HER2 Copy Research Articles

The Impact of 2013 Updated ASCO/CAP HER2 Guidelines on the Diagnosis and Management of Invasive Breast Cancer: A Single-Center Study of 1739 Cases

BackgroundThe purpose of this study was to determine the impact of revised ASCO/CAP 2013 HER2 guidelines on the clinical practice of pathologists and oncologists. Materials and MethodsRetrospective analysis of 1739 patients with invasive breast carcinoma who underwent reflex HER2 (fluorescence in situ hybridization [FISH]) testing, using both 2007 and 2013 guidelines (2007-2014). ResultsUsing 2013 guidelines, 255 (15%; 95% confidence interval [CI], 13%-16%) cases were classified as HER2+ as opposed to 186 (11%; 95% CI, 9%-12%) by 2007 guidelines (odds ratio [OR] 1.4; 95% CI, 1.2-1.8; P = .0005). Sixty-nine cases equivocal by 2007 guidelines (12% of all equivocal cases) were converted to HER2+ by 2013 guidelines. Sixty-two of these 69 cases shifted from HER2 equivocal to positive due to change in FISH ratio cutoff from 2.2 to 2.0. Six cases had FISH ratio < 2.0 but immunohistochemistry (IHC) score 3+ in 10% to 30% of tumor cells. One case had FISH ratio of 2.0 and IHC score 3+ in 10% to 30% of tumor cells. FISH and IHC test results were discordant in 5% (95% CI, 4%-6%) of cases using 2013 guidelines. No increase in HER2 FISH equivocal cases was observed. Reflex FISH testing of all IHC 1+ cases at our institution additionally detected 58 patients (5%; 95% CI, 4%-6%) with HER2 amplification. ConclusionsThe 2013 guidelines increase the detection of HER2+ cases, without introducing significant difference in discordance rate of the IHC and FISH assays. Inclusion of HER2 copy number criterion does not increase the number of FISH equivocal cases in our cohort. We recommend IHC 1+ cases should be offered reflex FISH testing because failure to test them will miss a small number (5%) of potentially treatable cases.

The role of HER2 overexpression in Middle Eastern papillary thyroid cancer

Background: HER2 oncogene is involved in many cancers and serves as a prognostic marker and therapeutic target in breast cancer. The purpose of this study was to learn more on the prevalence and clinical significance of HER2 overexpression and its association with clinical parameters in Middle Eastern papillary thyroid carcinoma (PTC). Methods: A tissue microarray (TMA) containing >1,000 PTC cases with follow-up data was used. TMA sections were analyzed on protein and DNA level using immunohistochemistry and fluorescence in situ hybridization (FISH). FISH analyses were performed to look for the gain or amplifications in HER2 gene. Results: Immunohistochemical analysis showed HER2 overexpression in 19.7% of our cases. Elevated expression was almost exclusively 2+ (194 tumors) and only rarely 3+ (1 tumor). No amplification was seen by FISH. Only 3% of our cases showed mildly elevated HER2 gene copy numbers not reaching the threshold for amplification. HER2 overexpression and copy number gains were unrelated to tumor stage, metastasis, patient survival and other clinical and pathological parameters. Conclusions: Our results demonstrated that HER2 overexpression occurs at relevant frequency in papillary thyroid cancer and in the absence of gene amplification. Additionally, expression of HER2 seems to hold no clinical value as prognostic factor in PTC.

Impact of an alternative chromosome 17 probe and the 2013 American Society of Clinical Oncology and College of American Pathologists guidelines on fluorescence in situ hybridization for the determination of HER2 gene amplification in breast cancer.

The dual-probe fluorescence in situ hybridization (FISH) assay for human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer provides an HER2:CEP17 (centromere enumeration probe for chromosome 17) ratio. Copy number alteration (CNA) in CEP17 may skew this ratio. The authors analyzed the impact of the 2013 American Society of Oncology/College of American Pathologists (ASCO/CAP) guidelines and an alternative chromosome 17 probe on HER2 status in tumor specimens with CEP17 CNA. Specimens with CEP17 CNA (n = 310) were selected from 3048 tumor samples that were received from January 2013 to June 2015 for testing with the alternative chromosome 17 probe D17S122. Reclassification of HER2 status was assessed using the 2007 and 2013 ASCO/CAP guidelines. The alternative chromosome 17 probe reclassified 82 of 310 (26.5%) and 87 of 310 (28.1%) tumors using the 2007 and 2013 guidelines, respectively. Of the 41 of 310 tumors (13.2%) that were reclassified from nonamplified to amplified according to 2007 guidelines, 28 of 41 (68.3%) had an average HER2 copy number ≥4.0 and <6.0. The 39 of 310 tumors (12.6%) that were reclassified from equivocal to amplified according to 2013 guidelines had a mean HER2 copy number between ≥4.0 and <6.0. Most of these patients had stage I, hormone receptor-positive, lymph node-negative tumors, which is an unusual clinicopathologic profile for HER2-amplified tumors, and most received HER2-targeted therapy in addition to endocrine therapy. Reflex testing with an alternative chromosome 17 probe using the 2013 ASCO/CAP guidelines reclassified 28.1% of tumor samples that had CEP17 CNA, converting nearly one-half from equivocal to amplified. The benefit of HER2-targeted therapy in this patient population requires further study. Cancer 2017;123:2230-2239. © 2017 American Cancer Society.

Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer

Fluorescence in situ hybridization (FISH) is one of the recommended techniques for human epidermal growth factor receptor 2 (HER2) status assessment on cancer tissues. Here we develop microfluidics-assisted FISH (MA-FISH), in which hybridization of the FISH probes with their target DNA strands is obtained by applying square-wave oscillatory flows of diluted probe solutions in a thin microfluidic chamber of 5 μl volume. By optimizing the experimental parameters, MA-FISH decreases the consumption of the expensive probe solution by a factor 5 with respect to the standard technique, and reduces the hybridization time to 4 h, which is four times faster than in the standard protocol. To validate the method, we blindly conducted HER2 MA-FISH on 51 formalin-fixed paraffin-embedded tissue slides of 17 breast cancer samples, and compared the results with standard HER2 FISH testing. HER2 status classification was determined according to published guidelines, based on average number of HER2 copies per cell and average HER2/CEP17 ratio. Excellent agreement was observed between the two methods, supporting the validity of MA-FISH and further promoting its short hybridization time and reduced reagent consumption.

RAI1 Alternate Probe Identifies Additional Breast Cancer Cases as Amplified Following Equivocal HER2 Fluorescence In Situ Hybridization Testing: Experience From a National Reference Laboratory.

-In 2013 the American Society of Clinical Oncology and College of American Pathologists updated the HER2 guidelines and changed the equivocal category for HER2 in situ hybridization testing to an average HER2 copy number of 4.0 to 5.9 with a HER2:CEP17 ratio of less than 2.0 and proposed retesting, with an option of using another control probe to avoid false-negative results. RAI1, located at band position 17p11.2, is a popular alternate probe locus for retesting equivocal changes. -To review experience with the RAI1 alternate probe in HER2 fluorescence in situ hybridization equivocal breast cancers. -Primary and metastatic breast cancers with equivocal HER2 fluorescence in situ hybridization, retested with an alternate (RAI1) probe, were identified. HER2, RAI1, and CEP17 copy numbers, HER2 to control probe ratios, and genetic heterogeneity were recorded. Hematoxylin-eosin-stained slides were reviewed for type and grade of cancer. -Of 876 cases tested with CEP17 as the reference probe, 97 (11.1%) had equivocal HER2 fluorescence in situ hybridization results. Additional testing with the RAI1 probe classified 39.2% cases (38 of 97) as amplified with a HER2:RAI1 ratio ranging from 2.0 to 3.2 (mean, 2.37); 3.1% (3 of 97) were still unclassifiable because of a deletion of RAI1. -RAI1 identified close to 40% of original HER2 fluorescence in situ hybridization equivocal cases as amplified, making these patients eligible for targeted therapies. It is not known whether guidelines for US Food and Drug Administration-approved probes can be extrapolated to alternate probes when an alternate control probe shows losses or gains. Because of the lack of guidelines for reporting HER2 status with alternate probes, laboratories face challenges in interpreting results.

Prognostic significance of equivocal human epidermal growth factor receptor 2 results and clinical utility of alternative chromosome 17 genes in patients with invasive breast cancer: A cohort study.

The 2013 testing guidelines for determining the human epidermal growth factor receptor 2 (HER2) status include new cutoff points for the HER2/chromosome enumeration probe 17 (CEP17) ratio and the average HER2 copy number per cell, and they recommend using a reflex test with alternative chromosome 17 probes (Ch17Ps) to resolve equivocal HER2 results. This study sought to determine the clinical utility of alternative Ch17Ps in equivocal cases and the effects of equivocal results and/or a change in the HER2 status on patients' outcomes. The University of Texas MD Anderson Cancer Center database of HER2 dual-probe fluorescence in situ hybridization results from 2000 to 2010 was searched for cases of invasive breast cancer with HER2/CEP17 ratios < 2 and average HER2 copy numbers < 6 per cell. Cases with HER2 copy numbers of 4 to < 6 (the definition of equivocal HER2 results) were analyzed with alternative Ch17Ps for Smith-Magenis syndrome and retinoic acid receptor α genes. Disease-free survival (DFS) and overall survival (OS) were evaluated with respect to the HER2 copy number with multivariate Cox proportional hazards regression. Among the 3630 patients meeting the inclusion criteria, 137 (4%) had equivocal HER2 results. With alternative Ch17Ps, 35 of 57 equivocal HER2 cases (61%) were upgraded to a positive HER2 status, and 22 cases (39%) remained unchanged. The 5-year DFS and OS adjusted hazard ratios (HRs) for copy numbers of 4 to < 6 versus < 4 were 0.6 (95% confidence interval [CI], 0.3-1.2) and 0.5 (95% CI, 0.2-1.0) with P values of .16 and .66, respectively. In comparison with HER2-negative cases, these CIs indicated that equivocal HER2 results were associated with either a protective effect (HR, < 0.5) or no effect (HR, 1.0). These findings rule out a significant deleterious effect of equivocal HER2 results. Alternative Ch17Ps may erroneously upgrade the HER2 status; therefore, they cannot be considered reliable in clinical practice. Cancer 2017;123:1115-1123. © 2016 American Cancer Society.

Clinical Overestimation of HER2 Positivity in Early Estrogen and Progesterone Receptor-Positive Breast Cancer and the Value of Molecular Subtyping Using BluePrint.

PurposeHuman epidermal growth factor receptor 2 (HER2) positivity is an important prognostic and predictive indicator in breast cancer. HER2 status is determined by immunohistochemistry and fluorescent in situ hybridization (FISH), which are potentially inaccurate techniques as a result of several technical factors, polysomy of chromosome 17, and amplification or overexpression of CEP17 (centromeric probe for chromosome 17) and/or HER2. In South Africa, HER2-positive tumors are excluded from a MammaPrint (MP; Agendia BV, Amsterdam, Netherlands) pretest algorithm. Clinical HER2 status has been reported to correlate poorly with molecular subtype. The aim of this study was to investigate the correlation of clinical HER2 status with BluePrint (BP) molecular subtyping.MethodsClinico-pathologic and genomic information was extracted from a prospectively collected central MP database containing records of 256 estrogen receptor–positive and/or progesterone receptor–positive tumors. Twenty-one tumors considered HER2 positive on immunohistochemistry or FISH were identified for this study.ResultsThe median age of patients was 56 years (range, 34 to 77 years), with a median tumor size of 16 mm (3 to 27 mm). Four (19%) tumors were confirmed HER2-enriched subtype, six (29%) were luminal A, and 11 (52%) were luminal B. The positive predictive values of HER2/CEP17 ratio ≥ 2 and HER2 copy number ≥ 6 were only 29% and 40%, respectively. The differences in means for HER2/CEP17 ratio were significant between BP HER2-enriched versus luminal (P = .0249; 95% CI, 0.12 to 1.21) and MP high-risk versus low-risk tumors (P = .0002; 95% CI, 0.40 to 1.06).ConclusionOf the 21 tumors considered clinically HER2 positive, only four were HER2-enriched subtype with BP, indicating an overestimation of HER2 positivity. FISH testing has a poor positive predictive value.

Abstract A019: Chimeric antigen receptors (CARs) as a low-impact treatment of pediatric ependymomas

Abstract Ependymoma is the third most common paediatric brain tumor and can occur anywhere along the neuroaxis, the most common location being the posterior fossa (PF). Surgery followed by radiation therapy (&amp;gt;4 years of age) is to date the most effective treatment regimen; chemotherapy is not part of the current standard of care as multiple clinical trials have failed to show any survival benefits. Despite extensive characterization of ependymoma, few credible oncogenes and tumor suppressor genes have been identified, in fact, up to 50% of PF ependymoma cases exhibit a balanced and bland genomic profile. Consequently, actionable mutations are not currently identifiable in the vast majority of cases. We are investigating candidate chimeric antigen receptor (CAR)- strategies to treat posterior fossa-A ependymoma (PFA) — a subtype that has the worst associated prognosis and is found predominately in infants. Probing of our extensive ependymoma gene expression dataset has identified several potential CAR target antigens, including HER2, EPHA2 and IL13Rα2, these have been further verified with tissue microarrays (TMAs). Using our orthotopic xenograft models of PFA ependymoma, we are confident from our preliminary analysis that HER2-positive PFA ependymomas respond to HER2-specific CAR treatment. Furthermore, the use of a TRI-CAR, tailored to target cells expressing EPHA2, IL13Rα2 and HER2, both collectively and independently, increases the efficacy and specificity as a therapy for both primary and recurrent ependymoma. To ensure we are using the most effective negative controls when confirming the therapeutic response of our CARs, we are silencing our genes of interest in our patient-derived ependymoma lines using shRNA and CRISPR-technology for HER2, EPHA2 and IL13Rα2. Whole-genome and whole-exome sequencing has demonstrated, that in comparison to PFB ependymomas, PFA tumors have more methylated CpG sites, and more genes that are transcriptionally silenced by CpG hypermethylation. To reduce this silencing and potentially improve therapeutic response to our CARs, we are using Azacitadine in combination with our CAR therapies. In parallel with our pre-clinical trials, we are performing safety trials to assess the administration of intravenous versus intrathecal HER2 CAR delivery. Patients presenting with a brain tumor frequently present with a leaky blood-CSF and blood-Brain barrier, therefore we aim to observe whether CARs injected intrathecally can be found within the blood and vice versa. Using a HER2 Copy Number Assay, we observed two significant results: firstly, HER2 CARs injected intrathecally, in the presence of a HER2-positive brain tumor, remain within the central nervous and are not found within the blood; secondly, HER2 CARs administered intraventrically in the presence of a HER2-positive brain tumor, migrate to the CSF (P = 0.009). Results from this project have the potential to create a paradigm shift in our approach to the treatment of ependymoma, including the initiation of a clinical trial for children with PFA ependymoma. This technology has the real potential to improve cure rates whilst also dramatically improving the quality of life for surviving patients by reducing the impact that current treatments have on normal brain development. Citation Format: Laura K. Donovan, Kevin J. Bielamowicz, Alex Manno, Nabil Ahmed, Michael D. Taylor. Chimeric antigen receptors (CARs) as a low-impact treatment of pediatric ependymomas [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A019.

Benchmarking a large regional UK HER2 testing service against current practice guidelines

AimsTo critically evaluate HER2 testing data for 3500 consecutive cases over 28 months for a single laboratory and review these findings in the light of current UK reporting guidelines.MethodsWe have...

Genomic Copy Number Analysis of HER2-Equivocal Breast Cancers.

Guidelines for HER2 testing define an equivocal range for HER2 using two approved testing methods, immunohistochemistry (IHC) and in situ hybridization (ISH). We investigated genome-wide copy number alterations in this subgroup. Ten breast cancers with equivocal HER2 status by both IHC and ISH were analyzed by single-nucleotide polymorphism cytogenomic microarray (SNP array). DNA ploidy analysis by flow cytometry was performed on nine cases with sufficient material remaining. SNP array analysis showed uniform gain of chromosome 17 (polysomy) in one case and segmental copy number gains encompassing HER2 and the centromere in five other cases. Flow cytometry revealed hyperdiploidy in six cases, all but one of which also had HER2 gains on SNP array. Although there was no evidence of HER2 amplification by SNP array, six cases showed amplification of other genomic regions, including known oncogenes in four cases. A combination of hyperdiploidy and segmental copy number gains contributes to HER2 ISH-equivocal results in most breast cancers. Cases in which HER2 copy number gain is not corroborated by genomic analysis suggest the presence of other contributing variables influencing ISH results. Genomic copy number analysis also implicates non-HER2 oncogenic drivers in many cases that are HER2 equivocal.

Copy Number Variation Analysis of Circulating Tumor Cells at a Single Cell Level Based on Hydrogel Encapsulation

Circulating tumor cells (CTCs) are defined as tumor cells circulating in peripheral blood of patients with metastatic cancer. CTCs have potential as a biomarker for cancer diagnosis or drug discovery. Recently, much attention has been paid to genomic analyses such as copy number variation (CNV) analysis of CTCs, because it will provide important information related to treatment for personalized therapy. These analyses should be performed at a single cell level because of the heterogeneity of cancer cells. However, CTCs are extremely rare cell in blood (1 in 109blood cells), so highly efficient technique for CTC recovery and single-cell isolation is required. Our group has developed “microcavity array system” on which rare CTCs can be efficiently captured from whole blood on the basis of differences in the size and deformability between tumor and blood cells. Furthermore, a novel technique for a single cell manipulation using a photo-polymerized hydrogel, “hydrogel-based cell encapsulation” has been demonstrated. In this study, to evaluate the performance of “microcavity array system” and “hydrogel-based cell encapsulation”, whole genome amplification (WGA) was performed using single-CTCs isolated by our proposed method. Furthermore, the utility of the proposed method in CNV analysis were evaluated. The microcavity array (φ= 8 μm, 4×103 cavities) was created by electroforming technique as microfabricated filter. The microcavity array was fabricated with PDMS structure equipped with a vacuum microchannel to apply negative pressure for cell entrapment. A549 cells (lung cancer cell line), SKBR-3 cells (breast cancer cell line) or MCF-7 cells (breast cancer cell line) were used as model CTCs. These cells were stained with fluorescent dyes, and suspended in PBS or healthy whole blood. These cell suspensions were introduced into the microcavity array at a flow rate of 150 µl/min. After cell recovery process, the number of cancer cells captured on the microcavity array was counted under a fluorescence microscope. As a results, more than 95% of cancer cells was successfully recovered on the microcavity array from PBS and whole blood. Then, poly (ethylene glycol) diacrylate (PEGDA) hydrogel was introduced onto the MCA. An excitation light (λ=365 nm) was irradiated to the targeted cancer cells for 30 seconds, leading to cross-linking reaction of PEGDA hydrogel and encapsulation of a single-cell. Solidified PEGDA hydrogel can be easily recovered from microcavity array by tweezers. Fluorescent microscopic analysis revealed that targeted single-cells were successfully encapsulated with PEGDA hydrogel. The hydrogel-based cell encapsulation process for 20 single-cells was completed within 10 min. To evaluate the effect of hydrogel on genome amplification, hydrogels with single-cells were subjected to the WGA, and the yields of WGA products were measured by PicoGreen assay. Genome amplification was also successfully carried out in all single-cells encapsulated on the hydrogel, and the yields of WGA products were the same with those obtained by using the micromanipulation as a conventional method. The amplification bias of WGA was evaluated based on the success rate of PCR amplification using 9 different primer sets (multiple loci). The PCR amplification of 9 fragments revealed that no significant bias was observed between WGA samples from single-cells isolated by hydrogel encapsulation and micromanipulation. Based on these results, we concluded that our single cell isolation method could be utilized for WGA at single cell level. Furthermore, quantitative PCR was performed to evaluate CNV analysis using SKBR-3 and MCF-7 cells, which have different HER2 status. As a result, the level of HER2 copy number in WGA products obtained from single-cells was similar with that from bulk analysis (103cells).Determination and monitoring of HER2 status in genome is well known to serve as a useful biomarker for diagnosis of breast cancer. Therefore, our proposed method for CNV analysis of CTCs is expected to be widely utilized towards real-time cancer diagnosis without repeating surgical biopsy.

Detection of HER-2 gene copy number variations as a molecular marker in the peripheral blood of women with endometriosis in Iranian population: Case-control study

Endometriosis is a common chronic and polygenic disease among women. Considering the importance of the relationship between endometriosis and the increasing risk of being diagnosed with various cancers among women with endometriosis, it has been demonstrated that genetic factors are clearly involved in half of these cases. The amplification/overexpression of growth factors as a dominant mechanism in developing cancer is of high importance in this progress. HER-2 is one of the main growth factors and contributors in tumorigenesis. The evident role of this biomarker in identifying and curing prevalent cancers among women is an indicator of its possible role in the cancer development process after being diagnosed with endometriosis. Thus, according to the distinctive genetic model of Iran's population, the aim of this study was to explore the relationship between HER-2 gene copy number variations and the risk of endometriosis in Iranian women. In a population-based case-control study conducted in Tehran, in-person interviews were completed for 100 women aged 15–49years and an equal number of controls frequency-matched to cases by age. All cases were newly diagnosed with endometriosis and all controls were with no laparoscopic evidence of disease between Dec 2014 and Aug 2015. We extracted the genomic DNA from peripheral blood and then HER-2 copy number was detected by real-time PCR. OR and 95% CI were calculated with Mann–Whitney test (P<0.05 was considered significant). An association was found between endometriosis and HER-2 copy number. Our study showed HER-2 copy number was increased by 2.89 fold (95% CI: 1.58–3.69) in patient group comparing to control group that was statistically significant. Regarding the significant difference between HER-2 gene copy number, it can be concluded that copy number variation, and as a result, variation in levels of HER-2 proto-oncogene expression could serve as a prognostic marker for investigating the chance of endometriosis -related cancers in women with endometriosis.

Comparison of HER2 Dual-Color and Fluorescence In Situ Hybridization in Breast Cancer

Human epidermal growth factor receptor 2 (HER2; ERBB2 gene) is of prognostic and predictive significance in breast carcinoma. Both fluorescence in situ hybridization (FISH) and dual-color in situ hybridization (DISH) methods are available. DISH and FISH are highly concordant in validation studies, but differences may be more prevalent in the equivocal range. Our goal was to compare FISH and DISH on a cohort enriched for equivocal cases, with respect to HER2 determination. The cohort was enriched for equivocal (2+) cases. DISH and FISH were evaluated using standard protocols and the results compared with respect to HER2 status, HER2 copy number, and HER2/chromosome 17 (Chr17) ratio. In total, 109 cases were identified. The agreement rate of DISH with FISH was 74%. The mean ± SD HER2/Chr17 ratio by DISH was 1.63 ± 0.08 vs 1.59 ± 0.26 by FISH (P = .45). The mean ± SD HER2 copy number by DISH was 4.56 ± 0.45 vs 4.75 ± 1.08 by FISH (P = .004). Individual signals were more easily resolved using FISH in cases with higher copy numbers. In our cohort enriched for equivocal cases, the numerical values of HER2 copy number were significantly lower using DISH, resulting in discordances. Although DISH is a valid method, variations with FISH may be expected in high-equivocal cases and in quality assurance activities.

Sticky Patches on Lipid Nanoparticles Enable the Selective Targeting and Killing of Untargetable Cancer Cells.

Effective targeting by uniformly functionalized nanoparticles is limited to cancer cells expressing at least two copies of targeted receptors per nanoparticle footprint (approximately ≥2 × 10(5) receptor copies per cell); such a receptor density supports the required multivalent interaction between the neighboring receptors and the ligands from a single nanoparticle. To enable selective targeting below this receptor density, ligands on the surface of lipid vesicles were displayed in clusters that were designed to form at the acidic pH of the tumor interstitium. Vesicles with clustered HER2-targeting peptides within such sticky patches (sticky vesicles) were compared to uniformly functionalized vesicles. On HER2-negative breast cancer cells MDA-MB-231 and MCF7 {expressing (8.3 ± 0.8) × 10(4) and (5.4 ± 0.9) × 10(4) HER2 copies per cell, respectively}, only the sticky vesicles exhibited detectable specific targeting (KD ≈ 49-69 nM); dissociation (0.005-0.009 min(-1)) and endocytosis rates (0.024-0.026 min(-1)) were independent of HER2 expression for these cells. MDA-MB-231 and MCF7 were killed only by sticky vesicles encapsulating doxorubicin (32-40% viability) or α-particle emitter (225)Ac (39-58% viability) and were not affected by uniformly functionalized vesicles (>80% viability). Toxicities on cardiomyocytes and normal breast cells (expressing HER2 at considerably lower but not insignificant levels) were not observed, suggesting the potential of tunable clustered ligand display for the selective killing of cancer cells with low receptor densities.

Impact of 2013 American Society of Clinical Oncology/College of American Pathologist guidelines on borderline immunostaining results for HER2: a retrospective study on HER2 FISH results in 1 780 cases of invasive breast cancers

To analyze the impact of the revised 2013 American Society of Clinical Oncology/College of American Pathologist(ASCO/CAP)HER2 testing guidelines on the status of HER2 and its clinical significance in invasive breast cancers by fluorescent in situ hybridization(FISH). One thousand seven hundred and eighty invasive breast cancer cases with equivocal 2+ immunostaining detected by FISH were retrospectively selected from 2010 to 2014, and the HER2/CEP17 dual-probe results were evaluated according to both the 2007 and 2013 ASCO/CAP guidelines for comparative analysis. Among the 1 780 IHC HER2 (2+ ) invasive breast cancers, the number of HER2 positive, equivocal and negative case were 310(17.41%), 66(3.71%)and 1 404(78.88%) respectively, basing on the 2007 guidelines; whereas basing on the 2013 ASCO/CAP HER2 guidelines, the number of HER2 positive, equivocal and negative case was 360 (20.22%), 182 (10.23%)and 1 238 (69.55%) respectively. Compared with the 2007 guidelines, the proportion of positive and equivocal cases were higher in the 2013 guidelines (17.41% versus 20.22%, 3.71% versus 10.23% respectively), while the proportion of negative cases was lower(78.88% versus 69.55%). Using the 2013 ASCO/CAP guidelines could lead to an increase in positive and equivocal cases, and a decrease in negative cases. The increase can probably be attributable to the inclusion of HER2 copy number besides HER2/CEP17 ratio as positive criteria, and it improves the accuracy and may be of important value for screening more population who benefit from HER2 targeting treatment; however the benefits for HER2 positive with low HER2 copy number and the clinical significance of the equivocal cases need to be further investigated.

HER2 gene copy number and breast cancer‐specific survival

HER2 amplification occurs in 10-15% of breast cancers. It is associated with poor breast cancer-specific survival (BCSS) and is an important prognostic and predictive marker. While it has been accepted that the HER2:chromosome 17 centromere (CEP17) ratio determines HER2 status, recent guidelines acknowledge the significance of HER2 copy number alone. The aims of this study were to assess BCSS according to mean HER2 copy number and HER2 status expressed as a HER2:CEP17 ratio with and without increased CEP17 copy number. The study population comprised breast cancer patients treated with surgery only and with long-term follow-up. In situ hybridization for HER2:CEP17 was performed on tissue microarrays and was successful in 679 cases. These were included in the study. Kaplan-Meier methods were used to estimate BCSS. A total of 47 cases had ≥4 < 6 HER2 copies; 16 were HER2+ and 31 were HER2- by ratio. Eighty-five cases had ≥6 copies of HER2 and only two of these were HER2- by ratio. The risk of death from breast cancer was increased among those with ≥6 HER2 compared to cases with 0-3.9 HER2 signals [hazard ratio (HR): 2.05; confidence interval (CI): 1.49-2.82 (unadjusted)]. After adjusting for stage, there was increased risk of death from breast cancer during the first 5 years after diagnosis in cases that were HER2- by ratio but with ≥4 < 6 HER2 (HR: 2.38; CI: 1.23-4.60). Increased copy number of HER2 may confer an increased risk of death from breast cancer despite negative HER2 status by ratio.

Effects of Estrogen Receptor and Human Epidermal Growth Factor Receptor-2 Levels on the Efficacy of Trastuzumab: A Secondary Analysis of the HERA Trial.

A number of studies suggest that response to antihuman epidermal growth factor receptor-2 (currently known as ERBB2, butreferred to asHER2 in this study) agents differs by estrogen receptor (ER) level status. The clinical relevance of this is unknown. To determine the magnitude of trastuzumab benefit according to quantitative levels of ER and HER2 in the HERceptin Adjuvant (HERA) trial. The HERA trial was an international, multicenter, randomized trial that included 5099 patients with early-stage HER2-positive breast cancer, randomized between 2001 and 2005 to receive either no trastuzumab or trastuzumab, after adjuvant chemotherapy. This is a secondary analysis of the HERA study. Local ER immunohistochemical (IHC) analyses, HER2 fluorescence in situ hybridization (FISH) ratio, and copy number results were available for 3037 patients (59.6%) randomized to observation and trastuzumab (1 or 2 years) (cohort 1). Transcript levels of ESR1 and HER2 genes were available for 615 patients (12.1%) (cohort 2). Patients were randomized to receive either no trastuzumab or 1 year vs 2 years of trastuzumab. Endocrine therapy was given to patients with hormone receptor-positive disease as per local guidelines. Disease-free survival (DFS) and overall survival (OS) were the primary and secondary end points in the intent-to-treat population (ITT). Analyses adjusting for crossover (censored and inverse probability weighted [IPW]) were also performed. Interactions among treatment, ER status, and HER2 amplification using predefined cutoffs were assessed in Cox proportional hazards regression models. Median follow-up time was 8 years. Levels of FISH and HER2 copy numbers were significantly higher in ER-negative patients (P < .001). In cohort 1, for DFS and OS, a significant treatment effect was found for all ER, IHC, and FISH levels, except for the ER-positive/HER2 low FISH ratio (≥2 to <5) group (DFS: 3-way ITT Pvalue for interaction = .07; censored = .02; IPW = .03; OS ITT Pvalue for interaction = .007; censored = .04; IPW = .03). In cohort 2, consistent with cohort 1, a significant predictive effect of the ESR1 gene for both end points was also observed (DFS Pvalue for interaction = .06; OS = .02), indicating that breast cancers with higher ESR1 levels also derive less benefit from trastuzumab. Patients with HER2-positive breast cancers that are ER-positive by IHC analyses with low FISH ratio (≥2 to <5), or with higher ESR1 levels derive significantly less benefit from adjuvant trastuzumab after chemotherapy. These data may explain heterogeneity in response to anti-HER2 agents in HER2-positive, ER-positive breast cancers as some may be more luminal-like than HER2 driven. clinicaltrials.gov Identifier: NCT00045032.

Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods

The National Institute of Standards and Technology (NIST) Standard Reference Materials 2373 is a set of genomic DNA samples prepared from five breast cancer cell lines with certified values for the ratio of the HER2 gene copy number to the copy numbers of reference genes determined by real-time quantitative PCR and digital PCR. Targeted-amplicon, whole-exome, and whole-genome sequencing measurements were used with the reference material to compare the performance of both the laboratory steps and the bioinformatic approaches of the different methods using a range of amplification ratios. Although good reproducibility was observed in each next-generation sequencing method, slightly different HER2 copy numbers associated with platform-specific biases were obtained. This study clearly demonstrates the value of Standard Reference Materials 2373 as reference material and as a calibrator for evaluating assay performance as well as for increasing confidence in reporting HER2 amplification for clinical applications.

Abstract 462: The role of Her2 overexpression in Middle Eastern papillary thyroid cancer

Abstract Thyroid cancer is the second most common malignancy among females in Saudi Arabia accounting for 7.4% of all cancers and 10.6% of all female malignant cancer. Among different subtypes, papillary thyroid cancer (PTC) is the most common subtype of thyroid cancer. Although the overall cancer related ten year survival rate for PTC has reached 90%, local, regional and distant metastasis does occur in 10% of cases. Therefore, in search for new therapeutic targets for PTC, we investigated the role of Her2 oncogene in PTC as this gene is known to be involved in other cancers such as breast cancer. The aim of this study was to determine the prevalence and prognostic role of Her2 overexpression in Middle Eastern papillary thyroid carcinoma by immunohistochemistry (IHC), Fluorescent In-Situ Hybridization (FISH) and clinical data. A tissue microarray (TMA) containing &amp;gt;1000 PTC cases with follow-up data was used. TMA sections were analysed at protein and DNA level using IHC and FISH. FISH analyses were performed to look for the gain or amplifications in Her2 gene. IHC analysis showed Her2 overexpression in 19.7% of our cases of PTC. Elevated expression was almost exclusively 2+ (194 tumors) and only rarely 3+ (1 tumor). Interestingly, no amplification of Her2 gene was visualized by FISH. Only 3% of our cases showed mildly elevated HER2 gene copy numbers not reaching the threshold for amplification. HER2 overexpression and copy number gains were unrelated to tumor stage, metastasis, patient survival and other clinical and pathological parameters. Our results demonstrated that Her2 overexpression occurs at relevant frequency in papillary thyroid cancer and in the absence of gene amplification. Additionally, expression of Her2 seems to hold no clinical value as prognostic factor in PTC Citation Format: Abdul K. Siraj, Shaham Beg, Rong Bu, Saif AlSobhi, Fouad Al-Dayel, Khawla Al-Kuraya. The role of Her2 overexpression in Middle Eastern papillary thyroid cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 462.

Abstract 509: HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant colorectal cancer

Abstract Background: Patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS benefit from epidermal growth factor receptor (EGFR)-targeted therapy. However, patients who are treated with anti-EGFR antibodies will eventually develop the resistance to those agents. We previously found that HER2 amplification was one of the mechanisms conferring resistance to anti-EGFR antibody therapy and could therefore be a potential therapeutic target (Yonesaka et al, STM 2011). The aim of this study was to detect HER2 amplification in circulating tumor DNA (ctDNA) from patients with CRC and acquired resistance to anti-EGFR antibody therapy. Methods: We analyzed plasma ctDNA using digital polymerase chain reaction (PCR) from 18 patients with CRC, who had been treated with anti-EGFR antibody-based therapy (cetuximab) and subsequently acquired resistant cetuximab. We aimed to obtain at least 100 droplets for HER2 and EFTUD2 to accurately assess the ratio. Digital PCR data were analyzed using the QuantaSoft analytical software package (Bio-Rad). The copy number concentration of each gene (HER2 and EFTUD2) was estimated from the Poisson distribution. HER2 amplification with digital PCR was defined as a HER2 ratio (HER2/EFTUD2 copy number ratio) of 1.25 accordingly (Gevensleven H et al, CCR 2013). HER2 gene copy number was analyzed using fluorescence in situ hybridization also in tumor samples before and after acquisition of resistance to cetuximab-based therapy. Results: Our data showed various HER2 copy number in plasma ctDNA from CRC patients (median: 1.09; range: 0.94-5.18). 22% (4/18) of patients in the cohort exhibited HER2 amplification. One of these patients was found to be positive for HER2 amplification in matched tumor specimens collected after cetuximab therapy, at which point the patient had acquired cetuximab resistance, despite being negative for HER2 amplification prior to therapy (pre: 1.14, post: 2.78). Conclusion: Analysis of plasma ctDNA by digital PCR could be useful for detecting HER2 amplification in patients with CRC who were resistant to anti-EGFR antibody therapy. We are now conducting another, large-scale study for evaluating HER2 amplification by ctDNA in CRC patients using digital PCR. Citation Format: NAOKI TAKEGAWA. HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 509.