3-1402 in HCC827GR5 cells. Specifically, 1.0 μM erlotinib reduced the viable cell proportion to 69% of control, and 1.0 μg/ml U3-1402 reduced it to 50% of control in HCC827GR5 cells, but both agents combined reduced it to 3% of control. In an effort to clarify the underlying mechanism by which EGFR-TKI resistant HCC827GR5 cells were more sensitive to U3-1402 than parental HCC827 cells were, we evaluated the HER3 mRNA expression in both cell lines. HCC827GR5 cells had significantly higher levels of HER3 mRNA than parental HCC827 cells did (1.85 vs 0.65, t-test; p = 0.003). Conclusions U3-1402 preclinically exhibited its efficacy in NSCLC with EGFR mutation. Its efficacy was enhanced by EGFR-TKI combination. Sensitivity to U3-1402 might depend on HER3 expression levels. These results provide a rationale for U3-1402 alone or in combination with EGFR-TKI to be investigated in patients with NSCLC with EGFR mutation and aberrant HER3 expression. Citation Format: Kimio Yonesaka, Koji Haratani, Kenji Hirotani, Kazuhiko Nakagawa. U3-1402, a novel HER3-targeting ADC, and a novel DNA topoisomerase I inhibitor inhibit the growth of non-small cell lung cancer with EGFR mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 44. doi:10.1158/1538-7445.AM2017-44

Pathological complete response in HER2 positive breast cancer treated with trastuzumab and chemotherapy: Predictive factors report.

e12133 Background: Achieving a pathological complete response (pCR) in HER2 positive disease is a surrogate of survival. Several factors were studied in order to predict pCR, but only negative estrogen receptor is correlated to pCR. Methods: 168 patients (March 2002-December 2016) with neoadjuvant trastuzumab-based treatment were retrospectively analyzed in order to find if common clinicopathological factors, HER2 expression and immune infiltrates could correlate with pCR. Results: Median age was 55 years (29-86), median tumor size of 39mm and 53% of tumors had initial nodal involvement. 45% of patients were estrogen and 71% progesterone receptor negative and median ki67 expression was of 43%. Otherwise HER2 expression was analyzed by immunohistochemistry, FISH amplification and mRNA expression; we also determined the presence of immune infiltrates stromal and intratumoral L45 lymphocytes, PD1 and PD-L1. A 38% pCR rate was found in whole population. In the univariate model we found and association with pCR and: estrogen receptor negative (p = 0.003; OR 2.5), high HER2 expression by mRNA in tissue > 3.9 (p = 0.012; OR 8.4), stromal L45 expression > 35% (p = 0.001; OR 5.3), stromal PD1 expression > 10 (p = 0.004; OR 4.5). The most powerful factor associated with pCR is HER2 mRNA expression > 3.9, with 82% of pCR rate in those tumors; followed of L45 stromal expression > 35%, with 76% of pCR rate. Conclusions: Factors related to pCR are mostly estrogen receptor negative and high HER2 expression by mRNA and high L45 stromal infiltrates. With those variables, we could design a predictive model in order to perform the best neoadjuvant treatment to increase the pCR rate.

HER2 activity in solid tumors.

e23121 Background: ERBB2 is a member of the human epidermal growth factor receptor family. HER2 expression that is immunohistochemistry (IHC) 3+ or IHC 2+ with copy number gain is an effective predictor for treatment with trastuzumab in breast and gastroesophageal cancers. Here we present an analysis on HER2 activity measured by IHC, mRNA expression and copy number variation (CNV) across a variety of solid tumors. Methods: The study population consists of patients diagnosed with solid tumors (n = 856) that underwent a Paradigm Diagnostic Cancer Test during 2016. We then analyzed tumor types that tested positive for HER2 by IHC (3+), CNV and/or mRNA expression. Results: We identified 365 (43%) patients positive for HER2 by IHC, 258 (30%) had high HER2mRNA expression and 41 (5%) had amplification by CNV. Seventy-five patients were HER2 IHC 3+ or 2+/CNV positive. The proportion of HER2 IHC 3+ or 2+/CNV positive tumors in each tumor type was as follows: breast cancer 41 (18.5%), NSCLC 10 (8.1%), colorectal 6 (6.7%), esophago-gastric 3 (10%,) urothelial/bladder 3 (16%), biliary 2 (28.6%), ovarian cancer 2 (5.1%) and pancreas 1 (3.1%). Using copy number gain as the gold standard, across all tumor types, an IHC of 3+ had a 90.2% sensitivity and a 95.6% specificity. In the same analysis with breast cancers omitted, the sensitivity was 75%, and specificity 96.8%. Whereas high mRNA expression had a sensitivity of 97.6% and specificity of 73.3%, omitting breast cancers, the sensitivity was 93.8% and specificity 73.2%. Conclusions: HER2 activity was identified in a wide variety of solid tumors and a small but significant proportion of these tumors maybe candidates for treatment with HER2 inhibitors such as trastuzumab. Our analysis also identified that HER2 activity in breast cancers has a distinctive pattern which was not seen in other tumor types. HER2 IHC 3+ expression was much less sensitive among other tumor types compared to breast cancer. mRNA expression, while remaining sensitive among other tumor types, is not specific, even among breast cancer patients. Our analysis also identified that HER2 activity in breast cancers has a distinctive pattern which was not seen in other tumor types.

Abstract P6-07-09: Biomarker analysis from the neoadjuvant KRISTINE study in HER2-positive early breast cancer (EBC)

Abstract Introduction KRISTINE (NCT02131064) is an open-label, phase 3 study of neoadjuvant trastuzumab emtansine + pertuzumab (T-DM1+P) vs docetaxel + carboplatin + trastuzumab + pertuzumab (TCH+P) in pts with HER2+ EBC (all pts, Table). In exploratory analyses we assessed whether HER2 expression and gene amplification levels and PIK3CA mutation were associated with pathologic complete response (pCR) in KRISTINE. Methods Treatment-naive pts with stage II–IIIC HER2+ EBC were randomized to receive 6 cycles of T-DM1+P or TCH+P and assessed for the primary endpoint, pCR (ypT0/is, ypN0). HER2 status was confirmed centrally as immunohistochemistry (IHC) status of IHC3+ and/or HER2/CEP17 gene ratio ≥2 by in situ hybridization (ISH) before study entry. Baseline HER2 mRNA, PIK3CA mutation, and hormone receptor (HR) status were evaluated. Rates of pCR were compared across biomarker subgroups, including: HER2 mRNA, HER2 IHC staining percentage, HER2 gene ratio, and PIK3CA mutation. Incidence of overlap between biomarkers was assessed. All analyses were descriptive. Results KRISTINE randomized 444 pts (data cutoff, Dec 3, 2015; TCH+P=221; T-DM1+P=223). The biomarker population was representative of the ITT population. Biomarker values and distribution were balanced across treatment arms. Lower pCR rates were seen in both treatment arms across biomarker subgroups with lower vs higher HER2 expression levels (Table). PIK3CA mutation was associated with numerically lower pCR with T-DM1+P but not TCH+P. Evaluation of 15 pts who progressed during T-DM1+P neoadjuvant therapy suggests that nonresponse to therapy in this arm may be associated with low HER2 levels (mRNA, IHC, and ISH). PIK3CA mutation rate was higher in tumors with ≤median HER2 mRNA (38%) vs >median (16%), and in focal (53%) and variable (41%) HER2 expression vs homogeneous (24%). A numerically higher proportion of HR positivity was seen in the HER2 IHC2+ (81%) subgroup vs IHC3+ (55%), and in the focal (83%) and variable (62%) HER2 subgroups vs homogeneous (53%). Table TCH+PT-DM1+P BiomarkernpCR (%)npCR (%)Difference in pCR rates (%)95% CIAll pts22155.722344.4-11.3-20.5, -2.0HER2 IHC3+19460.819549.7-11.1-20.9, -1.3IHC2+2520.0287.1-12.9-31.2, 5.5HER2 IHC 2+/3+ fraction* Focal (<30%)1533.3160-33.3-57.2, -9.5Variable (30–79%)2729.62722.2-7.4-30.7, 15.9Homogeneous (≥80%)17961.518051.7-9.8-20.0, 0.4HER2 mRNA expression ≤median10649.110937.6-11.4-24.6, 1.7>median10763.610851.9-11.7-24.8, 1.4HER2/CEP17 gene ratio ≥2 to <44533.35219.2-14.1-31.6, 3.4≥416662.015853.2-8.9-19.6, 1.8PIK3CA mutation Mutated5354.76131.1-23.6-41.3, -5.8Nonmutated16056.315151.0-5.3-16.3, 5.8*Categorization based on sum of IHC2+ and IHC3+ staining percentage. Conclusions None of the evaluated biomarker subgroups showed superior pCR rates for T-DM1+P vs TCH+P. Lower HER2 levels based on mRNA and protein expression and gene amplification were associated with numerically lower pCR in both treatment arms. PIK3CA mutation results differ from prior observations. Unfavorable biomarkers for pCR, like low HER2, PIK3CA mutation, and HR positivity, showed a tendency for co-expression, which should be considered when planning future trials in HER2+ BC. Citation Format: de Haas SL, Hurvitz SA, Martin M, Kiermaier A, Lewis Phillips G, Xu J, Helms H-J, Slamon D, Press MF. Biomarker analysis from the neoadjuvant KRISTINE study in HER2-positive early breast cancer (EBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-09.

Human Epidermal Growth Factor Receptor-3 mRNA Expression as a Prognostic Marker for Invasive Duct Carcinoma not Otherwise Specified.

Breast cancer is the most common cancer in women and the Erythroblastosis Oncogene B(ErbB) receptor family holds crucial role in its pathogenesis. Human Epidermal Growth Factor Receptor 3 (HER-3) gene over expression in breast tissue has been associated with aggressive clinical behaviour and bad prognosis. To evaluate HER-3 mRNA expression level as a prognostic marker for breast cancer and to correlate its level with other established prognostic parameters. This study was carried out on specimens of 100 cases that were divided into 40 patients presented with fibroadenoma and 60 patients presented with Invasive Ductal Carcinoma (IDC) not otherwise specified and underwent modified radical mastectomy. All specimens were investigated for HER-2/neu, ER and PR expression by Immunohistochemistry (IHC) and quantitative assay of HER-3 mRNA expression using real time PCR technique. There was a significant high HER3 mRNA level in carcinoma cases compared to fibroadenoma. In malignant cases, HER3 mRNA level was significantly associated with advanced T stage, advanced N stage, number of positive lymph nodes, large tumour size and cases associated with an adjacent in situ component. Moreover, HER-3 mRNA level was of highest values in Her-2/neu positive group followed by triple negative cases with the lowest level in luminal group (p<0.05). HER-3 gene is upregulated in IDC especially those carrying poor prognostic features. HER-3 mRNA level may identify a subset of patients with a poor prognosis, and who could undergo further evaluation for the efficacy of HER3 targeted anticancer therapy.

NanoString nCounter® Approach in Breast Cancer: A Comparative Analysis with Quantitative Real-Time Polymerase Chain Reaction, In Situ Hybridization, and Immunohistochemistry.

PurposeAccurate testing for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is essential for breast cancer treatment. At present, immunohistochemistry (IHC)/florescence in situ hybridization (FISH) are widely accepted as the standard testing methods. To investigate the value of NanoString nCounter®, we performed its comparative analysis with IHC/FISH and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the assessment of ER, PR, and HER2.MethodsData on IHC/FISH results for ER, PR, and HER2 in 240 patients from a single tertiary hospital in Korea were collected and compared with NanoString nCounter® and qRT-PCR results at a single institution.ResultsExpression levels for each gene using NanoString nCounter® showed good correlation with the corresponding data for protein expression by IHC (p<0.001) and gene amplification status for HER2 (p<0.001). Comparisons between gene expression and IHC data showed good overall agreement with a high area under the curve (AUC) for ESR1/ER (AUC=0.939), PgR/PR (AUC=0.796), and HER2/HER2 (AUC=0.989) (p<0.001).ConclusionThe quantification of ER, PgR, and HER2 mRNA expression with NanoString nCounter® may be a viable alternative to conventional IHC/FISH methods.

Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples

Background: Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. Methods: In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. Results: The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). Conclusion: Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression.

Heregulin-1ß and HER3 in hepatocellular carcinoma: status and regulation by insulin.

BackgroundThe heregulin-1ß/HER3-driven pathway is implicated in several epithelial malignancies and its blockade is currently undergoing clinical investigation. Paradoxically, the status and the regulation of this pathway is poorly known in hepatocellular carcinoma (HCC).MethodsUsing 85 HCC obtained after tumour resection, heregulin-1ß and HER3 expression was evaluated by real-time RT-PCR, ELISA and/or immunohistochemistry. Statistics were performed to analyze associations between gene expression and clinicopathological parameters. The effects of insulin on the heregulin-1ß/HER3 pathway was investigated in four HCC cell lines.ResultsHER3 mRNA was upregulated in 52 % of tumours, while heregulin-1ß mRNA was downregulated in 82 %. Hepatitis B and C viral infections were respectively associated with high and low HER3 mRNA expression. No association was seen between neither HER3 or heregulin-1ß mRNA and prognostic factors, survival or recurrence. Immunohistochemistry showed predominant cytoplasmic staining of HER3 in tumours but the staining was nonreproducible. HER3 mRNA and protein levels were not correlated in liver tissues. In HCC cells, insulin promoted HER3 proteasomal degradation and inhibited heregulin-1ß stimulation of cell migration. HER3 and insulin receptor co-immunoprecipitated in these cells. The loss of insulin receptor expression by RNA interference sensitized cells to heregulin-1ß-induced AKT phosphorylation.ConclusionsAutocrine heregulin-1ß loop is uncommon in HCC and HER3 mRNA expression is differentially influenced by hepatitis viruses. Insulin is a negative regulator of HER3 protein expression and function in HCC cells. Altogether these data may explain why HER3 and heregulin-1ß expression have no prognostic value and suggest that HCC patients are unlikely to derive benefit from HER3-targeted monotherapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0402-3) contains supplementary material, which is available to authorized users.

Detection of HER2 polymorphism and expression using circulating DNA and RNA as a tool in lung adenocarcinoma patients: a case control study.

Circulating DNA and RNA is an important prognostic tool for noninvasive malignant disease detection and in disease prognosis. Study aimed to evaluate the possible prognostic role of HER2 (-3444C/T) promoter polymorphism and its mRNA expression in Lung adenocarcinoma patients using circulating DNA and RNA. One hundred newly diagnosed lung adenocarcinoma patients and 100 age and sex matched healthy controls were included and allele specific (AS) polymerase chain reaction (PCR) was used for genotyping and expression was analyzed by quantitative real time PCR. Overall survival of patients was analyzed by Kaplan-Meier method. We observed a statistically significant difference in the frequency of HER2 CC, CT, and CT genotype among lung adenocarcinoma cases vs. healthy controls (P=0.001). Compared to the CC genotype, OR 2.51 (1.4-4.51), 5.97 (1.17-30.41) and RR 1.56 (1.17-2.07), 2.83 (0.82-9.73) for heterozygous CT and homozygous TT genotypes suggesting possible dominant effect on risk of lung adenocarcinoma. Cases with CC genotype showed 9.29 fold increased mRNA expression while cases with heterozygous CT and homozygous TT genotype showed 16.26, 16.72 fold increased mRNA expression (P<0.0001). We observed 13.92 fold increased HER2mRNA expression Lung adenocarcinoma patients. Patients in different TNM stages showed significant difference in HER2 mRNA expression which was found to be significantly associated (P<0.0001). Patients with distant metastases and without distant metastases had 17.44 and 11.16 fold increased HER2 mRNA expression was also found to be significantly associated (P<0.0001). It was also observed that patients with pleural effusion and without pleural effusion showed significant difference in HER2 mRNA expression (P=0.03). We also analysed patients with CC, TT, CT (P=0.02) and CT + TT (P=0.008) genotype showed 15.8, 7.9, 9.5 and 7.9 months of overall median survival time and found to be significantly associated, respectively. Patients with >13 and ≤13 fold increased HER mRNA expression also showed 7.9 and 11.5 months of overall median survival time was also found to be significantly associated (P=0.01). Our work provides evidence that circulating DNA and RNA may be a potential prognostic tool in Lung adenocarcinoma patients. Promoter polymorphism of HER2 (-3444C/T) gene had significant impact on higher HER2 mRNA expression could be a predictive factor for patients' worse overall survival and metastatic behaviour.

Differential responses of HER2-positive breast cancer cells to anti-HER2 treatment.

e12095 Background: HER2 is a membrane tyrosine kinase receptor which promotes cells growth by activating downstream kinase pathways: RAS/MEK/ERK and PI3K/AKT. Given the fact that HER2 gene is amplified in certain population of breast cancer patients, therapeutic strategy has evolved to target HER2 function. However, it has been long controversial as to which patients are truly benefited from such treatment. In this study, we evaluate impact of HER2 overexpression on response to anti-HER2 treatment using a panel of breast cancer cell lines, and compared FISH vs. RT-PCR for their accuracy in predicting drug responses. Methods: Human breast cancer lines were obtained from ATCC or from cell repository bank at MD Anderson Cancer Center. RhuMAB HER2 was provided by Genentech. Her2 gene amplification was evaluated by FISH method using the PathVysion HER2/NeuDNA probe kit form Abbott. HER2 mRNA expression was analyzed by RT-PCR method. Growth inhibitory and cytotoxic effects of anti-HER2 antibody were determined by MTS assay. Apoptotic cell death was visualized with DAPI staining and quantified by Annexin V-based flow cytometric detection. Results: A total of 12 cell lines are chosen for this study, 4 of them are HER2-negative and 8 are HER2-positive as determined by FISH testing. Anti-HER2 antibody treatment inhibits growth of all cell lines tested, among them, 3 HER2-positive cell lines are identified to undergo apoptotic cell death. Re-culture in fresh medium restores cell growth potential in those non-apoptotic cells, but not in the apoptotic cell lines. FISH score has poor correlation with apoptotic cell death; in contrast, cell lines those expressing more than 20,000 copies of HER2 mRNA, as measured in 1 ng of total RNA, all show apoptosis. More interestingly, apoptotic death appears more severe as HER2 mRNA number increases. Conclusions: Accurate measurement of HER2 expression is essential for improving the outcome of anti-HER2 treatment. Our study suggests that RT-PCR technique seems to better fulfill the aim, and warrants future clinical trials of using RT-PCT to predict a positive response to anti-HER2 therapies.

HER2 mRNA expression in breast cancers with equivocal immunohistochemical results using in situ mRNA hybridization

To investigate in situ mRNA expression of HER2 oncogene in breast cancers with equivocal immunohistochemical results, and to explore the potential feasibility of RNAscope technique in evaluating HER2 status in breast cancers. Sixty-nine FFPE samples of invasive ductal breast cancer with equivocal HER2 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. 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). 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 HER2 gene amplification or protein overexpression, and has potential clinical utility.

The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

IntroductionThe N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab.MethodsN9831 tested the addition of trastuzumab to chemotherapy in stage I–III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative <10.7, equivocal 10.7 to <11.5, and positive ≥11.5 log2 expression units). Cox regression was used to assess the association of HER2 expression with trastuzumab benefit in preventing distant recurrence.ResultsMedian follow-up was 7.4 years. Of 1,940 total patients, 901 had consent and sufficient tissue. HER2 by reverse transcriptase polymerase chain reaction (RT-PCR) was negative in 130 (14 %), equivocal in 85 (9 %), and positive in 686 (76 %) patients. Concordance between HER2 assessments was 95 % for RT-PCR versus central immunohistochemistry (IHC) (>10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels.ConclusionsConcordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays.Trial registrationClinicaltrials.gov NCT00005970. Registered 5 July 2000.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0643-7) contains supplementary material, which is available to authorized users.

Abstract LB-231: A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors

Abstract Antibody-drug conjugates are effective in the treatment of HER2-amplified breast cancer and Hodgkin's lymphoma, but current ADC technologies have faced limitations expanding the addressable patient population and target space. Ado-trastuzumab emtansine (T-DM1) is an ADC with 3-4 cytotoxic drugs per antibody that was recently approved for HER2 IHC 3+ or HER2-amplified breast cancer. Even within this high HER2-expressing population, several studies have now shown greater T-DM1 benefit in patients with HER2 mRNA expression above the median. These data suggest the need for more potent anti-HER2 ADCs to maximize benefit for HER2 IHC 3+ or amplified patients, and to extend HER2 ADC therapy to low HER2-expressing patients (HER2 IHC 1+/2+). XMT-1522 is an anti-HER2 ADC that uses a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery, and is non-competitive with trastuzumab or pertuzumab for HER2 binding. Each antibody is conjugated to ∼15 proprietary auristatin molecules using Fleximer, a biodegradable hydrophilic polymer. XMT-1522 shows nanomolar potency in cultured tumor cells with HER2 receptor densities as low as 10,000 per cell, and is typically 1-3 logs more potent than T-DM1 across a panel of 25 tumor cell lines. In mouse xenograft studies XMT-1522 has excellent pharmacokinetic properties and achieves complete tumor regressions at well-tolerated doses. In the high HER2-expressing N87 gastric cancer model (800,000 HER2 receptors/cell), complete regressions are achieved with a single 1 mg/kg dose of XMT-1522, while 10 mg/kg T-DM1 is required for comparable activity. In the same model, the XMT-1522/trastuzumab/pertuzumab triple combination results in tumor regressions where the same doses of XMT-1522 alone or the trastuzumab/pertuzumab doublet result in tumor stasis. In the low HER2-expressing JIMT-1 breast cancer (79,000 HER2/cell) and SNU5 gastric cancer (22,000 HER2/cell) models, complete regressions are achieved with single 1 mg/kg or 0.67 mg/kg doses of XMT-1522, respectively, while T-DM1 is inactive at doses ≥10 mg/kg. In non-human primates XMT-1522 demonstrates good stability of drug conjugate in plasma with t1/2 ∼5 days (comparable to antibody t1/2) and minimal exposure to free payload. Despite the high potency of XMT-1522 in low HER2 tumor models, there is no XMT-1522-related toxicity observed in critical HER2-expressing tissues including heart and lung. The preclinical data support testing XMT-1522 as a single agent in tumors with low HER2 expression where current HER2-directed therapies are not indicated. Furthermore, combination of XMT-1522 with trastuzumab and/or pertuzumab achieves efficient cytotoxic payload delivery while retaining the potential for full inhibition of HER2 signaling, which may be necessary to improve on current regimens in HER2-driven tumors. Citation Format: Donald A. Bergstrom, Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Michael DeVit, Mao Yin, Laura Poling, Joshua D. Thomas, Dmitry Gumerov, Dongmei Xiao, Elena Ter-Ovanesyan, LiuLiang Qin, Alex Uttard, Alex Johnson, Timothy B. Lowinger. A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-231. doi:10.1158/1538-7445.AM2015-LB-231

High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression.

BackgroundElevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.MethodsTwelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.ResultsHigh basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.ConclusionsHigh basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.

Highlights of This Issue

To augment existing CAR technology, Wang and colleagues transduced selected CMV-specific human T cells to express both a CD19 CAR and a truncated EGFR, and investigated the effects of restimulating CAR+ T cells through an endogenous CMV-specific T cell receptor. This study has demonstrated that the antitumor activity of CMV/CD19 bispecific T cells can be enhanced as a consequence of proliferation following CMV peptide vaccination. Additionally, the bispecific T cells could be ablated in vivo by anti-EGFR antibody cetuximab. This vaccine boost strategy can be extended to multiple malignancies by transducing various tumor-specific CARs into selected CMV patient T cells.Lapatinib inhibits the tyrosine kinases of both HER1 (EGFR) and HER2, although its clinical use in breast cancer is confined to tumors that overexpress HER2. Leary and colleagues report a study with 2 weeks of lapatinib treatment prior to surgery in newly diagnosed primary breast cancer and show that proliferation (Ki67) was significantly decreased in both HER2-negative and HER2-positive tumors. Among HER2-negative tumors, high HER3 mRNA expression identified Ki67 responders, with a trend for combined increase in levels of HER3/HER2 expression to predict response. HER2-HER3 heterodimers could identify patients with HER2-negative breast cancer that might benefit from lapatinib and merit further investigation.Wide excision of osteosarcoma is necessary to effect successful treatment. Bone morphogenetic protein-2 (BMP-2) could improve bone union following tumor reconstruction but is not currently used due to concerns it may prompt recurrence. Geller and colleagues developed an osteosarcoma model system to more clearly define the effect of both surgical margins and BMP-2 on local recurrence in the context of conventional chemotherapy. Margins greater then 997 μm yielded reliable local control using surgery alone. Single-agent and dual-agent chemotherapy lowered these margins to 36 μm and 12 μm, respectively. The application of BMP-2 did not increase local recurrence rates.The interleukin-11 receptor (IL11R) is an established molecular target in primary bone, tumors (such as osteosarcoma), and in secondary bone metastases (such as prostate cancer). In this study the authors evaluated the IL-11R as a target in preclinical models of leukemia and lymphoma and also validated their findings in human tumors. The results indicate that the IL-11R is a suitable cell surface target for ligand-directed applications against leukemia and lymphoma and that BMTP-11, an IL11R-targeting drug prototype (and its derivatives), have translational potential against this group of malignant diseases.

The relationship between the expression of VEGF, EGFR, and HER-2 mRNA in esophageal squamous cell carcinoma (ESCC) and clinicopathological features of different ethnic groups in Xinjiang.

The purpose of this study was to explore the relationship between the expression of vascular endothelial growth factor (VEGF), human epidermal growth factor receptor 2 (HER-2), and epidermal growth factor receptor (EGFR) mRNA in esophageal squamous cell carcinoma (ESCC) and the clinicopathological characteristics of the Han, Uyghur, and Kazakh in Xinjiang. Real-time quantitative polymerase chain reaction technology (RT-PCR) was used to detect the expression of VEGF, HER-2, and EGFR mRNA in esophageal squamous cancer tissue of 60 cases and 30 cases of VEGF, HER-2, and EGFR mRNA in esophageal cancer adjacent tissues, and analyze its relationship with clinicopathological features. The results were as follows: (1) There was no statistically significant difference in esophageal tissue VEGF, HER-2, and EGFR mRNA gene expression levels of the Han, Uyghur, and Kazakh patients (P > 0.05). (2) The expression levels of VEGF, HER-2, and EGFR mRNA in ESCC group were higher than those in adjacent esophageal tissue group (P < 0.05). (3) The expression levels of VEGF and HER-2 mRNA in ESCC of the Han patients were higher than those of the Uyghur and Kazakh patients (P < 0.05). (4) The expression levels of VEGF, HER-2, and EGFR mRNA in lymph node metastases were higher than those without lymph node metastasis (P < 0.05). (5) The expression level of HER-2 mRNA was related with the degrees of pathological differentiation, and the higher pathologic degree, the lower expression level in HER-2 mRNA (P < 0.05). Therefore, the following conclusions were drawn: (1) There were ethnic differences in the VEGF and HER-2 gene mRNA expression levels of the Uyghur, Han, and Kazakh patients in Xinjiang. (2) The expressions of VEGF, HER-2, and EGFR mRNA were related to the lymph node metastasis in ESCC and pathologic differentiation degree.

Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

Canine mammary tumors (CMTs) share many features with human breast cancer (HBC), specifically concerning cancer-related pathways. Although the human epidermal growth factor receptor 2 (HER2) plays a significant role as a therapeutic and prognostic biomarker in HBC, its relevance in the pathogenesis and prognosis of CMT is still controversial. The aim of this study was to investigate HER2 expression in canine mammary hyperplasic and neoplastic tissues as well as to evaluate the specificity of the most commonly used polyclonal anti HER2 antibody by multiple molecular approaches. HER2 protein and RNA expression were determined by immunohistochemistry (IHC) and by quantitative real-time (qRT) PCR. A strong cell membrane associated with non-specific cytoplasmic staining was observed in 22% of carcinomas by IHC. Adenomas and carcinomas exhibited a significantly higher HER2 mRNA expression when compared to normal mammary glands, although no significant difference between benign and malignant tumors was noticed by qRT-PCR. The IHC results suggest a lack of specificity of the FDA-approved antibody in CMT samples as further demonstrated by Western immunoblotting (WB) and reverse phase protein arrays (RPPA). Furthemore, HER2 was not detected by mass spectrometry (MS) in a protein-expressing carcinoma at the IHC investigation. This study highlights that caution needs to be used when trying to translate from human to veterinary medicine information concerning cancer-related biomarkers and pathways. Further investigations are necessary to carefully assess the diagnostic and biological role specifically exerted by HER2 in CMTs and the use of canine mammary tumors as a model of HER2 over-expressing breast cancer.

Evaluation of the prognostic significance of HER family mRNA expression in high-risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study

BackgroundThe aim of the study was to evaluate the prognostic ability of the transcriptional profiling of the HER family genes in early breast cancer, as a validation analysis of another previously published HeCOG study.MethodsRNA was extracted from 663 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples of high-risk early breast cancer patients enrolled in the randomized HE10/00 trial. Relative mRNA expression of all four HER family members was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).ResultsIn compliance with our previous study, the overall agreement between qRT-PCR and IHC/FISH for HER2 status determination was good (69%). Likewise, the overall concordance between qRT-PCR and IHC for EGFR status was high (81%). In line with our previously reported data, we demonstrated a positive association between HER2 and HER3 mRNA expression. Similarly, mRNA expression of HER3 and HER4 was positively associated with each other and negatively associated with EGFR. Regarding relationships with clinico-pathological parameters, our findings are also in agreement with our previous results. Generally, increased EGFR and HER2 mRNA expression was related to unfavorable, whereas high HER3 and HER4 mRNA expression was associated with favorable clinico-pathological parameters. In univariate analysis, no significant association between EGFR, HER2 and HER3 mRNA expression and overall survival (OS) or disease-free survival (DFS) was demonstrated. However, high EGFR protein expression was associated with significantly shorter OS (log-rank, p = 0.015). In compliance with our previously published data, increased HER4 mRNA expression had a significantly favorable prognostic value in terms of OS (p = 0.044) and DFS (p = 0.047). In multivariate analysis, among all HER receptors, only EGFR protein expression was found to affect OS (Wald’s p = 0.028) and DFS (p = 0.015) independently. Concerning the combined expression of all four HER family receptors, the combination of high EGFR, high HER2, low HER3 and low HER4 mRNA expression was associated with a trend for shorter OS (log-rank, p = 0.065) and significantly worse DFS (p = 0.033), compared with all other co-expression profiles.ConclusionsThese data indicate that qRT-PCR may represent a valid alternative method for evaluating the expression of HER family members in FFPE breast carcinoma tissue samples.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12609001036202

Efficacy and safety of chemotherapy (CT) ± pertuzumab (P) for platinum-resistant ovarian cancer (PROC): AGO-OVAR 2.20/ENGOT-ov14/PENELOPE double-blind placebo-controlled randomized phase III trial.

5504 Background: Adding P to gemcitabine (GEM) for PROC improved progression-free survival (PFS) in a subset of patients (pts) with low tumor HER3 mRNA expression [Makhija 2010]. Methods: Eligible ...

Gemcitabine treatment enhances HER2 expression in low HER2-expressing breast cancer cells and enhances the antitumor effects of trastuzumab emtansine.

Trastuzumab emtansine (T-DM1), trastuzumab-conjugated with a cytotoxic agent, has shown promising antitumor effects in breast cancer. Since a good therapeutic response using T-DM1 treatment requires high human epidermal growth factor receptor 2 (HER2) expression, breast cancers with low or no HER2 expression have not been used for T-DM1 treatment. The aim of the present study was to show that treatment of low HER2-expressing breast cancer cells with gemcitabine (GEM) enhanced HER2 expression using RT-qPCR, immunoblot and flow cytometric analysis. The results showed that GEM treatment significantly enhanced HER2 expression in MDA-MB-231, MCF7 and BT-20 breast cancer cells, while paclitaxel (PTX) treatment induced lower or no enhancement in HER2 expression. The expression of HER2 mRNA was also enhanced in GEM-treated MCF7 cells. Treatment with an inhibitor for nuclear factor-(NF)-κB suppressed GEM-induced HER2 upregulation, indicating that NF-κB activation by GEM may be associated with HER2 upregulation. T-DM1 binding to HER2 on MCF-7 cells was enhanced by GEM pretreatment and the combined treatment of GEM and T-DM1 synergistically inhibited the proliferation of MCF7 cells. Thus, the combined treatment with GEM and T-DM1 may be a promising therapeutic modality for low HER2-expressing breast cancers, which was facilitated by the unique HER2-upregulating effect of GEM.