HER2 Probe Research Articles

Efficient generation of recombinant anti-HER2 scFv with high yield and purity using a simple method.

We developed a method to produce a soluble form of a single-chain fragment variable (scFv) targeting human epithelial growth factor receptor 2 (HER2) in Escherichia coli. By optimizing the orientations of the variable heavy (VH) and variable light (VL) domains and the His-tag, we identified the HL-His type antibody with the highest HER2-binding activity. Purification of HL-His yielded 40.7mg from a 1 L culture, achieving >99% purity. The limit of detection was determined to be 2.9ng, demonstrating high production yield, purity, and sensitivity. Moreover, we successfully labeled HER2+ cell lines with fluorescent dye-conjugated scFv, resulting in a significantly higher observed signal-to-background ratio, compared to that of HER2- cell lines. This highlights the potential of these fluorescent scFvs as valuable probes for HER2+ breast cancer diagnostics. Notably, the process for the complete scFv production was streamlined and required only 4-5 days. Additionally, the product maintained its activity after freeze storage, allowing for large-scale production and a wide range of practical applications.

HER2 Overexpression and Cytogenetical Patterns in Canine Mammary Carcinomas.

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical-pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical-pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns.

Abstract 3676: Race-specific methylation profiles and epigenetic age acceleration differentiates estrogen receptor status breast cancer

Abstract Breast cancer (BrCa) is the second most common cancer among women in the U.S.. Black women with BrCa, on average, have an earlier age of onset, more aggressive subtypes and experience higher mortality compared with White women. Although the underlying causes remain unknown, genetic and lifetime exposures are hypothesized. Here, we utilize blood-based whole-genome methylation arrays to: (1) discover CpG probes related to BrCa receptor status, (2) identify biological pathways driving BrCa subtype disparities, and (3) quantify epigenetic age acceleration differences by race and receptor status. DNA methylation levels were generated for 95 blood samples from women diagnosed with BrCa using the Illumina EPIC array. After quality control, M-values for 91 samples and 808,329 methylation probes were evaluated using a modified Student t-test. BrCa estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status were collected from health records. Stratified analyses by race adjusting for age were applied. We calculated the association of age acceleration, based on Horvath’s epigenetic clock, by race and receptor status. Statistical significance, corrected for multiple testing, was defined as a p-valueadj <0.05. Among the 91 BrCa samples, nearly half were Black women (40.7%) and the overall mean age at diagnosis was 56.5 years ±11.3. The majority of BrCa tumors were ER-, 60.4% (N=55), and HER2-, 76.9% (N=70). Overall, two methylation probes significantly differentiated ER+/- BrCa. The most significant methylated gene was FOXK2 (p-valueadj=0.03, logFC=0.63). The race-stratified analysis identified 188,398 probes for ER+/- and five probes for HER2+/- among White women as marginally significant (p<0.05). Two genes (FAM107B, PRKCZ) for ER+/- BrCa and four genes (ZNF430, TTC27, SEC1 and NOS1AP) for HER2+/- BrCa remained statistically significant after correcting for multiple comparisons (p-valueadj < 0.05; |logFC| >1). The mean age acceleration is estimated as 3.7 years greater for Black women compared to White women. Among White women, the mean age acceleration was 1.9 years greater for ER+ than ER- subtypes, and those HER2- were 2.2 years older than HER2+. Similarly, among Black women the mean age acceleration difference for ER- was 3.2 years greater than ER+, and HER2- was 3.8 years older than HER2+. Our study confirmed (e.g. FOXK2) and identified several novel methylation sites associated with BrCa subtypes. Furthermore, we demonstrated in our study that epigenetic age acceleration of BrCa varies by race, with Black women biologically aging faster than White women. Although this is a limited sample size, stratified analyses revealed biological age differences by race and receptor status. In-depth pathway analyses and measuring additional samples are underway. Lastly, differential methylation profiles for known risk factors of aggressive tumors will be evaluated. Citation Format: Yanning Wu, Cheryl L. Thompson, Fredrick R. Schumacher. Race-specific methylation profiles and epigenetic age acceleration differentiates estrogen receptor status breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3676.

Targeting HER2 protein in individual cells using ICP-MS detection and its potential as prognostic and predictive breast cancer biomarker

The human epidermal growth factor receptor 2 (HER2) is a transmembrane protein that has become one of the most specific prognostic and predictive biomarker of breast cancer. Its early detection is key for optimizing the patient clinical outcome. This work is focused on the detection of HER2 in individual cells using an antibody containing lutetium (Lu) as reporter group that is monitored by introducing the individual cells into the inductively coupled plasma mass spectrometer (ICP-MS). This Lu-containing antibody probe is used to label different breast cancer cell lines considered HER2 negative (MDA-MB-231) and positive (SKBR-3 and BT-474). Optimizations regarding the amount of the probe necessary to ensure complete labelling reactions are conducted in the different cell models. Concentrations in the range of 0.006 fg Lu/cell and 0.030 fg Lu/cell could be found in the HER2 negative and HER2 positive cells, respectively. In addition, the selectivity of the labelling reaction is tested by using two different metal-containing antibody probes for HER2 (containing Lu) and for transferrin receptor 1 (containing Nd), respectively, within the same cell population. Finally, the methodology is applied to the targeting of HER2 positive cells in complex cell mixtures containing variable amounts of BT-474 and MDA-MB-231 cells. The obtained results showed the excellent capabilities of the proposed strategy to discriminate among cell populations. This finding could help for scoring HER2 positive tumors improving existing technologies.

HER2 Overexpression and Amplification in Feline Pulmonary Carcinoma.

HER2 is overexpressed, amplified, and mutated in a subset of human lung cancer. The aim of this study was to investigate HER2 protein overexpression and gene amplification in feline pulmonary carcinomas. Thirteen pulmonary carcinomas were selected and TTF-1 and HER2 expression was evaluated by immunohistochemistry. Fluorescence in situ hybridization (FISH) was performed with a HER2 probe and a BAC probe for the feline chromosome E1p1.12-p1.11 region. Twelve adenocarcinomas and 1 squamous cell carcinoma were diagnosed. TTF-1 was positive in 7 carcinomas (58%). HER2 was overexpressed in 2 (15%), equivocal in 5 (38%), and negative in 6 cases (46%). FISH analysis of HER2 was indeterminate in 2 cases. Three pulmonary carcinomas (27%) had HER2 amplification and 8 cases were not amplified (73%). The significant correlation between HER2 protein overexpression and gene amplification are promising preliminary data, but study of additional cases is needed to confirm HER2 as a target for possible innovative treatments.

Abstract P4-09-09: Characteristics of stage 1 to 3 breast cancer patients with amplification of centromere of chromosome 17

Abstract Background: Amplification of the centromeric region of chromosome 17 (CEP17) as measured by In Situ Hybridization (ISH) of the CEP17 probe is used clinically as part of the ISH assay for HER2 status determination in breast cancer. The value of amplification of CEP17 beyond its use in the HER2 ISH test and characteristics of cases bearing CEP17 amplification, have not been fully explored. Methods: A retrospective review of charts of patients with stage 1 to 3 breast cancer that had a dual probe HER2/ CEP17 FISH test in our cancer center during an eight-year period was performed. Data on demographic, and cancer-specific characteristics of the included patients were extracted. The group of patients with an amplified CEP17 defined as mean copy number ≥3 per nucleus was compared with the group without amplification. Results: Two hundred and two patients were included in the analysis. Amplification of CEP17 was observed in 36 patients (17.8%). All patients in the amplified group had a concomitant amplification of HER2 (mean copy number ≥3 per nucleus). In the CEP17 non-amplified group 78 of 166 patients (47%) had an amplified HER2 status. More patients in the amplified group had a clinical HER2+ status according to the 3-protein classifier (33.3% versus 11.5% in the non-amplified group) and less patients in the amplified group had a clinical ER+/ HER2- status (63.9% versus 81.8% in the non-amplified group, x2 p=0.0003). Other significant differences between the amplified and CEP17 non-amplified groups were observed in their lymph node (LN) status (55.6% of patients in the amplified group versus 36.8% in the non-amplified group were lymph node positive, p= 0.04) and in the nuclear heterogeneity component of grade (90.9% of patients in the amplified group were nuclear grade 3 versus 66.9% in the non-amplified group, p= 0.02). There were no statistically significant differences between the groups in overall stage, grade, menopause status or histology. Conclusion: Patients with amplification of CEP17 had a co-amplified HER2 and were more commonly HER2+, LN positive and grade 3 in the nuclear component of grade. Citation Format: Ioannis Voutsadakis, Vanessa Davies. Characteristics of stage 1 to 3 breast cancer patients with amplification of centromere of chromosome 17 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-09.

Herceptin: A First Assault on Oncogenes that Launched a Revolution

This year's Lasker Clinical Research Award honors H. Michael Shepard, Dennis J. Slamon, and Axel Ullrich for their invention of Herceptin, the first monoclonal antibody that blocks a cancer-causing protein, and for its development as a life-saving therapy for women with breast cancer.

Equivocal (HER2 IHC 2+) breast carcinomas: gene-protein assay testing reveals association between genetic heterogeneity, individual cell amplification status and potential treatment benefits.

Genetic heterogeneity can pose a challenge to identifying eligible cases for targeted therapy in the human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) 2+ breast carcinoma group. In this study, we characterised this subset of tumours according to clinicopathological parameters. We assessed 1000 tumour cells per case and recorded the number of HER2 and chromosome enumeration probe 17 (CEP17) copies using gene-protein assay slides. HER2 status was determined based on ASCO/CAP 2013 guidelines. Tumours with 5-50% of cancer cells with amplification were considered to be heterogeneous, whereas those with >50% were considered to be non-heterogeneous. In a study cohort of 110 HER2 IHC 2+ carcinomas, 93 (84.5%) were non-amplified, 12 (10.9%) were amplified and five (4.5%) were ISH-equivocal. All the HER2-amplified and two of ISH-equivocal cases (12.7%) corresponded to non-heterogeneous tumours, with highly significant differences evident in the average HER2/CEP17 ratio (P=0.0002) and the proportion of cells with HER2 >6 copies (P<0.0001) compared with heterogeneous lesions. NST grade 3 and HER2-amplified carcinomas average HER2/CEP17 ratio correlated with an increased number of cells with HER2/CEP17 ≥2.0 (P<0.014). Triple-negative CEP17 polysomic carcinomas showed increased metastatic capacity (P=0.003) compared with other tumour types. Non-heterogeneous HER2 IHC 2+ tumours tend to be HER2-amplified. Adding the percentage of cells with HER2 >6 copies to the average HER2/CEP17 ratio may facilitate assessment of amplification status in ISH-equivocal cases. The proportion of cells with HER2/CEP17 ≥2.0 contributes information concerning the actual average HER2/CEP17 ratio, depending on tumour type.

Aptamer based fluorescent probe for serum HER2-ECD detection: The clinical utility in breast cancer

The transmembrane protein HER2 is overexpressed in approximately 30% of breast cancer patients. HER2-positive breast cancers tend to spread more aggressively, which result in increased mortality in women. Nowadays, the real-time monitoring of HER2 status is important in clinical diagnosis and treatment for HER2-positive patients. Although IHC and FISH assay are standard methods to evaluate the tissue HER2 status, both approaches which required high quality tissue samples and are not suitable for monitoring the status of HER2 in real time. Since extracellular domain (ECD) of the HER2 receptor can be shed into the circulation, the serum test of HER2 ECD has been developed as an additional approach to probe HER2 overexpression. The serum test will be able to monitor the dynamic changes of HER2 status. In this paper, we detected serum HER2 ECD using Cy5-labeled HB5 aptamer as a result of its specific binding ability to HER2 ECD. This aptamer-based fluorescent probe is easily synthesized and modified and as sensitive as anti-HER2 antibodies. We believe that Cy5-HB5 may have application potentials in serum HER2 test for clinical utility of breast cancer, such as recurrence and metastases.

HER2 equivocal breast cancer that is positive by alternative probe HER2 FISH are classified as HER2 negative by Oncotype DX.

In breast cancer, human epidermal growth factor receptor 2 (HER2) status is determined by immunohistochemistry (IHC) and/or insitu hybridization. Oncotype DX also reports HER2 status by an rt-PCR-based assay. Assay concordance between IHC and fluorescent insitu hybridization (FISH) (including alternative probe HER2 FISH) vs Oncotype DX HER2 rt-PCR has not been described in the post-2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 Guideline revision setting. We performed a retrospective review of HER2 equivocal invasive breast carcinoma from 2014 to 2016 with the Oncotype DX HER2 result. Fifteen patients with HER2 equivocal invasive breast cancer had Oncotype DX performed. Of these, 13 underwent alternative probe HER2 FISH yielding 4 negative, 6 equivocal and 3 positive results. All 15 cases were classified as HER2 negative by Oncotype DX rt-PCR, including the three cases which were positive by alternative probe HER2 FISH, yielding a discordance rate for Oncotype DX rt-PCR HER2 of 20% (3/15). All three patients with HER2-positive breast cancer on the basis of alternative probe HER2 FISH received anti-HER2-targeted therapy. Treatment decisions based on HER2 status should utilize the IHC/FISH result as Oncotype DX results may incorrectly disqualify some patients from being eligible for anti-HER2 therapy based on the current 2013 ASCO/CAP HER2 Guidelines.

Impact of 2013 American Society of Clinical Oncology/College of American Pathologists Guidelines on HER2 Fluorescent In Situ Hybridization Testing in Breast Cancers : Experience From a National Reference Laboratory.

We compared the impact of 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) testing results on breast cancers. HER2 FISH testing performed between May 2015 and April 2016 following 2013 ASCO/CAP guidelines was included. HER2 to control probe ratios, mean HER2, and control probe copy numbers were used to reassign HER2 status using 2007 ASCO/CAP and US Food and Drug Administration (FDA) guidelines. HER2 FISH results were available in 2,017 cases. A total of 342 (17.0%) cases were amplified, 301 (14.9%) were equivocal, and 1,374 (68.1%) were nonamplified. After additional testing with the alternate probe, amplified cases increased to 21.6%. HER2 positivity rates following the 2013 ASCO/CAP guidelines were significantly higher compared with the 2007 ASCO/CAP and FDA guidelines. The 2013 ASCO/CAP guidelines lead to a higher number of HER2 FISH positive and equivocal cases. In a reference laboratory setting where an alternative control probe was used to resolve equivocal FISH cases, 31.2% of patients with initial equivocal results became HER2 positive.

HER2 testing in advanced gastric and gastro-oesophageal cancer: analysis of an Australia-wide testing program

This Australian human epidermal growth factor receptor 2 (HER2) testing program aimed to analyse >800 cases tested in a coordinated setting to further evaluate the criteria to establish HER2 status in advanced gastric and gastro-oesophageal junction (GOJ) cancer. Heterogeneity, and minimum number of biopsy fragments for reliable HER2 assessment were also examined in a subset of samples. Five laboratories tested 891 samples referred to determine HER2 status for potential anti-HER2 treatment. Cancer site, specimen type (endoscopic biopsy/resection/metastases), immunohistochemistry (IHC) score, HER2 gene and CEP17 copy number (CN) and HER2:CEP17 ratios were recorded. Samples were derived from stomach (53.1%), GOJ (28.2%) or metastases (18.5%). IHC for HER2 and dual probe HER2:CEP17 in situ hybridisation (ISH) were performed in parallel. A stringent definition (SD) of HER2 positivity was used (IHC2+/3+plus CN>6 and ratio>2) and compared with other published criteria. HER2 positive rate was 13.9% (114/820) by SD, and 12.9-16.0% using other definitions. There was higher concordance between IHC and HER2 CN by ISH than with ratio. The HER2 positive rate was significantly higher in GOJ samples than others (p=0.03) and in endoscopic biopsies than resections (p=0.047). In a subset of 98 positive cases, 39 (39.8%) showed heterogeneity, and in 282 endoscopic biopsies positivity rate plateaued at five tumour fragments, suggesting this is the minimum number of biopsies that should be examined.

Impact of 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on HER2 fluorescent in situ hybridization (FISH) testing in breast cancers: Experience from a national reference laboratory.

e23188 Background: Accurate HER2 testing is expected to identify patients who will benefit from HER2 directed therapy (HDT) minimizing false positive and negatives. Like many biologic processes, HER2 protein expression and gene copy numbers are a continuum, however these tests are expected to be reported dichotomously. In 2013, ASCO/CAP revised the guidelines in an attempt to decrease false negative results and reverted to FDA approved ratio of ≥ 2.0 for HER2 positivity. We reviewed HER2 FISH testing results after the implementation of 2013 ASCO/CAP guidelines to determine the effects on HER2 reporting from our national reference laboratory. Methods: HER2 FISH testing performed between 5/2015-4/2016 at ARUP Labs following current 2013 ASCO/CAP guidelines was included. HER2 to control probe ratios, mean HER2 and control probe copy numbers were used to reassign HER2 status using 2007 ASCO/CAP, and FDA guidelines for each case. Results: HER2 FISH results were available in 2,017 cases. 342 (17.0%) cases were amplified, 301 (14.9%) were equivocal, and 1374 (68.1%) were non-amplified. After additional testing with alternate probe, 93 (31.2%) of the equivocal cases were reclassified as amplified increasing amplified cases to 21.6%. All of the equivocal cases which were reinterpreted as amplified with alternate probe showed low level amplification (range: 2.0-3.6; mean: 2.3). HER2 positivity rates following 2013 ASCO/CAP guidelines, both at initial testing and after additional testing to resolve equivocal results were significantly higher compared to 2007 ASCO/CAP guidelines and FDA criteria. Conclusions: 2013 ASCO/CAP guidelines lead to higher number of HER2 FISH positive and equivocal cases. In a reference laboratory setting where alternative control probe was used to resolves equivocal FISH cases, 31.2% of patients with initial equivocal results become HER2 positive. However, it is not known if these patients benefit from targeted therapies as they would not be included in the original adjuvant or metastatic trials.

Updated 2013 College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) guideline recommendations for human epidermal growth factor receptor 2 (HER2) fluorescent in situ hybridization (FISH) testing increase HER2 positive and HER2 equivocal breast cancer cases; retrospective study of HER2 FISH results of 836 invasive breast cancers.

For dual probe HER2 FISH assay, the 2013 CAP/ASCO guideline recommendations lowered the HER2/CEP17 ratio cut off for HER2 amplification to ≥2.0 and introduced an average HER2 copy number criterion for HER2 amplification (≥6.0/cell) and HER2 equivocal categories (≥4 and <6/cell). The HER2/CEP17 equivocal category is eliminated. The aim of this study is to assess the impact of 2013 HER2 FISH testing guideline recommendations update on the assignment of HER2 status with dual probe HER2 FISH assay. Dual probe HER2 FISH assay results on breast cancers from 09/2009 to 07/2015 that underwent reflex HER2 FISH testing after equivocal HER2 (2+) immunohistochemistry (IHC) were reviewed. HER2 copy number, CEP17 signals, and HER2/CEP ratios were noted. HER2 status was assigned as HER2 negative (HER2-), HER2 equivocal (HER2e), and HER2 amplified (HER2+) by applying both 2007 and 2013 CAP/ASCO HER2 FISH guideline recommendations and results were compared. New guidelines reclassified HER2 FISH status in a significant proportion of cases (8.3%, 69/836; p=.021). There were 22 (2.6%) more HER2+, 17 (2.1%) more HER2e, and 39 (4.1%) fewer HER2- tumors. Change of HER2 status correlated significantly with ≥3 CEP17 signals (38 vs. 2%; p<.001).The 2013 CAP/ASCO guideline recommendations for HER2 FISH testing by dual probe assay increased the HER2 amplified and HER2 equivocal tumors. Increase in HER2 equivocal tumors would potentially increase the frequency of repeat HER2 testing. Tumors with ≥3 CEP17 signals, so-called chromosome 17 polysomy, are more likely to be impacted and classified as HER2 equivocal.

Clinical role of HER2 gene amplification and chromosome 17: a study on 154 IHC-equivocal cases of invasive breast carcinoma patients.

Accurate evaluation of human epidermal growth factor receptor 2 (HER2) status is quite crucial for invasive breast tumor patients in order to select anti-HER2 therapy for effective clinical outcomes. Immunohistochemistry (IHC) assay is routinely used to evaluate the HER2 oncoprotein overexpression but is unable to explain the chromosomal and genetic alterations and has been considered as a hot issue in IHC-equivocal cases. We investigated these molecular aberrations in correlation with prognostic factors. A cohort of 154 IHC-equivocal (+2) cases was selected and retrospectively analyzed by dual-probe fluorescence in situ hybridization (FISH) assay by using locus-specific HER2 and centromere enumeration probes (CEP17) for the identification of HER2 proto-oncogene amplification and chromosomal copy number per cell, respectively. The data were analyzed by SPSS 16.0 version using chi-square test (p < 0.05). We identified 36 out of 154 cases (23.4%) showing HER2 gene amplification (average HER2 gene copies per cell >4 or <4 with HER2/CEP17 ratio >2) in concordance with HER2 oncoprotein overexpression, and significant correlation was observed with prognostic parameters including histological type, tumor grade II to III, histology and pathological type, lymphatic invasion, ductal carcinoma in situ (DCIS), and estrogen-positive and progesterone-negative receptors. Of the 154 cases, 18 cases (11.7%) showed polysomy 17 with CEP17 probe signals per cell ≥3 and 22 cases (14.3%) presented monosomy 17 (CEP17 probe signals per cell ≤1). Our data indicate that the use of anti-HER2 therapy should not be suggested unless true evaluation of HER2 protein expression is made regarding gene amplification essentially in IHC-ambiguous invasive breast tumors.

Development of RNA aptamers as molecular probes for HER2(+) breast cancer study using cell-SELEX.

Development of molecules that specifically recognize cancer cells is one of the major areas in cancer research. Human epidermal growth factor receptor 2 (HER2) is specifically expressed on the surface of breast cancer cells. HER2 is associated with an aggressive phenotype and poor prognosis. In this study we aimed to isolate RNA aptamers that specifically bind to HER2 overexpressing TUBO cell line. Panel of aptamers was selected using cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). Binding studies showed that selected aptamers can identify TUBO cell line with high affinity and selectivity. Our preliminary investigation of the target of aptamers suggested that aptamers bind with HER2 proteins on the surface of TUBO cells. We believe the selected aptamers could be useful ligands for targeted breast cancer therapy.

Abstract LB-212: HER2-overexpressing breast cancer xenografts amplify FGFR signaling upon acquisition of resistance to dual blockade with trastuzumab and lapatinib

Abstract We sought to discover mechanisms of acquired resistance to the combination of the HER2 inhibitors trastuzumab and lapatinib in HER2 gene amplified BT474 xenografts. The combination of lapatinib and trastuzumab completely eliminated BT474 tumors established in nude mice after &amp;lt;3 weeks of treatment. However, some tumors recurred as early as 9 weeks after the combination was stopped. Three recurrent tumors did not respond to retreatment with the combination of lapatinib and trastuzumab. More than 90% of tumors derived from these recurrent tumors did not respond to lapatinib/trastuzumab or the combination of trastuzumab and pertuzumab. Both the parental drug-sensitive xenografts and the lapatinib/trastuzumab-resistant (LTR) tumors exhibited 5 to 6-fold HER2 gene amplification as measured by FISH using probes for HER2 and chromosome 17. The LTR cancers maintained levels of P-HER2, P-AKT and P-ERK in the presence of lapatinib and trastuzumab as measured by IHC of tumor sections whereas treatment of parental BT474 xenografts reduced P-HER2 levels. LTR tumors exhibited markedly reduced in situ levels of lapatinib compared to BT474 sensitive tumors (2.157 µg lapatinib/gm tissue versus 4.701 µg lapatinib/gm tissue, p=0.02) as assessed by liquid chromatography-mass spectrometry (LC-MS). Quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation from tumor lysates revealed enriched P-Y877 HER2 in LTR compared to parental BT474 xenografts. We next performed next generation sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes. There were no mutations in HER2 but we observed amplification of FGF3, FGF4, and FGF19 genes in LTR xenografts. Consistent with increased autocrine activation of FGFR, we observed markedly elevated levels of P-FGFR in LTR xenografts compared to parental xenografts as assessed by IHC. We speculate that ligand-induced FGFR signaling may induce resistance to dual HER2 blockade by 1) a tumor cell autonomous mechanism resulting in transactivation of HER2 and, 2) an alteration in the tumor microenvironment that reduces lapatinib uptake and/or retention by the LTR tumors. In conclusion, we show amplification of FGFR signaling upon acquired resistance to combinations of HER2-targeted agents. Studies with FGFR tyrosine kinase inhibitors to reverse drug resistance are in progress. Citation Format: Joan T. Garrett, Jennifer Becker, Mónica Valeria Estrada, Carlos L. Arteaga. HER2-overexpressing breast cancer xenografts amplify FGFR signaling upon acquisition of resistance to dual blockade with trastuzumab and lapatinib. [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 LB-212. doi:10.1158/1538-7445.AM2014-LB-212

HER2 In Situ Hybridization in Gastric and Gastroesophageal Adenocarcinoma

Patients with gastric and gastroesophageal junction (GEJ) adenocarcinomas that are HER2 positive by immunohistochemistry (IHC) or in situ hybridization show a significant survival benefit with trastuzumab therapy. In situ hybridization is traditionally done by fluorescence in situ hybridization (FISH), despite some limitations. An alternative is the dual in situ hybridization (Dual ISH) technique that is fully automated and uses differentially labeled CEP17 and HER2 probes that can be read by light microscopy on 1 slide. The aim of this study was to assess the utility of Dual ISH in gastric/GEJ cancer and to compare the results with those obtained by IHC and FISH. Cases of gastric/GEJ adenocarcinoma were analyzed by IHC, FISH, and Dual ISH and the correlation between methods calculated. Results for 50 patients were available. There was a 98% (49/50) concordance rate between Dual ISH and FISH. One discrepant case was nonamplified by FISH but showed focal amplification by Dual ISH. Discrepancy was attributed to tumor heterogeneity, which was a frequent finding (78% of HER2-positive cases). There was excellent correlation between Dual ISH and FISH for assessment of HER2 amplification. Dual ISH was rapid, easy to interpret, and maintained cell morphology, which was valuable in identifying tumor heterogeneity.

Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue

Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

Abstract P1-07-01: Attainment of extremely high concordance rates between fluorescence in situ hybridization and immunohistochemistry in testing for human epidermal growth factor receptor 2 (HER2) in breast cancer using a normalized scoring system for immunohistochemistry: a four year experience with over 9000 cases

Abstract Background: The 2007 ASCO-CAP guidelines mandate a high level (95%) of concordance between different testing modalities such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for demonstration of the human epidermal growth factor receptor 2 (HER2) status in breast cancer. As few laboratories had been able to attain this level of concordance, the ASCO-CAP guidelines mandated regulation of pre-analytical variables such as fixation time in an attempt to improve concordance. We wished to test our hypothesis that the major reason for discordance between IHC and FISH results is false positive IHC, i.e., IHC 3+ immunostaining in cases which are non amplified by FISH, and that the vast majority of these false positives can be eliminated by using a normalized IHC scoring system. Materials and Methods: A total of 9,022 breast cancer cases, representing all those submitted to PhenoPath Laboratories for FISH testing from pathology laboratories around the United States between September 2008 and May 2012, had parallel sections immunostained with the rabbit monoclonal antibody SP3 to the HER2 gene product, with localization via a polymer based detection system. Tumor was scored according to ASCO-CAP criteria, as 0, 1+, 2+ or 3+; however, scoring was tabulated in two ways. The first was an absolute HER2 score based solely on analysis of the carcinoma, and the second was a normalized HER2 score, obtained by subtracting the score of the non-neoplastic breast epithelium from that of the infiltrating cancer. FISH was performed using the Vysis PathVysion combined HER2 and CEP17 probe kit with morphometric analysis performed using a MetaSystems image analysis system that incorporated the Metafer software. A score of &amp;gt; 2.2 was required for classification as amplified, and &amp;lt;1.8 for classification as non amplified. Results: Using the standard scoring system, negative concordance (0 or 1+ IHC, non-amplified by FISH) was obtained in 3448/3496 (98.6%) of cases, and positive concordance (3+ IHC, amplified by FISH) was obtained in 502/666 (75.3%) of cases, indicating the presence of significant numbers of falsely IHC positive cases. However, using the normalization method, the negative concordance rate remained unchanged at 3903/3960 (98.6%) but the positive concordance rate improved to 450/463 (97.2%). The absolute number of false IHC positive cases, using the normalization method, was reduced by over 90%, from 164 to 13. Discussion: Extremely high concordance rates, with elimination of the vast majority of false IHC positive cases, can be attained in a clinical laboratory examining tissues from divergent sources, even in the absence of controlling pre-analytical variables, by employing a normalized scoring system for IHC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-01.