HER2 Copy Number Research Articles

A Validation Study of Human Epidermal Growth Factor Receptor 2 Immunohistochemistry Digital Imaging Analysis and its Correlation with Human Epidermal Growth Factor Receptor 2 Fluorescence In situ Hybridization Results in Breast Carcinoma

Background: The Visiopharm human epidermal growth factor receptor 2 (HER2) digital imaging analysis (DIA) algorithm assesses digitized HER2 immunohistochemistry (IHC) by measuring cell membrane connectivity. We aimed to validate this algorithm for clinical use by comparing with pathologists’ scoring and correlating with HER2 fluorescence in situ hybridization (FISH) results. Materials and Methods: The study cohort consisted of 612 consecutive invasive breast carcinoma specimens including 395 biopsies and 217 resections. HER2 IHC slides were scanned using Philips IntelliSite Scanners, and the digital images were analyzed using Visiopharm HER2-CONNECT App to obtain the connectivity values (0–1) and scores (0, 1+, 2+, and 3+). HER2 DIA scores were compared with Pathologists’ manual scores, and HER2 connectivity values were correlated with HER2 FISH results. Results: The concordance between HER2 DIA scores and pathologists’ scores was 87.3% (534/612). All discordant cases (n = 78) were only one-step discordant (negative to equivocal, equivocal to positive, or vice versa). Five cases (0.8%) showed discordant HER2 IHC DIA and HER2 FISH results, but all these cases had relatively low HER2 copy numbers (between 4 and 6). HER2 IHC connectivity showed significantly better correlation with HER2 copy number than HER2/CEP17 ratio. Conclusions: HER2 IHC DIA demonstrates excellent concordance with pathologists’ scores and accurately discriminates between HER2 FISH positive and negative cases. HER2 IHC connectivity has better correlation with HER2 copy number than HER2/CEP17 ratio, suggesting HER2 copy number may be more important in predicting HER2 protein expression, and response to anti-HER2-targeted therapy.

Correlation of HER2, MDM2, c-MYC, c-MET, and TP53 Copy Number Alterations in Circulating Tumor Cells with Tissue in Gastric Cancer Patients: A Pilot Study

Background:The analysis of the gene copy number alterations in tumor samples are increasingly used for diagnostic and prognostic purposes in patients with GC. However, these procedures are not always applicable due to their invasive nature. In this study, we have analyzed the copy number alterations of five genes (HER2, MDM2, c-MYC, c-MET, and TP53) with a fixed relevance for GC in the CTC of GC patients, and, accordingly, as a potential approach, evaluated their usage to complete primary tumor biopsy. Methods:We analyzed the status of the copy number alterations of the selected genes in CTC and matched biopsy tissues from 37 GC patients using FISH. Results: HER2 amplification was observed in 2 (5.41%) samples. HER2 gene status in CTC showed a strong agreement with its status in 36 out of 37 patients’ matched tissue samples (correlation: 97.29%; Kappa: 0.65; p < 0.001). MDM2 amplification was found only in 1 (2.70%) sample; however, the amplification of this gene was not detectable in the CTC isolated from this patient. c-MYC amplification was observed in 3 (8.11%) samples, and the status of its amplification in the CTC indicated a complete agreement with its status in the matched tissue samples (correlation: 100%; Kappa: 1.0). Conclusion:Our work suggests that the amplification of HER2 and c-MYC is in concordance with the CTC and achieved biopsies, and, consequently, CTC may act as a non-invasive alternative for recording the amplification of these genes among GC patients.

Longitudinal HER2 amplification tracked in circulating tumor DNA for therapeutic effect monitoring and prognostic evaluation in patients with breast cancer

sBackgroundHuman epidermal growth factor receptor 2(HER2) status is a crucial predictive factor for prognostic assessment and targeted therapy selection, which may be influenced by intratumor heterogeneity and molecular divergence between the primary site and different metastases. Therefore, we performed a prospective study to confirm the concordance of HER2 amplification in circulating tumor DNA(ctDNA) with primary tumor tissue and verified its clinical implications.MethodsA total of 105 breast cancer patients were enrolled, and dynamic monitoring of HER2 copy numbers in ctDNA was conducted in 31 participants during the treatment. Totally 186 plasma samples were prospectively obtained and blinded to test HER2 copy numbers in ctDNA based on low-coverage whole genome sequencing(WGS) by next-generation sequencing(NGS).ResultsComparing HER2 copy numbers in ctDNA collected before the initiation of next line of anticancer treatment with primary tumor tissue, the concordant rate of HER2 amplification was 86.5%(χ2 = 52.901, p < 0.001), with a positive and negative predictive value of 94.9% and 80.7%, respectively. Histopathologically positive, high-level amplification of HER2 copy numbers in the baseline was significantly correlated with best objective response during the anticancer therapy(p = 0.010). Moreover, HER2 copy numbers fluctuated with HER2-targeted therapeutic response, and the patients with a constantly positive level after 6 weeks of treatment appeared to suffer from significantly reduced progression free survival(p < 0.001).ConclusionsHER2 amplification in ctDNA, with a concordance rate of over 80% with primary tumors, may be a predictive index for prognostic evaluation and therapeutic response monitoring in a noninvasive, repeatable and practical method for breast cancer patients.

Impact of the 2018 American Society of Clinical Oncology/College of American Pathologists HER2 Guideline Updates on HER2 Assessment in Breast Cancer With Equivocal HER2 Immunohistochemistry Results With Focus on Cases With HER2/CEP17 Ratio

The American Society of Clinical Oncology/College of American Pathologists HER2 testing guideline in breast cancer was updated in 2018 to address issues on interpretation of uncommon results using dual-probe in situ hybridization according to the 2013 guideline. To assess impact of the 2018 guideline on breast cancer with equivocal HER2 immunohistochemistry results. We retrospectively reviewed HER2 fluorescence in situ hybridization (FISH) data (HER2/CEP17 ratio and average HER2 copy number per cell) of HER2 immunohistochemistry-equivocal (2+ or 1+ to 2+) breast cancers at our center between January 2014 and May 2018 and compared HER2 FISH results according to 2013 and 2018 guidelines. A total of 1666 HER2 FISH results from 1421 patients with equivocal HER2 immunohistochemistry were reviewed. Based on the 2013 guideline, HER2 FISH results were amplified in 346 cases (20.8%), equivocal in 242 (14.5%), and nonamplified in 1078 (64.7%). Using the 2018 guideline, 258 cases (16%) were reclassified, including 242 previously equivocal test results (15%) and 16 previously positive results (1%) reclassified as negative. The subset of 2013 HER2-equivocal and 2018 HER2-nonamplified cases with HER2/CEP17 ratio lower than 2.0 and average HER2 copy number 4.0 or higher and lower than 6.0 showed higher incidence of micropapillary morphology compared with HER2-amplified cases. Despite most patients in this group not receiving HER2-targeted treatment, 96% had no evidence of disease at follow-up. The 2018 guideline eliminated HER2 FISH-equivocal cases by reclassifying HER2-equivocal cases and cases with nonclassical amplification without HER2 overexpression as HER2 negative. As a consequence, we observed a considerable increase in HER2 FISH-negative cases and a slight decrease in HER2 FISH-positive cases.

779P - HER2 copy number as predictor of disease-free survival in HER2-positive resectable gastric cancer

BackgroundThe identification of HER2 overexpression in a subset of gastric cancer (GC) patients represents a significant step forward in unveiling the molecular complexity of this disease. The predictive and prognostic value of HER2 amplification in advanced HER2 inhibitor-treated GC patients has been investigated. However, its predictive value in early stage resectable patients remains unclear. In this study we interrogated the correlation between HER2 copy number (CN) and disease-free survival (DFS). MethodsWe enrolled 98 treatment-naïve resectable (stage I-III) Chinese GC patients with HER2 overexpression assessed using IHC. Capture-based targeted sequencing using a panel consisting of 41 gastrointestinal cancer -related genes was performed on FFPE samples. Furthermore, we also investigated the correlation between HER2 CN and DFS. This cohort had 81 males and 17 females, with a median age of 64. 12, 62 and 24 patients had diffuse, intestinal and mixed GC respectively. ResultsOf the 98 HER2-overexpressed patients assessed using IHC, 91 had HER2 CN amplification assessed using next-generation sequencing, achieving 93% concordance. In addition, 10 had concurrent HER2 missense mutations, including 4 with S310X. All patients except for two had at least one concurrent mutation. The most commonly seen concurrent mutations were TP53, EGFR and PIK3CA, occurring in 85%, 13% and 12% patients, respectively. Furthermore, 23 patients had mutations in EGFR, MET, PDGFRA or KIT, which may participate in the activation of bypass pathways. We also revealed that patients with concurrent TP53 (p=0.05) or PI3KCA (p=0.02) mutations were significantly older than patients without. Next, dichotomous analysis was performed to investigate the association between HER2 CN and DFS. Stage I patients did not undergo adjuvant chemotherapy and all stage II-III patients underwent adjuvant chemotherapy. Our study revealed that patients with high HER2 CN had a significantly shorter DFS than patients with low HER2 CN (p=0.002). ConclusionsWe elucidated the mutation spectrum of HER2-amplified resectable Chinese GC patients and the association between HER2 CN and DFS. Our study revealed the predictive value of HER2 CN and allowed us to further stratify HER2-positive patients. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

1168P - Trastuzumab plus docetaxel in patients with advanced HER2-positive salivary duct carcinoma: Exploratory biomarker analyses

BackgroundA phase II trial of trastuzumab plus docetaxel in patients with advanced HER2–positive salivary duct carcinoma (SDC) showed 70% of the overall response rate. However, biomarkers which predict survival in this population remain unknown. MethodsA total of 91 patients with HER2–positive SDC treated with trastuzumab plus docetaxel were included. Age, sex, ECOG performance status (PS), status of visceral metastases, previous treatment, previous docetaxel exposure, previous combined androgen blockade, the best overall response to the treatment, pretreatment serum C-reactive protein (CRP) level, modified Glasgow Prognosis Score (mGPS), HER2 status (immunohistochemistry [IHC] score, HER2/CEP17 ratio, HER2 copy number, overall positivity according to ASCO/CAP Guidelines), mutational status of PIK3CA, HRAS, and TP53, IHC score of AR, Ki-67, CK5/6, p53, HER3, Akt, PI3K, FOXA1, adipophilin, mTOR and PTEN were assessed and correlated with progression-free survival (PFS) and overall survival (OS). ResultsTreatment response (PR or CR vs. SD or PD) and PTEN IHC score (1+ – 3+ vs. 0) were related with favorable PFS (hazard ratio [HR], 0.25 and 0.39, respectively) and OS (HR, 0.29 and 0.36, respectively). CRP (≥ 0.50mg/dL) was related with shorter PFS (HR, 2.28) and OS (HR 4.46). Presence of previous treatment and mTOR IHC (1+ – 3+ vs. 0) had significant predictive value of better PFS (HR 0.47 and 0.22, respectively) but not significantly related with OS. ECOG PS of 1-2 (vs. 0), presence of visceral metastasis, and mGPS of 1-2 (vs. 0) had significant relationship with shorter OS but not with PFS. Neither previous docetaxel exposure nor previous combined androgen blockade did not affect the survival outcome. HER2, PIK3CA, HRAS and TP53 status did not related with survival. ConclusionsAlthough HER2 status did not correlated with survival, its downstream factors PTEN and mTOR can serve as predictive biomarkers in patients with advanced SDC treated with trastuzumab plus docetaxel. Serum CRP level may predict survival of this population. Clinical trial identificationUMIN000009437, Released on 03/12/2012. Legal entity responsible for the studyThe authors. FundingMEXT/JSPS. DisclosureAll authors have declared no conflicts of interest.

P2.14-49 Molecular Characteristics of HER2 Mutations in Non-Small Cell Lung Cancer

Earlier clinical trials targeting on HER2 exon 20 mutations show promising results. However, target therapy also had shown a favorable effect on non-tyrosine kinase domain (non-TKD) mutations in preliminary studies and case report, while no systemic study was reported about non-TKD mutations in HER2. Hence, the study aims to comprehensively outline the mutation landscape of HER2 in NSCLC. HER2 profile data (patients, N=5,222) from thirteen NSCLC studies in the cBioPortal for Cancer Genomics was screened. Finally, after excluding duplicated data (n=2,725) and no HER2 profile data (n=563), 1,934 individuals were enrolled in the analysis. The mutation subtype, mutation type, mutation region, biological effect of mutations referred to OncoKB, and HER2 copy number variation were described. 4.3% (84/1934) of NSCLC patients were detected with HER2 mutation, and three patients carried double HER2 mutations, totally eighty-seven HER2 mutations were identified in the study. Fifty-three HER2 mutation subtypes were identified, and the most common mutation subtypes were Y772_A775dup (24%, 21/87), S310F (6%, 5/87), G776delinsVC (5%, 4/87) and G778_P780dup (5%, 4/87) respectively. HER2 fusion was identified in 8% (7/87) of mutations with a tendency to concurrent with HER2 copy number increased (5 amplification, 2 copy number gain). As for mutation region, 43% (37/87) of mutations occurred in TKD, while biological effect was not validated in 16% (6/37) of TKD mutations. Notably, biological effect of 56% mutations was inclusive or unknown. In gain of function subset, 82% (31/38) of mutations located at TKD, 13% (5/38) and 5% (2/38) were located at furin-like cysteine rich region and transmembrane domain. Mutation subtypes were diverse in HER2. Though accounting for more than half of HER2 mutations, the effect of non-TKD mutations was fewly understood rather than TKD mutations. Biological effect and clinical implication of non-TKD mutations need to be further investigated in the near future.

Clinicopathologic Factors Associated With Response to Neoadjuvant Anti-HER2–Directed Chemotherapy in HER2-Positive Breast Cancer

BackgroundHER2-targeted neoadjuvant therapy has high efficacy in treating HER2-positive breast cancer. Response to neoadjuvant therapy helps clinicians make treatment decisions and make estimates about prognosis. This study examined clinicopathologic features to determine which may be most predictive of response to neoadjuvant therapy in HER2+ breast cancer. Patients and MethodsPatients with HER2+ breast cancer (n = 173) who had an initial biopsy performed between 2010 and 2016 were identified at our institution. Tumor response was evaluated on excisional specimens using the MD Anderson residual cancer burden (RCB) classification. Tumors with pathologic complete response (defined as no residual invasive carcinoma in the breast and lymph nodes) and RCB-I were classified as having response and tumors with RCB-II and -III as having no response. Patient age, tumor size, nuclear grade (1/2 vs. 3), mitosis, Nottingham grade, HER2 immunohistochemistry (1/2+ vs. 3+), HER2/CEP17 (chromosome enumeration probe 17) ratio, HER2 copy number, estrogen receptor, progesterone receptor, Ki-67, and tumor-infiltrating lymphocytes (TIL) were evaluated and correlated with response. TILs were evaluated for an average and also for the hot spot/total tumor stromal ratio. ResultsSmall tumor size, low estrogen receptor and progesterone receptor expression, HER2 immunohistochemistry 3+, high Ki-67, high HER2/CEP17 ratio, and high HER2 copy number were significantly associated with response (all P < .05). TIL hot spot was associated with RCB in univariate (P < .05) but not multivariate analyses. ConclusionClinicopathologic features may help predict HER2+ breast cancer response to neoadjuvant therapy. Larger studies would be useful to confirm these associations, which may have relevance to clinical practice.

Quantitative assessments and clinical outcomes in HER2 equivocal 2018 ASCO/CAP ISH group 4 breast cancer

We quantified human epidermal growth factor receptor 2 (HER2) RNA and protein expression in 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) in situ hybridization (ISH) group 4 (HER2/centromeric probe 17 (CEP17) ratio <2.0, average HER2 copy number ≥4.0 and <6.0, and 2013 ASCO/CAP ISH equivocal) breast cancers. Breast cancers in 2018 ASCO/CAP ISH group 4 between 2014 and 2017 were identified from the Yale archives. Sixty-three patients (34 with HER2 immunohistochemistry (IHC) 0/1+ and 29 with HER2 IHC 2+) were included. We compared patient characteristics, systemic treatments, and outcomes. We assessed HER2 by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and quantitative immunofluorescence (QIF). Among ISH group 4 cancers, higher HER2 mRNA (P < 0.0001) but similar HER2 protein levels were observed in IHC 2+ compared to IHC 0/1+ cancers. The distribution of RT-qPCR and QIF scores were independent of fluorescence in situ hybridization (FISH) ratio/copy number. Concordance between HER2 RT-qPCR and QIF was 69.8% (r = 0.52). Among 29 patients with IHC2+ results, 16 were HER2 positive by RT-qPCR and 12 were HER2 positive by QIF. Systemic treatment, recurrence, and survival outcomes were comparable among ISH group 4 cancers regardless of IHC 0/1+ or 2+ results. ISH group 4 cancers appear to form a distinct group with intermediate levels of RNA/protein expression, close to positive/negative cut points. Therefore, adjudication into positive or negative categories may not be meaningful. Our results support the 2018 ASCO/CAP recommendation to refrain from routine additional testing of these samples. Additional outcome information after trastuzumab treatment for patients in this special group might help to guide treatment decisions in these patients.

Precision Medicine Tools to Guide Therapy and Monitor Response to Treatment in a HER-2+ Gastric Cancer Patient: Case Report

Trastuzumab, has played a major role in improving treatment outcomes in HER-2 positive gastric cancer. However, once there is disease progression there is a paucity of evidence for second line therapy. Patient-derived xenografts (PDXs) in combination with liquid biopsies can help guide individual therapeutic decisions and have now started to be studied. In the present case we established a PDX model from a metastatic HER-2+ gastric cancer patient and after the first engraftment passage we performed a mouse clinical trial to test T-DM1 as an alternative therapy for the patient. The PDX tumor response served as a guide to administer T-DM1 therapy to the patient who responded to treatment before relapsing 6 months later. Throughout out the clinical follow up of the patient, ctDNA levels of HER-2 copy number and a PIK3CA mutation were monitored and we found their correlation with drug response and disease progression to outperform that of CEA levels. This study highlights the utility of applying precision medicine tools combining PDX models to guide therapy with circulating tumor DNA (ctDNA) to monitor treatment response and disease progression.

Comprehensive assessment of HER2 alteration in a colorectal cancer cohort: from next-generation sequencing to clinical significance.

PurposeHuman epidermal growth factor receptor 2 (HER2) is an emerging therapeutic target in colorectal cancer (CRC). Currently, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) have been used to determine HER2-positive CRCs; however, the clinical utility of next-generation sequencing (NGS)-based techniques for determining HER2 status in CRC has been limited. Here, we detail our experience regarding the assessment of HER2 alterations in a CRC cohort.Materials and methodsWe prospectively enrolled 73 CRC patients who underwent surgery and received adjuvant oxaliplatin treatment. We then examined HER2 alterations using the Oncomine Comprehensive Assay version 1, as well as clinical outcomes, in this cohort.ResultsUsing the NGS-based assay, HER2 copy number gains in 12 of 73 CRCs were determined to range from 2.74 to 92.62. Of these 12 tumors, 6 had HER2 high-level copy number gain (92.6, 57.9, 57.0, 52.0, 35.2, and 8.42) and were all defined as HER2-positive CRC using HERACLES Diagnostic Criteria. Nevertheless, other 6 patients with low-level copy number gain (ranging from 2.74 to 3.04) and the remaining 61 patients without increase in HER2 copy number were all HER2-negative. Among the 6 HER2-positive CRCs, KRAS and PIK3CA mutations were detected in 1 (17%; G13D) and 2 (33.3%; 1 Q546R and 1 H1047R) patients, respectively. Moreover, 2 of the 6 (33.3%) HER2-positive patients had recurrent disease, while one patient had a partial response after anti-HER2 therapy.ConclusionNGS-based tools could assist in the simultaneous detection of HER2 and other genomic alterations in patients with CRC. Only CRCs with HER2 high-level copy number gain were HER2-postive by current diagnostic criteria.

Abstract 1414: Durable response to first-line Trastuzumab in HER2 amplified colorectal cancer

Abstract Background: Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality with limited precision strategies. The human epidermal growth factor receptor (HER2) is amplified in 5% of patients with metastatic CRC. Therapeutic studies have demonstrated activity of HER2 targeting regimens in the late-line treatment setting though little characterization has been done in the setting of not having prior anti-epidermal growth factor receptor antibody therapy or in the first-line treatment setting. Methods: Patient with metastatic colorectal cancer underwent next-generation sequencing (NGS) panel testing by Strata Oncology through the University of Wisconsin Precision Medicine Molecular Tumor Board. Patients were treated per multi-disciplinary recommendation across multiple lines of therapy and tracked by prospective registry (IRB#UW15068). HER2 copy numbers &amp;gt;/= 10 were used to determine amplification. A subset of patients with HER2-amplified CRC were treated with single agent trastuzumab. Results: HER2 amplifications were identified in 4 of 78 cases of metastatic CRC (5.1%). All biopsies were performed prior to exposure to anti-epidermal growth factor receptor inhibitors. Median estimated copy number (CN) was 55.5 [range 13-178]. There was no concurrent alterations in extended RAS, BRAF, PIK3CA, AKT or MTOR. All cases included concurrent mutations in TP53 at R175H (2), C135W, or C135F. Single agent trastuzumab was administered to two patients at standard dosing (trastuzumab 8mg/kg intravenous day 1 cycle 1 followed by 6 mg/kg intravenous day 1 cycle 2+ on a 21 day cycle). One patient was treated in the treatment refractory setting. They had resolution of bowel obstructive symptoms and a minor response on imaging. Additionally, a patient with a HER2 amplification (CN 178) was treated in the first-line setting with single agent trastuzumab. The patient had grade 1 fever with the first cycle and no other treatment related adverse events thereafter. The patient’s CEA decreased from 7,506 to 2,525 ng/mL with the first cycle of therapy and overall reduced to 6.5 ng/mL after 12 cycles of therapy. The patient has had a partial response per RECIST v1.1 response criteria with a reduction of cross-sectional diameter of their disease by 60%. The patient currently continues on this therapy without evidence of disease progression. Conclusions: HER2 amplified colorectal cancers are an important subtype of colorectal cancer. Here we describe the potential benefit of HER2 targeting with single agent trastuzumab and even demonstrate single agent activity in the first-line setting. Further studies should be done to further characterize the benefit of this low toxicity treatment option for patients with metastatic HER2 amplified colorectal cancer. Citation Format: Jeremy Kratz, Nataliya Uboha, Kayla Lemmon, Hannah Houtler, Mark Burkard, Dustin Deming. Durable response to first-line Trastuzumab in HER2 amplified colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1414.

Abstract 3986: Digital droplet PCR measurement for plasma HER2 amplification in patients with AGC

Abstract Introduction: With the recent introduction of human epidermal growth factor receptor 2 (HER2) targeted therapies for patients with advanced gastric cancer (AGC), determining HER2 status is essential to select the patients who may benefit from this treatment. The current standard method assessing HER2 positivity is immunohistochemistry (IHC) or in situ hybridization assays, but this way of HER2 assessment has weaknesses especially when considering tumor heterogeneity. Using digital droplet polymerase chain reaction (ddPCR) technique, we evaluated HER2 amplification in both tissue and plasma samples collected from AGC patients and analyzed clinical implication of HER2 amplification in circulating tumor DNA. Method: Biopsied tissue and plasma samples were collected from patients with metastatic or recurrent AGC who were enrolled in AGC biomarker study conducted at Asan Medical Center. Samples were obtained before the start of anti-cancer treatment. HER2 amplification in DNA from tissue and cell-free DNA from plasma were determined by ddPCR, performed in Kindai University. Results: Samples of 63 patients with HER2 positive GC and 37 patients with HER2 negative GC enrolled between April 2014 and October 2017 were included in the analysis. As expected, the median HER2 copy number (CN) was higher in patients diagnosed as HER2 positive than negative, both for tissue (4.54 vs 0.95, P &amp;lt; 0.001) and plasma (1.49 vs 1.07, P &amp;lt; 0.001). ROC curve analysis showed 83.8% specificity and 69.8% sensitivity for tissue HER2 positivity at 1.17 CN of plasma HER2. In patients receiving anti-HER2 therapy (n=57), there was a trend for worse progression-free survival (PFS) outcome in patients with higher plasma HER2 CN (&amp;gt;1.5 vs ≤1.5), although no statistical significance was seen (median PFS 6.67 vs 8.19 months, P = 0.172). In multivariate Cox regression analysis including the age (&amp;gt;60 vs ≤60), HER2 IHC intensities (2+ vs 3+), and risk groups determined by Koo et al.’s prognostic model (Good vs Moderate vs Poor), higher plasma HER2 CN (&amp;gt;1.5 vs ≤1.5) was significantly associated with poor PFS (HR 2.22, 95% CI 1.14-4.32, P = 0.019). However, when tumor burden (sum of largest diameter of all measurable lesions) was combined as another factor in patients with measurable disease (n=39), plasma HER2 CN was no longer an independent factor (HR 1.38, 95% CI 0.58-3.27, P = 0.466) and larger tumor burden (&amp;gt;6.5cm vs ≤6.5cm) was found to be an independent prognostic factor for poor PFS (HR 2.63, 95% CI 1.12-6.21, P = 0.027), instead. Of note, plasma HER2 CN showed a positive relationship with tumor burden multiplied by tissue HER2 CN in linear regression (β=0.32, P = 0.002). Conclusion: Using the ddPCR technique, we found that plasma HER2 CN was significantly increased in HER2 positive GC patients compared to negative patients. However, plasma HER2 CN did not provide more information on predicting therapeutic effects than the tumor burden in patients receiving anti-HER2 therapy. Citation Format: Kyoungmin Lee, Kazuko Sakai, Min-Hee Ryu, Jae-Joon Kim, Young Soo Park, Young-Soon Na, Jungeun Ma, Hana Na, Kazuto Nishio, Yoon-Koo Kang. Digital droplet PCR measurement for plasma HER2 amplification in patients with AGC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3986.

A combinatorial biomarker predicts pathologic complete response to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2+ breast cancer.

HER2-positive (+) breast cancers, defined by HER2 overexpression and/or amplification, are often addicted to HER2 to maintain their malignant phenotype. Yet, some HER2+ tumors do not benefit from anti-HER2 therapy. We hypothesize that HER2 amplification levels and PI3K pathway activation are key determinants of response to HER2-targeted treatments without chemotherapy. Baseline HER2+ tumors from patients treated with neoadjuvant lapatinib plus trastuzumab [with endocrine therapy for estrogen receptor (ER)+ tumors] in TBCRC006 (NCT00548184) were evaluated in a central laboratory for HER2 amplification by fluorescence in situ hybridization (FISH) (n = 56). HER2 copy number (CN) and FISH ratios, and PI3K pathway status, defined by PIK3CA mutations or PTEN levels by immunohistochemistry were available for 41 tumors. Results were correlated with pathologic complete response (pCR; no residual invasive tumor in breast). Thirteen of the 56 patients (23%) achieved pCR. None of the 11 patients with HER2 ratio <4 and/or CN <10 achieved pCR, whereas 13/45 patients (29%) with HER2 ratio ≥4 and/or CN ≥10 attained pCR (P = 0.0513). Of the 18 patients with tumors expressing high PTEN or wild-type (WT) PIK3CA (intact PI3K pathway), 7 (39%) achieved pCR, compared with 1/23 (4%) with PI3K pathway alterations (P = 0.0133). Seven of the 16 patients (44%) with HER2 ratio ≥4 and intact PI3K pathway achieved pCR, whereas only 1/25 (4%) patients not meeting these criteria achieved pCR (P = 0.0031). Our findings suggest that there is a clinical subtype in breast cancer with high HER2 amplification and intact PI3K pathway that is especially sensitive to HER2-targeted therapies without chemotherapy. A combination of HER2 FISH ratio and PI3K pathway status warrants validation to identify patients who may be treated with HER2-targeted therapy without chemotherapy.

Evaluation of next generation sequencing for detecting HER2 copy number in breast and gastric cancers.

e13004 Background: Amplicon-based next generation sequencing (NGS) approaches have been preferentially adopted by the clinical laboratories on the basis of a short turnaround time (TAT) and small DNA input needs. However, little work has been done to assess the amplicon-based NGS methods for copy number variation (CNV) detection in comparison with current standard methods like immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The correlation between NGS based CNV detection and the later standard methods has remained unexplored. Methods: We developed an amplicon-based panel to detect human epidermal receptor growth factor (HER2) amplification in formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 280 breast cancer and 50 gastric cancer patients. Assessment by IHC and FISH was conducted in parallel, and descriptive statistics were used to assess the concordance. Results: The copy number detected by NGS was correlated with either the average HER2 copy number (signals/cell) (r = 0.844; p &lt; 0.001) or the HER2/CEP17 ratio (r = 0.815; p &lt; 0.001). We determined a cut-off value for NGS to categorize HER2 amplification status by using 131 HER2 non-amplified FFPE samples. In breast cancer patients, the cut-off value was 2.910, with 95.28%, 99.23% and 97.29% sensitivity, specificity and concordance, respectively. However, this cut-off value displayed low sensitivity in gastric cancer patients (64.71%), and the following macro-dissection procedure was not effective for increasing sensitivity (57.14%). Thus indicated that HER2 copy number correlation between NGS and FISH in gastric cancer was different from breast cancer. Conclusions: Evaluation of HER2 copy number with NGS in our study was comparable with IHC and FISH in breast cancer patients, but concordance in gastric cancer was only moderate. NGS-based HER2 assessment may decrease the equivocal HER2 determinations in breast cancer patients assessed by FISH/IHC. The greater discordance in gastric cancer may reflect the underlying biological mechanisms, and further study is warranted.

Genomics of HER2+ breast cancer in young women before and after exposure to chemotherapy (chemo) plus trastuzumab (H).

554 Background: HER2+ breast cancer (BC) is particularly common in young women. Genomic features of HER2+ tumors before and after H-based therapy have not been described in a population of young women and may point to clinically targetable mechanisms of resistance. Methods: From a large prospective cohort of women diagnosed with BC age ≤40 years, we identified those with HER2+ BC and tumor tissue available for sequencing before and after chemo+H. Whole exome sequencing (WES) was performed on each tumor and on germline DNA from blood. Tumor-normal pairs were analyzed for mutations and copy number (CN) changes. Evolutionary analysis was performed for patients with both pre- and post-treatment (tx) samples. Results: 22 women had successful WES samples from at least one timepoint; 13 of these had paired sequencing results both before and after chemo+H. For the majority of women, post-tx sample was following neoadjuvant chemo + H, though post-tx timepoint for other women represented locoregional or distant metastasis (Table). TP53 was the only gene that was significantly recurrently mutated in both pre- and post-tx samples. Comparison of matched pre-tx and post-tx samples demonstrated that large changes in HER2 CN over the course of tx were uncommon, only 2/13 pts had &gt; 2-fold change in HER2 CN. Other clonal and subclonal genomic alterations were found to be acquired in the post-tx sample compared to the pre-tx sample. One patient acquired a putative activating mutation in ERBB2. Another patient acquired a clonal hotpsot mutation in TP53. MYC gene amplification was observed in 4 post-tx tumors. NOTCH2 alterations were found in post-tx biopsies from 2 different patients, and mutations in STIL were also found in post-tx biopsies from 2 patients, though the function of these mutations is not known. Conclusions: HER2+ breast tumors in young women display genomic evolution following tx with chemo+H. HER2 CN changes are uncommon, but we identified several genes that warrant exploration as potential mechanisms of resistance to therapy in this population.[Table: see text]

HER2 cluster amplification as a factor of an especial sensitivity for anti-HER2 neoadjuvant therapy with biosimilar of trastuzumab in Russian women with breast cancer stage II-III.

e12102 Background: Effect of neoadjuvant chemotherapy (NACT) is extremely important for patients with HER2-positive breast cancer stage II-III as it correlates with long-term outcomes. However, predictive factors for achieving complete pathologic response (pCR) remain unclear. Aim of the study: Assess an impact of clinical, morphological and genetic factors on pCR achievement in patients with HER2-positive breast cancer stage II-III treated by biosimilar of trastuzumab. Methods: We studied treatment results in 73 patients with HER2-positive breast cancer (BC) stage II-III (aged 29-71, median – 51 years), treated with NACT with anti-HER2 therapy and radical surgery in “N.N. Blokhin National Medical Research Center of Oncology” оf the Ministry of Health of the Russian Federation in 2015-2018. After radical surgery pathologic response was assessed. Initially operable tumors were observed in 45,2% patients, locally advanced - in 53,4%. Luminal HER2-positive BC was diagnosed in 41,1%, non-luminal HER2-positive in 58,9%. Ki67 was high (≥ 20%) in 91,5%. NACT included anthracycline and non-antracycline regimens with addition of biosimilar of trastuzumab ± pertuzumab. Radical mastectomy was performed in 78,8% patients, 21,2% had breast-conserving surgery. We studied biopsies obtained before the start of treatment in all women, HER2 amplification was detected by HER2 IQFISH pharmDx (DAKO) kit in accordance with ASCO/CAP 2018 guidelines. In 87,1% HER2+ status was defined as ASCO/CAP 2018 category 1; cluster amplification was detected in 30,1% patients. We analyzed the rate of bpCR и tpCR achievement depending on clinical, pathological data and amplification of HER2. Results: Pathologic complete response in primary tumor (bpCR) was achieved in 57,4%, both in primary tumor and lymph nodes (tpCR) – in 48,9% patients; bpCR achievement depended on age, NACT regimen, addition of pertuzumab and HER2 copy number ( &lt; 0,05). The highest rate of bpCR was noted in women aged 50 and older (71,9%, p = 0,026); in patients received TCH±Р regimen (80,0%, p = 0,045); with addition of pertuzumab (88,9%, p = 0,049); and if cluster amplification was detected (81%, p = 0,013). Cluster amplification was the only one significant predictive factor for achieving tpCR: with cluster amplification - 68,8%, without - 38,7% (p = 0,049). Conclusions: Cluster amplification of HER2 is the most significant factor of especial sensitivity for NACT with biosimilar of trastuzumab in HER2-positive BC stage II-III.

Reassignment of HER2 status for subgroups of breast cancer according to the 2018 updated American Society of Clinical Oncology and College of American Pathologists guidelines: The impact of combined immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) reflex testing in a large national reference laboratory.

3144 Background: Updated ASCO/CAP Guidelines for HER2 testing in breast cancer have been most impactful on the resolution of certain challenging groups of FISH results. We review the change in assignment of HER2 status in a large series of breast cancers referred to a large national reference laboratory for FISH testing since the introduction of the 2018 updated guidelines. Methods: Patient samples submitted to the Mayo Clinic Cytogenetics Laboratory (N = 2208) were analyzed by FISH. Samples with Group 2, Group 3, or Group 4 FISH results were reflexed to immunohistochemistry (IHC) in our central laboratory; FISH slides for those cases with equivocal 2+ IHC results were re-scored in the regions of invasive cancer showing more intense membranous staining. A subset of 202 samples with Group 4 FISH results were also reflexed to the previously employed reflex FISH assay (HER2/D17S122), and these were also re-analyzed according to the new reflex IHC/FISH process. Results: 382 of 2208 breast cancer samples tested (17.3%) had FISH results categorized as Group 2 (N = 17, 0.8%), Group 3 (N = 34, 1.5%), or Group 4 (N = 331, 15%) and required reflex IHC testing, and of those, 75% were 2+ equivocal and required targeted re-analysis of the FISH slide according to the 2018 updated guidelines. Re-analysis of the FISH slide resulted in switching between Groups 1-5 in 19.4% of cases, but HER2 status was changed by FISH re-scoring in only 7.7% of cases re-scored (1.0% of all samples), generally due to only minor shifts in HER2 copy number and HER2/control ratios between the initial and IHC-guided reflex FISH scores. In the subset of 202 cases tested by both reflex methods, the previously employed HER2/D17S122 reflex probe set was positive in 123 cases (60.9%), whereas reflex IHC/FISH was positive in only 10 cases (7.9%). Including positive reflex IHC (0.4%) and positive reflex FISH results (2.1%), the overall assignment of positive HER2 status on our series of 2208 cases was 11.5%. Conclusions: Overall rates of HER2 positive FISH results have declined under the most recent ASCO/CAP guideline update as a consequence of new recommendations for reflex testing for Groups 2-4. This change is largely due to reassignment of Group 2 and Group 4 results as negative in the absence of positive IHC.

Monitoring treatment efficacy and resistance in breast cancer patients via circulating tumor DNA genomic profiling.

e12502 Background: Circulating tumor DNA (ctDNA) from liquid biopsy provides a valuable assessment of invasive breast cancer (BC). We evaluated the utility of ctDNA to reflect the efficacy of HER2-targeted trastuzumab in treating HER2+ BC patients, as well as chemotherapy in treating HER2- BC patients, to monitor trastuzumab and chemotherapy resistance mechanisms. Methods: Targeted next-generation sequencing (NGS) of 416 cancer-relevant genes was performed in 41 plasma biopsy samples from 19 HER2+ and 12 HER2- BC patients in a retrospective study. We compared ctDNA somatic mutations and germline mutations for analyzing acquired and innate resistance, respectively. Results: ERBB2 somatic copy number of HER2+ BC patients who developed progressed diseases to HER2-target therapy was significantly higher than those benefited from HER2-target therapy. HER2+ BC patients who developed acquired resistance to trastuzumab showed frequent genomic alterations on ERBB2, TP53, EGFR, NF1 and SETD2 genes. Specifically, in longitudinal analyses, somatic mutations found in the original breast tumor can be detected in the liver metastasis and plasma ctDNA with increased allele frequencies. Newly emerged deleterious mutations occurred when the patient was benefiting from trastuzumab, predicting the poor prognosis. From these newly emerged somatic mutations, EBBB2L869R was further investigated in vitro and contributed to trastuzumab resistance. In the HER2- BC patients with chemotherapy resistance, frequently genetic alterations on TP53, PIK3CA and DNA damage repair genes were observed in ctDNA mutation profiling. Conclusions: liquid biopsy ctDNA, particularly longitudinal analyses, provides insights into targeted therapy efficacy and gene alterations underlying trastuzumab resistance and chemotherapy resistance in HER2+ and HER2- BC patients, respectively.

Plasma HER2 (ERBB2) Copy Number Predicts Response to HER2-targeted Therapy in Metastatic Colorectal Cancer.

ERBB2 (HER2) amplification is an emerging biomarker in colon cancer, conferring sensitivity to combination anti-HER2 therapy. Measurement of HER2 copy number is typically performed using surgical specimens, but cell-free circulating tumor DNA (ctDNA) analysis may be a noninvasive alternative. We determined the sensitivity of plasma copy number (pCN) for detecting ERBB2 amplifications and whether pCN correlated with tissue-detected copy number. We also assessed response to HER2-targeted therapy based on pCN and suggest a pCN threshold predictive of response. Forty-eight pretreatment and progression plasma samples from 29 HER2-positive patients in the HERACLES A clinical trial were tested using the Guardant360 cfDNA assay. We correlated ERRB2 pCN with progression-free survival (PFS) and best objective response (BOR) and applied an adjustment method based on tumor DNA shedding using the maximum mutant allele fraction as a surrogate for tumor content to accurately determine the pCN threshold predictive of response. Forty-seven of 48 samples had detectable ctDNA, and 46 of 47 samples were ERBB2-amplified on the basis of cfDNA [2.55-122 copies; 97.9% sensitivity (95% confidence interval, 87.2%-99.8%)]. An adjusted ERBB2 pCN of ≥25.82 copies correlated with BOR and PFS (P = 0.0347). cfDNA is a viable alternative to tissue-based genotyping in the metastatic setting. The cfDNA platform utilized correctly identified 28 of 29 (96.6%) of pretreatment samples as ERBB2-amplified and predicted benefit from HER2-targeted therapy. In this study, an observed pCN of 2.4 and an adjusted pCN of 25.82 copies of ERBB2 are proposed to select patients who will benefit from HER2-targeted therapy.