HER2 Copy Number Research Articles

Clinicopathologic Characteristics of HER2-positive Metastatic Colorectal Cancer and Detection of HER2 in Plasma Circulating Tumor DNA

BackgroundTherapy targeting human epidermal growth factor receptor 2 (HER2, also known as ERBB2) is an effective approach for HER2-positive metastatic colorectal cancer (mCRC). HER2 status is typically determined using immunohistochemistry and fluorescence in situ hybridization. Circulating tumor DNA (ctDNA) enables noninvasive detection of gene mutations and copy number alterations including HER2 amplification. Materials and MethodsWe screened 351 patients with mCRC and studied the clinicopathologic characteristics of HER2-positive mCRC. HER2 expression in tumor samples measured with immunohistochemistry and fluorescence in situ hybridization was compared with HER2 copy number variation in plasma ctDNA detected by targeted sequence capture covering exons of 170 genes. We also examined the correlation between changes in tumor burden in ctDNA and antitumor response by imaging evaluation during the treatment course. ResultsPositive HER2 status was observed in 12 (3.4%) patients (7 males and 5 females), with a median age of 56 years. The HER2 concordance rate between tumor samples and ctDNA was 66.7% (20/30). Changes in tumor burden in ctDNA during the treatment course correlated with responses on imaging. ConclusionsDetection of HER2 copy number variation in ctDNA may be an alternative option for noninvasive determination of HER2 status. Tumor burden changes in ctDNA were consistent with imaging evaluation.

Abstract P6-17-38: Clinical and pathological features of breast cancer with 'polysomy' of Chromosome 17

Abstract Background: Anti-HER2 therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely evaluated using immunohistochemical stain and/or florescence in situ hybridization (FISH). Interpretation of FISH results may be challenging in tumors with 'polysomy' of chromosome 17 ('polysomy'17), which is defined by increased chromosome enumeration probe 17 (CEP17) signal number. There is evident that 'polysomy'17 might account for anti-HER2 therapy response in breast cancer (BC) with normal HER2/CEP17 ratio. This study aims to study the clinical and pathological features of BC with 'polysomy'17. Method: Primary BC were selected based on elevated CEP17 count (≥3.0) in HER2 FISH performed at MD Anderson Cancer Center between April 2014 and March 2018 (n=385). Patient charts were reviewed for detailed clinical and pathological features. These cases were further divided into four groups according to HER2/CEP17 and HER2 copy number, based on ASCO-CAP guidelines: group 1 (HER2+) – HER2/CEP17≥2.0, HER2 copies≥4.0; group 3 (HER2+) – ratio<2.0, HER2 copies≥ 6.0; group 4 (HER2 equivocal) – ratio <2.0, HER2 copies ≥4.0 and <6.0; group 5 (HER2-) – ratio<2.0, HER2 copies<4.0. Chi-square tests were performed to study the difference of these characters. Results: In comparison with groups 1 and 3, BC in groups 4 and 5 are more commonly seen in elder patients (p=0.001). Also, these tumors show higher pathological category (p<0.001) and higher rate of lymph nodes metastasis (p<0.05). The clinical and pathological features are summarized in [Table 1]. Conclusion: Understanding the clinical and pathological features of BC with 'polysomy'17 may help clinically to choose patients who might be benefit from anti-HER2 therapy. Further study is to follow up the therapy response of those who received anti-HER2 treatment in this cohort of patients. Clinical and pathological characteristics of breast cancer with 'polysomy' of chromosome 17 Total (N=385)Group 1 ( N=131)Group 3 (N=25)Group 4 (N=69)Group 5 (N=160)P valueAge (year)Mean (range)56.8 (25-94)53.9 (25-83)52.8 (26-75)58 (33-92)59.3 (29-94)0.001Race RaceBlack (%)37 (9.6)14 (10.7)4 (16)5 (7.2)14 (8.8)0.873 Hispanic (%)61 (15.8)23 (17.6)3 (12)12 (17.4)23 (14.4) White (%)251 (65.2)82 (62.6)14 (56)44 (63.8)111 (69.4) Other (%)36 (9.4)12 (9.1)4 (16)8 (11.6)12 (7.4) GenderFemale (%)383 (99.5)131 (100)25 (100)68 (98.6)159 (99.4)0.478 Male (%)2 (0.5)0 (0)0 (0)1 (1.4)1 (0.6) Histology typeIDC, NOS (%)350 (90.9)121 (92.4)20 (80)66 (95.7)143 (89.4)0.471 ILC (%)9 (2.3)3 (2.3)1 (4)0 (0)5 (3.1) Others (%)26 (6.8)7 (5.3)4 (16)3 (4.3)12 (7.5) Nuclear gradeI (%)4 (1.2)2 (1.8)0 (0)1 (1.8)1 (0.7)0.044 II (%)122 (37)30 (26.3)12 (50)19 (33.3)61 (45.5) III (%)203 (61.8)82 (71.9)12 (50)37 (64.9)72 (53.8) NA561711226 Pathological tumor categorypT0+Tis (%)61 (21)36 (36.3)3 (20)5 (10.9)17 (13.1)<0.001 pT1 (%)125 (43.2)39 (39.4)7 (46.7)20 (43.5)59 (45.4) pT2+T3+T4 (%)104 (35.8)24 (24.3)5 (33.3)21 (45.6)54 (41.5) NA9532102330 Pathological lymph node categorypN0 (%)187 (65.4)81 (82.7)11 (73.3)22 (47.8)73 (57.5)0.048 pN1+N2+N3 (%)99 (34.6)17 (17.3)4 (26.7)24 (52.2)54 (42.5) pNx (%)9933102333 Citation Format: Sun H, Chen H, Lim B, Sahin AA. Clinical and pathological features of breast cancer with 'polysomy' of Chromosome 17 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-38.

Abstract P3-10-25: Clinicopathologic factors associated with pCR to neoadjuvant anti-HER2-directed chemotherapy

Abstract Background: While the use of neoadjuvant therapy has become the standard of care in certain locally advanced breast cancers, questions remain regarding the optimal chemotherapeutic regimen as well as biologic and patient-specific predictors of pathologic complete response. In this study, we examined patient and tumor-specific characteristics associated with increased prediction of pathologic complete response (pCR) to neoadjuvant anti-HER2-directed chemotherapy. Methods: 204 patients who received neoadjuvant anti-HER2-directed chemotherapy at our institution from 2006-2016 were included in this retrospective study. Univariate analyses were performed to analyze the relationships of multiple clinical and pathologic features to pCR rate. Multivariate analysis was also performed to evaluate the relative impact of specific pathologic characteristics on pCR rate. Results: Among 204 patients with HER2 overexpressing breast cancers treated with neoadjuvant chemotherapy, 52.7% achieved pCR. Pathologic complete response was positively associated with high tumor grade (61.0% grade 3 in pCR group vs 39.0% in non-pCR group, P=0.23) and high Ki67 index (mean Ki67 59.1 in pCR group vs 47.4 in non-pCR; P= 0.015). It was also associated with HER2 IHC 3+ (57.1% in pCR group vs 42.9% in non-pCR, p=0.029), HER2 copy number (mean copy number 16.8 in pCR group vs 12.4 in non-PCR, p-0.004), and HER2/CEP 17 ratio (mean ratio 6.42 in pCR group vs 5.17 in non-pCR; P= 0.046). Rates of HER2 FISH positivity were equal in the pCR and non-pCR groups (50.8% vs 49.2%, p=0.062). Multivariate analysis demonstrated that higher Ki67 index and higher HER2/CEP17 ratio are significant predictors of pCR after adjusting for other covariates (odds ratio for Ki67 1.03 (1.06-1.49), p=0.002; for HER2/CEP17 ratio OR 1.26 (1.06-1.49), p=0.009). Conclusions: In HER2 positive breast cancers, a higher Ki67 index as well as higher HER2/CEP17 ratios are associated with an increased pCR rate and may be useful as predictors of response prior to neoadjuvant therapy. Our results also demonstrate HER2 IHC to be a stronger predictor than HER2 FISH of response to upfront therapy. Larger studies would be useful as this association, if confirmed, may have relevance to clinical practice. Citation Format: Meisel JL, Suo A, Taylor CE, Zhang C, Li X. Clinicopathologic factors associated with pCR to neoadjuvant anti-HER2-directed chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-25.

Abstract ES1-1: What is a HER2-positive in breast cancer in 2018?

Abstract Remarkable progress has been made in the treatment of patients with HER2-positive breast cancer since trastuzumab was approved by the U.S. Food and Drug Administration 20 years ago. Among patients with HER2-positive disease, HER2-targeted therapy has been associated with substantial improvements in overall survival in the adjuvant setting, a high rate of pathologic complete response (pCR) after neoadjuvant therapy, and improvements in survival in patients with metastatic disease. While much of these improvements in outcome is due to more effective therapies, improvements in patient selection as a result of more accurate HER2 testing has also contributed to the better prognosis of patients with this disease. Data from clinical trials examining the benefit of trastuzumab in the adjuvant setting and published in the early 2000s (e.g., NSABP B31; NCCTG 9831) demonstrated that up to 25% of community-based assays reported as HER2-positive by immunohistochemistry (IHC) and up to 15% reported as HER2-positive by fluorescence in situ hybridization (FISH) could not be confirmed upon central laboratory testing. To address this high frequency of false-positive results and to improve the accuracy of HER2 testing, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2007 published guideline recommendations for HER2 testing. These guidelines were subsequently updated in 2013 and, more recently, in 2018. Since the implementation of these guidelines, the accuracy of HER2 testing has improved. In fact, in current clinical practice, when HER2 IHC and FISH testing are performed according to the ASCO-CAP guidelines, approximately 95% of breast cancers can ultimately be categorized as either unequivocally HER2-positive or HER2-negative. In the remainder of the cases, combined IHC and FISH assay results provide a less straightforward picture of the HER2 status and, in turn, create uncertainty regarding the likelihood of benefit from HER2-targeted therapy. These include cases which on dual-probe FISH testing show: 1) HER2/CEP17 ratio >2.0 and an average HER2 copy number <4.0 signals/cell (monosomy); 2) HER2/CEP17 ratio <2.0 and an average HER2 copy number >6.0 signals/cell (polysomy); and 3) HER2/CEP17 ratio <2.0 and an average HER2 copy number >4.0 and <6.0 signals/cell. However, the most recent (2018) update of the ASCO-CAP guidelines provides pragmatic recommendations to resolve the HER2 status of such cases, and this, in turn, should result in even fewer cases in which the HER2 status remains in doubt. It should be noted, however, that not all patients whose tumors are HER2-positive by IHC and/or FISH respond similarly to HER2-targeted therapy and that some patients who initially respond subsequently develop resistance. Active efforts are underway to understand the mechanisms of de novo and acquired resistance to HER2-targeted treatments and to identify biomarkers that can predict resistance to therapy. Thus, the clinically relevant question in 2018 is not “what is a HER2-positive breast cancer ”? Rather, the question that now needs to be addressed is “which HER2-positive breast cancers will respond to HER2 targeted-treatments and to which HER2-targeted treatments are they most likely to respond? ”. Citation Format: Schnitt S. What is a HER2-positive in breast cancer in 2018? [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES1-1.

Abstract P4-01-13: Molecular characterization of circulating tumor DNA in Chinese metastatic breast cancer (mBC) patients

Abstract Background: Circulating tumor DNA (ctDNA) have been increasingly used as a minimal invasive test to detect genomic alterations of cancer patients. Methods: We performed a retrospective analysis of blood samples from mBC patients (pts) collected at baseline, on-treatment, and disease progression. A highly sensitive, plasma-derived ctDNA-based NGS assay (PredicinePLUS, a comprehensive 180 key cancer gene panel) was used to detect somatic mutations and copy number variations in ctDNA with an in-house propriety algorithm. Results: In this study, we evaluated 162 blood samples from 109 mBC pts who underwent systematic therapy at Beijing Cancer Hospital with approval from Research Ethics Committee. 129 samples from 92 pts passed NGS quality check, and were included in the analysis. Among these pts, the percent of HR+, HER2+ and Triple-Negative pts is 36.8%, 29.5% and 31.5%, respectively. About 86% samples contain somatic mutations and/or copy number variations. Collectively, 372 somatic mutations (SNV and Indels) were detected on 76 genes, among which TP53 (44.2%), PIK3CA (25.3%), BRCA2(10.5%) and ATM (10.5%) were frequently altered with frequency varying across different subtypes. ESR1 hotspot mutations ((D538G and Y537S/N/C) were also detected in a subset of HR+ patients. Copy number gain or loss was detected on 32 genes, including amplification of ERBB2, PIK3CA, FGFR1 and deletion of CDKN2A, ATM, RB1 etc. Importantly, ERBB2 copy number amplifications were only detected in HER2 IHC positive cases for base-line samples, leading to 68% sensitivity and 100% specificity. Interestingly, ctDNA yield increased as disease progressed, suggesting ctDNA yield may serve as a potential biomarker for predicting treatment response and monitoring disease progression. Additional genomic alterations that change dynamically along with the course of treatment or associate with drug response and/or resistance were also identified. Conclusions: This study demonstrated ctDNA-based genomic analysis was highly sensitive and specific in detecting various genomic alterations, consistent with other published studies. It also suggests that HER2 copy number amplification could be robustly assessed in a non-invasive manner. Citation Format: Yu J, Li H, Zhang S, Wang A, Zhao Z, Liu X-R. Molecular characterization of circulating tumor DNA in Chinese metastatic breast cancer (mBC) patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-13.

Comparison of HER2 expression by immunohistochemistry and HER2 copy number by digital PCR in tumor tissue during treatment with trastuzumab in the prospective cohort of metastatic HER2-positive gastric cancer patients.

58 Background: Trastuzumab (Tmab) is an active molecular-targeted drug for HER2-positive gastric cancer (GC) patients. HER2 expression is known to change during the treatment of Tmab and HER2 amplification in blood is widely investigated as a new biomarker of the treatment candidate or the monitoring of HER2-target therapy. We evaluated the change of the HER2 expression by immunohistochemistory (IHC) and HER2 copy number by degital PCR during the treatment with Tmab in metastatic HER2-positive GC patients. Methods: Metastatic HER2-positive GC patients treated with Tmab were registered prospectively, and tissues were obtained by biopsy from primary lesions at the following points: (1) pre-treatment, (2) post-treatment, and (3) disease progression during chemotherapy with Tmab. Formalin-fixed paraffin-embedded tissue slides were prepared, and the expression of HER2 expression were scored by IHC and HER2 copy number were evaluated by digital-PCR. Results: Among 20 enrolled patients, HER2 expression was evaluated by IHC in all patients. HER2 copy number was evaluated by digital PCR in 15 patients. A patient was excluded because HER2 expression were not detected by re-evaluation. The median of HER2 copy number of 33 sampling points during the treatment was 6.37 (range: 2.12 - 85.8). The median of HER2 copy number of IHC 3+, IHC 2+, IHC 0-1 were 22.4 (3.37 – 85.8), 2.88 (2.65 – 3.6), 2.37 (2.12 – 3.85), respectively. High level of HER2 copy number was detected in tumor tissues of HER2 IHC 3+ compared with that of IHC≦2+. HER2 expression by IHC was disappeared after treatment of Tmab in 42% of HER2-positive patients. Among these patients, HER2 copy number at pre-treatment were relatively low (2.63 - 3.6). Conclusions: High copy number of HER2 was detected in tumor tissues with HER2 IHC3+ in HER2-positive GC patients during the treatment of Tmab. In addition, low copy number of HER2 in tumor tissues at pre-treatment may be associated with the loss of HER2 expression after treatment of Tmab. Further research by large number of patients to confirm our results is required.

Distribution of Cholesterol and HER2 in Patient Breast Cancer Cells using Quantitative Single Molecule Localization Microscopy

The nano-scale organization of cholesterol in the plasma membrane is important for cell signaling and function. Cholesterol content of the outer membrane has been assessed previously with domain 4 of the Perfringolysin O toxin (PFO-D4). Here, we used PFO-D4 (C459A)-Alexa Fluor 647 to image cholesterol with quantitative single molecule localization microscopy (qSMLM). We first assessed cholesterol in cultured breast cancer cells. SK-BR-3 cells exhibited cholesterol-enriched domains with an average domain size of 29 nm. We then assessed tumors from breast cancer patients using a combination of qSMLM and tissue touch preparation (touch prep-qSMLM). Patient tumor samples exhibited cholesterol-enriched domains; their sizes ranged from ∼20 to 110 nm. Additionally, the tumor samples were assessed for human epidermal growth factor receptor 2 (HER2) using fluorescently labeled trastuzumab. Importantly, HER2 copy numbers from traditional testing (e.g. fluorescence in situ hybridization) showed a significant positive correlation with detected densities from touch prep-qSMLM. In the future, these single molecule studies on HER2 and cholesterol may help clarify mechanisms of oncogenic signaling and, ultimately, resistance to therapy.

HER2 immunohistochemical scores provide prognostic information for patients with HER2-type invasive breast cancer.

Invasive breast cancer patients with human epidermal growth factor receptor 2 (HER2) immunohistochemical (IHC) scores of 3+ or 2+ with reflex in-situ hybridisation (ISH) positivity are suitable for anti-HER2 therapies. The aim of this study is to investigate whether the prognoses between IHC 3+ patients and IHC 2+/ISH+ patients are different. We analysed the clinicopathological information of 886 consecutive cases of HER2-positive early breast cancer. The influences of the patients' age, cancer stage, hormone receptor status and anti-HER2 treatment were adjusted using a multivariate Cox regression model. Both HER2 copy numbers and HER2 ISH ratios of the IHC 3+ group were significantly higher than those of the IHC 2+/ISH+ group. The outcomes of IHC 3+ patients were significantly better than those of IHC 2+/ISH+ patients in the univariate and multivariate analyses. HER2 copy numbers of ≥8 represented the best prognostic value, and it was chosen to be the cut-off value. The reflex ISH for IHC 2+ patients with high HER2 copy numbers (≥8) predicted a better overall survival than that for those with low HER2 copy numbers. HER2 IHC scores and HER2 copy numbers can provide prognostic information for patients with HER2-positive invasive breast cancer. Both IHC 3+ and IHC 2+ patients with high HER2 copy numbers had a better prognosis.

Assembled Step Emulsification Device for Multiplex Droplet Digital Polymerase Chain Reaction.

Digital PCR is a powerful method for absolute nucleic acid quantification with unprecedented accuracy and precision. To promote the wider use and application of digital PCR, several major challenges still exist, including reduction of cost, integration of the instrumental platform, and simplification of operations. This paper describes a reusable microfluidic device that generates nanoliter droplet arrays based on step emulsification for the on-chip multiplex digital PCR of eight samples simultaneously. The device contains two glass plates that can be quickly assembled with prefilled mineral oil. Droplets are simply generated through the arrays of step emulsification nozzles driven by a single pressure controller and are self-assembled into monolayer droplet arrays in U-shaped chambers. The use of mineral oil eliminates bubble generation; thus, no overpressure is required during thermocycling. Moreover, the device can be reused many times after disassembly and a brief cleaning procedure, which significantly reduces the cost of the device per dPCR assays. The device was able to detect template DNA at concentrations as low as 10 copies/μL with a dynamic range of approximately 4 logs. We applied this device in the quantitative assessment of HER2 copy number variation, which is important for targeted therapy and prognosis of breast cancer. The performance was validated by 16 clinical samples, obtaining similar results to commercial digital PCR. We envision that this low-cost, reusable, and user-friendly device can be broadly used in various applications.

Role of clustered HER2/neu amplification as a marker for a special sensitivity to neoadjuvant anti-HER2 therapy with trastuzumab in patients with stage II-III breast cancer

To evaluate the influence of clinical and morphological factors and HER2 copy numbers on pathologic complete response (pCR) rates in patients with HER2-positive stage II-III breast cancer (BC). Treatment results were studied in 73 patients with HER2-positive Stage II-III BC, who received treatment at the N.N. Blokhin National Medical Research Center of Oncology in 2015 to 2018. Treatment included neoadjuvant chemotherapy (NACT) with HER2-blockade and radical surgery followed by the evaluation of a pathologic response in the primary tumor and regional lymph nodes. The patients` age varied from 29 to 71; its median was 51.5; 45.2% of patients had primary operable stages (T1-3N0-1) and 54.8% had locally advanced tumors. All the patients had grade 2-3 anaplasia; luminal HER2-positive BC was diagnosed in 41.4% of patients; hormone-negative tumors were seen in 58.9%; 91.5% of patients had Ki-67 ≥20% in 75.3% of patients, preoperative systemic therapy included anthracycline-containing regimens (4AC + 4 x paclitaxel 175 mg/m2/12 × weekly administrations of paclitaxel 80 mg/m2; trastuzumab therapy was simultaneously performed with the administration of taxanes in the standard regimen) and anthracycline-free regimen TCH ± Pertuzumab regimen in 24.7% of cases. After NACT patients underwent surgery (radical mastectomy in 78.1%, breast-sparing treatment in 21.9%) with the assessment of morphological findings. Biopsy specimens obtained before the treatment was restudied; HER2 amplification was detected using a Dako HER2 IQFISH pharmDx kit according to its instruction and the 2018 ASCO/CAP guidelines. In 87.1% of cases, the HER2-positive status corresponded to the first category of the 2018 ASCO/CAP criteria for HER2-positive BC; clustered HER2 amplification was found in 30.1% of cases. The authors analyzed the frequency of bpCR and tpCR attainment by various clinical and morphological factors, as well as the impact of a HER2 amplification level on pCR rates. A breast pCR (bpCR) was achieved in 57.4% patients; bpCR and lymph node CR (lnCR) were noted in 48.9% patients. The rates of bpCR significantly depended on female age, chemotherapy regimen, addition of Pertuzumab, and HER2 copy number. That of bpCR in women less than 35 years of age, in those aged 36-50 years, and in those aged older than 50 years was 22.2, 57.7 and 71.9%, respectively (p=0.026). The maximum bpCR rate observed with the TCH±P regimen was 80.0%, that with anthracycline-containing regimes was 52.8% (p=0.045), and the addition of Pertuzumab increased complete response rates up to 88.9% (that with Trastuzumab was 54.2% (p=0.049). The relationship of bpCR rates to the detection of cluster amplification turned out to be highly significant (81% in its detection and 48.9% in its absence (p=0.013). In addition, clustered HER2 amplification was the only significant predictive factor for complete regression in the primary tumor and lymph nodes: in its presence, the tpCR rate reached 68.8% versus 38.7%. Clustered amplification of the HER2 gene is the most significant factor of sensitivity to anti-HER2 therapy for Stage II-III BC, and is associated with the maximum rate of both bpCR and total pCR. Further study of this factor may assist in optimizing the treatment algorithm for HER2 + BC.

Hormone Receptors and Human Epidermal Growth Factor (HER2) Expression in Fine-Needle Aspirates from Metastatic Breast Carcinoma - Role in Patient Management.

Introduction:Estrogen receptors (ER), progesterone receptors (PR), and epidermal growth factor (HER2) are prognostic and predictive factors for breast carcinoma. We determined them by immunohistochemistry (IHC) on cell blocks from fine-needle aspirates (FNA) of metastatic breast carcinoma to axillary lymphnodes and compared them with that reported in the primary breast carcinoma (PBC) to document any change in their expression for future management.Materials and Methods:ER, PR, and HER2 by IHC and HER2 oncogene by fluorescent in-situ hybridization (FISH) were studied on cell blocks of FNA of axillary lymphnodes in 53 of 94 PBC cases from 2012 to 2016.Results:In 25 of 38 (65.8%) ER, PR negative PBC the metastasis on FNA was ER, PR+, whereas the 15 (28.3%) ER, PRPBC remained negative. In 10 of 11 (91%) of HER2-IHC+, PBC the metastatic tumor was HER2-IHC+. 7 of 32 (21.9%) HER2-IHC negative PBC were HER2-IHC+ in metastatic tumor. HER2-FISH was performed in 37 cases on FNA. Six of 37 were HER2 amplified/positive, whereas 9 and 19 remained equivocal and negative for HER2 copy number, and 3 were not interpretable. All the 6 HER2-FISH+ cases were positive by IHC. In our study, 34.2% of ER, PR+ cases of PBC became ER, PR– in the metastatic tumor and 21.9% of HER2-IHC negative PBC became HER2-IHC+ in the metastatic aspirate.Conclusion:ER, PR, and HER2 by IHC in cell blocks of metastatic lymphnodes are reliable. Change in receptor (34.2%) and HER2 status (21.9%) was documented, which is of clinical significance as these patients warrant a change of management.

Liquid biopsy: a powerful tool to monitor trastuzumab resistance in HER2-positive metastatic gastric cancer

Human epidermal growth factor receptor 2 (HER2) overexpression/amplification affects about 6.1%–23.0% of gastric cancer patients [1, 2]. All major guidelines recommend HER2 testing to guide the selection for trastuzumab treatment in metastatic gastric cancer (mGC) [3], because doublet chemotherapy with trastuzumab significantly improved overall survival (OS) in the crucial trastuzumab for gastric cancer trial (the ToGA trial) [4]. However, the unsatisfactory gain of median progression-free survival (PFS) stresses important issues of intrinsic and acquired resistance to HER2-targeted therapies [4]. Currently, the underlying molecular mechanism of trastuzumab resistance in mGC is still unclear, and the strategies to real time monitor resistance-specific aberrations are urgently needed. As circulating tumor DNA (ctDNA) carries the same genomic alterations that are present in the primary tumor [5, 6], liquid biopsy-based ctDNA profiling offers a unique opportunity for serially monitoring treatment response in a non-invasive manner, and has opened the door for identification the mechanisms of drug resistance. In a study recently published in Gut, titled “Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer”, Wang et al. [7] demonstrated for the first time that longitudinal ctDNA sequencing could promisingly predict tumor shrinkage and progression in HER2 + mGC patients under trastuzumab treatment. Based on the detected molecular alterations from the ctDNA profiling, they further dissected various mechanisms of trastuzumab resistance (Fig. 1). Longitudinal circulating tumor DNA sequencing provides novel insights into gene alterations underlying trastuzumab resistance in HER2-positive metastatic gastric cancer. HER2+: HER2-positive; mGC: metastatic gastric cancer; ctDNA: circulating tumor DNA: Copy No.: copy number In this study, the authors sequenced 416 clinically relevant genes to determine the genomic profiles of GC patients. The molecular alterations, including copy number alterations, mutations and structural variations, detected in 78 paired plasma and tissue samples (46 from HER2+ patients and 32 from HER2− patients) were used to evaluate the consistency of detection between the “liquid” and “solid” biopsies. As expected, the alterations detected in plasma provided a good representation of the status in the tumor tissues. In particular, HER2 amplification status, the major marker of HER2 + GC, detected by plasma-based ctDNA sequencing was highly consistent with that by tissue-based sequencing and fluorescence in situ hybridisation. These findings indicated that plasma-based ctDNA profiling might provide insights into HER2 amplification in HER2 + GC. Further longitudinal analysis were perform in 97 time-course plasma samples collected from 24 HER2 + mGC patients to track the resistance during trastuzumab treatment. As compared to plasma carcinoembryonic antigen, the copy number variations (CNVs) of HER2 in ctDNA were better at tracking trastuzumab treatment response. Generally, the HER2 copy number decreased during treatment compared with baseline and levels of progressive disease (PD), indicating tumor shrinkage. Additionally, most patients with intrinsic resistance-related progression presented higher HER2 copy numbers at PD than baseline, while the patients with acquired resistance often had lower HER2 copy numbers at PD. From the same ctDNA profiles, the authors found evidence for reported mechanisms of trastuzumab resistance in 76.5% patients. They found that the patients with multiple resistance mechanisms (PIK3CA/R1/C3 and ERBB2/4 mutations) displayed much shorter PFS during trastuzumab treatment. These findings suggested that liquid biopsy-based ctDNA profiling can effectively capture the heterogeneity information about acquired resistance. Furthermore, they also observe several novel resistance mechanisms. One of them is the ERBB4 S774G mutation. This novel mutation can increase the sensitivity to trastuzumab treatment, suggesting for the first time that the mutation of ERBB4 play a critical role in mediating resistance to ERBB2 inhibitors in HER2 + GC. Another novel resistance mechanism is the emergence of NF1 mutations. Using in vitro and in vivo experiments, the authors demonstrated that NF1 was a resistance-related gene and could be used as a potential resistance marker of trastuzumab. NF1 lost (mutation or deletion) contributes to driving resistance of HER2-targeted therapy via elevating pMEK/ERK activation, whereas the combination of HER2 and MEK/ERK inhibitors might overcome trastuzumab resistance. This study has some obvious limitations. First, other rare genomic or even nongenomic mechanisms of trastuzumab resistance may have been missed by the 416-gene panel. Second, the relatively small number of patient enrolled does not allow drawing definitive conclusion on the predictive value of this liquid biopsy approach. Nevertheless, the study provides helpful information to monitor the treatment response of trastuzumab and develop therapeutic strategies for the HER2 + mGC patients with trastuzumab resistance. The author read and approved the final manuscript. Not applicable. The author declares no competing interests. Not applicable. Not applicable. Not applicable. Not applicable.

Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2

All breast cancers are assessed for levels of human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) and immunohistochemistry are currently used to determine if a patient is eligible for anti-HER2 therapy. Limitations of both tests include variability and relatively long processing times. Additionally, neither test determines whether HER2 contains the extracellular domain. While truncated in some tumors, this domain is required for binding of the therapeutic antibody trastuzumab. Here, trastuzumab was used to directly detect HER2 with quantitative single molecule localization microscopy (qSMLM). In proof of concept studies, our new method rapidly quantified both HER2 density and features of nano-organization. In cultured cells, the method was sensitive to subtle variations in HER2 expression. To assess patient samples, we combined qSMLM with tissue touch preparation (touch prep-qSMLM) and examined large areas of intact membranes. For cell lines and patient samples, HER2 copy numbers from FISH showed a significant positive correlation with detected densities from qSMLM and trended with HER2 cluster occupancy.

Droplet digital PCR using HER2/EIF2C1 ratio for detection of HER2 amplification in breast cancer tissues

Breast cancers with amplification and overexpression of human epithelial growth factor receptor 2 (HER2) are associated with poor prognosis, and targeted for anti-HER2 therapy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently the recommended methods to asses HER2 overexpression/amplification. Droplet digital PCR (ddPCR), a highly accurate method to quantify DNA copy number, is potentially a robust alternative for HER2 diagnostics. In the FISH assay and most of previous ddPCR reports, chromosome 17 centromere (CEP17) has been used as the reference control to determine HER2/CEP17 ratio. Nevertheless, miss-classification could occur when HER2 is co-amplified with CEP17. To avoid this inherent defect, in the present study, we employed ddPCR assay using the human eukaryotic translation initiation factor 2C1 (EIF2C1) gene located at chromosome 1p34.3 as the reference control to quantify HER2 copy number in 31 frozen breast cancer tissues. HER2 status of these samples had been determined by FISH and classified as HER2-amplified and HER2-non-amplified breast cancers. The results showed that HER2 determined by ddPCR using HER2/EIF2C1 ratio was in good concordance with HER2 determined by FISH using HER2/CEP17 ratio, the concordance rate 87.1% (27/31), Kappa = 0.719. The sensitivity and specificity of ddPCR assay was 90% (9/10) and 85.7% (18/21), respectively. The median HER2/EIF2C1 copy number ratio in HER2-amplified cancers (6.55, range 1.3–17.3) was significantly higher than in HER2-non-amplified cancers (1.05, range 0.6–3.6, p < 0.001). This study demonstrated that ddPCR using HER2/EIF2C1 ratio could accurately assess HER2 status in frozen breast cancer tissues. Thus, our findings warrant further studies into breast cancer with HER2-equivocal by IHC/FISH.

Intratumor heterogeneity defines treatment-resistant HER2+ breast tumors.

Targeted therapy for patients with HER2‐positive (HER2+) breast cancer has improved overall survival, but many patients still suffer relapse and death from the disease. Intratumor heterogeneity of both estrogen receptor (ER) and HER2 expression has been proposed to play a key role in treatment failure, but little work has been done to comprehensively study this heterogeneity at the single‐cell level. In this study, we explored the clinical impact of intratumor heterogeneity of ER protein expression, HER2 protein expression, and HER2 gene copy number alterations. Using combined immunofluorescence and in situ hybridization on tissue sections followed by a validated computational approach, we analyzed more than 13 000 single tumor cells across 37 HER2+ breast tumors. The samples were taken both before and after neoadjuvant chemotherapy plus HER2‐targeted treatment, enabling us to study tumor evolution as well. We found that intratumor heterogeneity for HER2 copy number varied substantially between patient samples. Highly heterogeneous tumors were associated with significantly shorter disease‐free survival and fewer long‐term survivors. Patients for which HER2 characteristics did not change during treatment had a significantly worse outcome. This work shows the impact of intratumor heterogeneity in molecular diagnostics for treatment selection in HER2+ breast cancer patients and the power of computational scoring methods to evaluate in situ molecular markers in tissue biopsies.

Breast cancers with a HER2/CEP17 ratio of 2.0 or greater and an average HER2 copy number of less than 4.0 per cell: frequency, immunohistochemical correlation, and clinicopathological features

The 2013 American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) guidelines classified breast cancers with a fluorescence in situ hybridization dual-probe HER2/CEP17 ratio of 2 or greater as "amplified," inclusive of cases with a HER2 copy number less than 4. The 2018 ASCO/CAP update assigns HER2/neu status for the latter group in a fashion that is highly dependent on the associated immunohistochemical findings. Herein, the authors define the frequency, immunohistochemical correlates, and other clinicopathological features of breast cancers with HER2/CEP17 ratio of 2 or greater and HER2/neu copy number less than 4 (group A), based on an analysis of an institutional cohort assessed for HER2/neu status by both florescence in situ hybridization and immunohistochemistry and scored using 2013 ASCO/CAP criteria. Group A cases were compared with a group B of HER2/neu-amplified breast cancers with a HER2/neu copy number of 4 or greater regarding a variety of clinicopathological features. One hundred sixty-nine (14%) of 1201 cases were HER2/neu amplified, 18 (10.7%) in group A and 151 (89.3%) in group B. By immunohistochemistry, 61.1% of group A cases were HER2/neu negative, 7 (38.9%) were equivocal, and none were positive. In contrast, 66.9% of group B cases were HER2 positive (3+). We could not demonstrate statistically significant differences between the 2 groups regarding standard clinicopathological variables. In summary, our group A cases account for 1.5% of breast cancers, and 10.7% of all HER2/neu-amplified cancers classified as such based on 2013 ASCO/CAP criteria. They are predominantly HER2/neu negative by immunohistochemistry, which suggests that they are biologically different from classically HER2/neu-amplified cases and which validates the 2018 ASCO/CAP guideline against automatically classifying such cases as HER2/neu amplified.

Trastuzumab and paclitaxel in patients with EGFR mutated NSCLC that express HER2 after progression on EGFR TKI treatment

BackgroundHER2 expression and amplification are observed in ~15% of tumour biopsies from patients with a sensitising EGFR mutation who develop EGFR TKI resistance. It is unknown whether HER2 targeting in this setting can result in tumour responses.MethodsA single arm phase II study was performed to study the safety and efficacy of trastuzumab and paclitaxel treatment in patients with a sensitising EGFR mutation who show HER2 expression in a tumour biopsy (IHC ≥ 1) after progression on EGFR TKI treatment. Trastuzumab (first dose 4 mg/kg, thereafter 2 mg/kg) and paclitaxel (60 mg/m2) were dosed weekly until disease progression or unacceptable toxicity. The primary end-point was tumour response rate according to RECIST v1.1.ResultsTwenty-four patients were enrolled. Nine patients were exon 21 L858R positive and fifteen exon 19 del positive. Median HER2 IHC was 2+ (range 1–3). For 21 patients, gene copy number by in situ hybridisation could be calculated: 5 copies/nucleus (n = 9), 5–10 copies (n = 8), and >10 copies (n = 4). An objective response was observed in 11/24 (46%) patients. Highest response rates were seen for patients with 3+ HER2 IHC (12 patients, ORR 67%) or HER2 copy number ≥10 (4 patients, ORR 100%). Median tumour change in size was 42% decrease (range −100% to +53%). Median duration of response was 5.6 (95% confidence interval [CI], 3.8 to 7.3) months. Treatment toxicity was mild with four patients experiencing grade ≥3 toxicity, including fatigue, neuropathy, neutropaenia, urinary tract infection, and pneumonitis.ConclusionsTrastuzumab-paclitaxel induces objective tumour responses in 46% of EGFR TKI pretreated patients with an activating EGFR mutation and HER2 expression. The treatment was well tolerated. The relation between response rate and HER2 expression level and copy number suggests effective HER2 targeting by trastuzumab, although the combination with paclitaxel does not allow to determine the relative contribution of the individual drugs in terms of treatment efficacy.

Abstract 4776: Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer

Abstract Purpose: Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-HER2 drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. Among HER2 targeting drug, afatinib and neratinib inhibit the activation of all HER family protein, and are called pan-HER inhibitors. In this study, we examined the effect of these pan-HER inhibitors to gastric cancer cells. Materials and Methods: We determined the molecular profiles of 12 gastric cancer cell lines. Protein level of HER2 and down-signal pathway molecules were analyzed by Western blotting, and copy number assay or gene expression assay were performed using qPCR. To detect HER2 mutation, we also performed direct-sequence of these cell lines. And next, we examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines, in vitro and in vivo. In addition, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. Results: HER2 was amplified in 5 out of 12 gastric cancer cell lines. Gene expression or protein level of HER2 were generally correlated with the copy number of HER2. HER2 mutation was found in one cell line, ECC10, at kinase domain (L755S). In the drug sensitivity analysis, both afatinib and neratinib showed an anti-tumor effect in all the HER2 amplified cell lines both in vitro and in vivo except MKN7 cell line. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene by inhibiting the activation of IGF-1R, were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, demonstrated a notable synergistic effect. Regarding HER2 alteration in 123 clinical samples, we found 19 cases of HER2 amplification and 3 cases of oncogenic mutations. Conclusion: Afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer. Citation Format: Takahiro Yoshioka, Kazuhiko Shien, Yuta Takahashi, Eisuke Kurihara, Kei Namba, Yusuke Ogoshi, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Toshiyoshi Fujiwara, Shinichi Toyooka. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4776.

Abstract 5592: HER-2 copy number measurement in tissue and cell-free plasma DNA of gastric cancer patients using droplet digital PCR method

Abstract In around 10% of gastric cancer, amplification and overexpression of the human epidermal growth factor receptor 2 (HER2) is observed and trastuzumab is adopted as a target therapy agent for HER2-positive gastric cancer. Because of efficiency and accuracy, droplet digital PCR (ddPCR) is one of the best methods for measuring HER2 amplification, especially in cell-free plasma samples. In this study, we aimed to investigate the correlation between tissue and plasma HER2 status by using ddPCR method in gastric cancer patients. We collected formalin-fixed and paraffin-embedded tissue (FFPE) and cell-free plasma samples of 81 gastric cancer patients, including 30 HER2 immunohistochemistry (IHC)-negative (0 or 1+) cases and 51 HER2 IHC-positive cases (IHC 2+, n=20; IHC 3+, n=31), with informed consents. We performed ddPCR of a target gene HER2 and a reference gene EIF2C1 in both tissue and cell-free plasma samples, and gene copy number (GCN) of HER2 was calculated. The median HER2 GCN of tissue DNA ddPCR was 2.61 (1.47-49.00) and the median GCN of plasma ddPCR was 1.83 (0.97-7.67). HER2 IHC results showed moderate correlation with HER2 GCN of tissue ddPCR (r=0.455, p&amp;lt;0.001) and weak correlation with plasma ddPCR (r=0.321, p=0.003). A ratio &amp;gt;2.405, which showed the best discriminating level from a receiver operating characteristic curve, was defined as a cut-off for tissue HER2 amplification using ddPCR method. Comparing the HER2 amplification results of tissue DNA ddPCR to HER2 IHC results, the concordance rate was 86.4% with Cohen's kappa of 0.715. Sensitivity and specificity of tissue ddPCR were 86.3% and 86.7%, respectively. HER2 amplification by tissue ddPCR was significantly associated with advanced gastric cancer and lymphatic invasion (p&amp;lt;0.05). HER2 GCN of tissue ddPCR was weakly correlated with plasma ddPCR (r=0.250, p=0.025). Sensitivity and specificity of plasma ddPCR for HER2 IHC positivity were 35.3% and 93.3%, respectively. However, sensitivity and specificity of plasma ddPCR were 66.7% and 60.0% with another cut-off of 1.715. In 51 HER2 IHC-positive cases, intratumoral heterogeneity was observed in 100% (5/5) of both tissue and plasma ddPCR-negative cases, 100% (2/2) of tissue-negative and plasma positive cases, 64.3% (18/28) of tissue-positive and plasma-negative cases, and 50.0% (8/16) of both positive cases (p=0.026). In conclusion, HER2 GCN of plasma ddPCR showed a weak correlation with tissue ddPCR, and the sensitivity of plasma ddPCR was not high. Intratumoral heterogeneity is suggested to be one of the possible causes, and these findings may contribute to developing plasma HER2 testing useful in daily practice. Citation Format: Boram Kim, Soo Kyung Nam, Soo Hyun Seo, Sang-Hoon Ahn, Do Joong Park, Hyung-Ho Kim, Kyoung Un Park, Hye Seoung Lee. HER-2 copy number measurement in tissue and cell-free plasma DNA of gastric cancer patients using droplet digital PCR method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5592.

Abstract 2177: Sensitive single cell copy number profiling using a novel microfluidic droplet based platform

Abstract Intratumoral genetic heterogeneity may relate to aggressiveness and treatment-resistance. However, the genomic abnormalities underlie this heterogeneity remain poorly characterized. To better describe the picture of tumour heterogeneity, single cell approaches to accommodate larger number of cells are highly in demand. Here, we present results of single cell DNA sequencing by using a novel microfluidic droplet system. We applied this approach to discover copy number profiles in two common and therapeutically challenging cancers, isocitrate dehydrogenase wild-type glioblastoma (GBM) and breast cancer. The GBM cells were extracted from patients operated on site and cultured for 2 months in restricted media to isolate glioma stem cells. The breast cancer cells were extracted from patient-derived xenografts (PDXs). A novel microfluidic droplet based platform (10x GenomicsTM) was used to perform sensitive copy number profiling of thousands of single cells simultaneously. Briefly, the platform integrates single cell encapsulation, cell lysis, genomic amplification, and barcoding with oligonucleotides. The barcoded genomic DNA fragments from individual cells were pooled and converted into libraries that are compatible with Illumina sequencers. Thus far, we sequenced one glioma stem cell sample and two breast cancer PDXs. Uniquely mapped reads were counted in variable-bins with GC bias corrected by lowess fit. The Circular Binary Segmentation was performed and the integer copy number was estimated by least square fitting. The single cell copy number profiles were clustered by hierarchical clustering. We achieved approximately 2% and 6% coverage of the genome per cell for the glioblastoma and the breast tumors, respectively. The uniformity of coverage was comparable to other whole genome amplification and single cell multiplex approaches, while requiring 700k-2.1mi reads/single cell with the median absolute deviation of pair-wise differences in read counts between neighboring bins between 0.17-0.21 vs 0.16)1. We identified two major clonal subpopulations in the glioblastoma sample. One subclone had chr1q amplification and the other subclone had a 9.8Mb deletion on chr4. The clonal CNVs observed were validated using single-cell RNA-seq-based CNV analysis, and the frequent CNVs using fluorescent in situ hybridization. We also identified the HER2 copy number profile within a basal like triple-negative breast cancer as 40% of 2.5N and 25% of 3N. This was previously validated by fluorescence in situ hybridization2. By applying this new microfluidic system, our work will provide more insights into genomic heterogeneity for malignant properties of cancers. 1. Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90-94 (2011). 2. Savage P. et al. A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer. Cell Rep. 21, 1140-1149 (2017). Citation Format: Rui Li, Charles Couturier, Paul Savage, Jean Monlong, Guillaume Bourque, Kevin Petrecca, Morag Park, Jiannis Ragoussis. Sensitive single cell copy number profiling using a novel microfluidic droplet based platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2177.