ERBB2 Variants Research Articles

Novel ERBB2 Variant Potentially Associated with Resistance against Anti-HER2 Monoclonal Antibody-Based Therapy in ERBB2-Amplified Metastatic Colorectal Cancer.

HER2-targeted therapies in ERBB2-amplified metastatic colorectal cancer (mCRC) are effective; however, a notable portion of patients do not respond to treatment, and secondary resistance occurs in most patients receiving these treatments. The purpose of this study was to investigate determinants of treatment efficacy and resistance in patients with ERBB2-amplified mCRC who received HER2-targeted therapy by analyzing multiomics data. We investigated genomic data from a nationwide large cancer genomic screening project, the SCRUM-Japan project. We analyzed paired genome and transcriptome data of tissue and genomic data of ctDNA collected pre- and postprogression in patients enrolled in the related trial, TRIUMPH, in ERBB2-amplified mCRC. In 155 patients with ERBB2-amplified solid tumors who received HER2-targeted therapy based on the SCRUM-Japan project, the objective response rate was 50%, 51%, and 35% in ERBB2 wild-type, variant of unknown significance, and pathogenic variant groups, respectively. In the paired genome and transcriptome data analyses in TRIUMPH, we identified the novel splicing-associated variant c.644-66_-2del in one of the 11 patients with paired whole-exome sequencing and whole-transcriptome sequencing data sets, which lacks the binding domain of pertuzumab, in progressed metastatic tumor as a variant with potential pathogenicity. The time-course ctDNA analysis detected c.644-66_-2del as an acquired variant. This study highlighted the importance of ERBB2 genomic status when evaluating the efficacy of HER2-targeted therapies in ERBB2-amplified mCRC. The identification of a novel splicing-associated variant may provide insights into potential mechanisms of treatment resistance. Furthermore, we demonstrated the utility of ctDNA to follow the acquired genomic status of mCRC tumors.

Extra-appendiceal mucinous neoplasms: A tumour with clinicopathologic similarities to low- and high-grade appendiceal counterpart

AimsAppendiceal mucinous neoplasms feature neoplastic mucinous epithelium with pushing borders and densely fibrotic walls. We have identified five examples of analogous colorectal tumours. Methods and resultsSlides, pathology reports, and clinical data were reviewed. Whole genome sequencing was performed in two cases. Three were women and the mean age was 70. Associated GI conditions included Crohn's disease [1], diverticulosis [2], and sarcoma of the terminal ileum [1]. Signs/symptoms included obstruction [2], nausea, vomiting, abdominal pain [1], and positive faecal immunohistochemical test [1]. Colonoscopic findings included narrowing [1], “fullness” [1], and caecal lesion concerning for GIST [1]. Tumours involved the rectosigmoid [2], sigmoid [1], transverse colon [1], and cecum [1] and ranged from 1.5 cm to 8.5 cm. All but one tumour arose in the setting of faecal stream abnormalities related to obstruction, diverticulosis, or bowel diversion. All cases showed columnar, variably mucinous epithelium associated with little-to-no lamina propria. All but one case showed fibrosis of the submucosa. Three cases had high-grade areas. Neoplastic glands and/or mucin dissected through the muscularis propria or subserosa in 3 examples. No extracolonic neoplastic cells/mucin, infiltrative invasion, or desmoplastic response were identified. Three patients with available follow-up [5.5–28 months] are alive. Whole genome sequencing identified pathogenic TP53 and ERBB2 variants, as well as ERBB2 copy number amplification in one high-grade example. ConclusionsThough these tumours share clinicopathologic characteristics with their appendiceal counterparts, our cohort is too small to draw solid conclusions. We propose the term “extra-appendiceal mucinous neoplasm [EAMN]” for these rare lesions.

Validation of a 56 gene panel for the detection of somatic variants in metastatic breast cancer.

e13131 Background: The use of ctDNA and next generation sequencing has unprecedented potential to improve genomic profiling and predicting actionable drug treatment in patients with metastatic breast cancer (MBC). Liquid biopsy is uniquely suited for patients where tissue biopsy is not feasible. We specifically focus on actionable variants found in liquid biopsy for MBC including bTMB and MSI. Here we report on the validation of a 56 gene sub panel for the determination of somatic mutations in MBC. Methods: Commercially available, well-characterized, biological ctDNA reference samples with allele frequency ranging from 0.5 -2.5% and bTMB scores (7 – 20) were obtained to analytically evaluate sensitivity, specificity and precision of a range of clinically significant variants (SNV, indels, fusions and copy number variants) in the TSO-500 ctDNA NGS assay. Additionally, 48 Whole blood samples from well characterized metastatic breast cancer patients and 13 healthy donors were obtained from commercial sources. cfDNA was extracted from the plasma separated from whole blood and analyzed on both TS0-500 ctDNA assay and PGDx Elio Complete. TSO-500 ctDNA sequencing data was further analyzed with the DRAGEN pipeline for variant calling and all variants were annotated and curated with the Clinical Genomic Workspace from PierianDx. PGDx utilized a proprietary pipeline and reporting of all variants detected. Results: Analytical validation determined excellent sensitivity (PPA: 99.4 – 100%), specificity (NPA: 99.6 -99.8%) and reproducibility (95 – 98.6%) for SNV, indels, fusions and Copy number variants. bTMB, with a cutoff for bTMB high as >20 mut/Mb, exhibited a sensitivity of 75% and specificity of 93.3%. Limit of detection (LOD) was established at 0.5 % VAF for SNV, Indels and fusions at a minimum read depth of 1000x. LOD for Copy number amplifications was established at 1.26-fold change or 3 copies. Clinical samples also exhibited excellent concordance across both TS0-500 ctDNA and PGDx Elo Complete. SNV, indels and copy number variants exhibited 100% (PPA) sensitivity and 99-100% Specificity (NPA). bTMB exhibited 87.5 % sensitivity and 96.22% specificity. As expected, fusions and microsatellite instability were observed in the clinical population but were rare. No reportable mutations were found in the healthy donors. Actionable mutations were reported for PIK3CA, ESR1, BRCA1 and BRCA2, TP53 and ERBB2 variants. Rare mutations in ATM, CHEC2, PALB2, FGFR1 and CCND1 were also observed. Conclusions: Clinical utility of liquid biopsy assays requires excellent sensitivity, specificity and precision. The Epic 56 gene panel has established clinical validity and high sensitivity and specificity across all relevant variants including bTMB in MBC. This clinically available NGS panel for metastatic breast cancer allows for routine genomic profiling, specifically in patients where tissue biopsy cannot be obtained.

Characteristics of long-term survivors with EGFR mutant (EGFRm) metastatic non-small cell lung cancer (mNSCLC).

9116 Background: Although osimertinib has become standard first-line (1L) therapy for patients (pts) with EGFRm mNSCLC, a subset treated with earlier-generation EGFR-targeted TKIs and chemotherapy have had long-term survival. We sought to characterize clinical features of long-term survival in pts with EGFRm mNSCLC treated prior to the osimertinib era. Methods: Data were abstracted from electronic medical records at 12 cancer centers participating in the Academic Thoracic Oncology Medical Investigator’s Consortium (ATOMIC). We included patients with mNSCLC with sensitizing mutations in EGFR who started 1L systemic therapy before 2015. Survival distributions and predictors were assessed using Kaplan-Meier estimates and a multivariable Cox proportional hazards model including time-dependent brain metastasis (met) development, age, 1L therapy (targeted vs. chemotherapy), sex, race, and smoking status. Multivariable logistic regression was used to evaluate baseline predictors associated with 5+ year survival vs. not for those with known survival status at 5 years after start of 1L. Results: We identified 304 patients (69% female, 56% White, 29% Asian, mean age 61.2). First-line targeted therapy was given in 70% of patients. With a median follow-up of 81.5 months, median overall survival was 63.5 months (95% CI 59.4-71.9). 135 (44%) pts survived 5+ years; among those with baseline next-generation sequencing (NGS), presence of a baseline pathogenic ERBB2 variant was higher in 5+ year survivors (4/51 [8%], 2x amplification, y772_A775dup, S310F) v others (1/65 [2%], amplification). Among 161 pts with baseline brain imaging, both baseline and on-treatment development of brain mets were associated with worse survival (HR 1.29, 95% CI 1.21-1.37; HR 1.59, 95% CI 1.47-1.72; both P < 0.001). Excluding 22 patients lost to follow-up before 5 years, a history of smoking (OR 2.99, 95% CI 1.35-6.90, P = 0.008) and baseline brain metastases (OR 3.57, 95% CI 1.64-8.13, P = 0.002) were associated with death before 5 years. Conclusions: Long-term survivors with EGFRm mNSCLC treated before the osimertinib era were more likely to be nonsmokers and have no baseline brain metastases. This highlights a subset of EGFRm patients who had excellent outcomes with older treatment strategies and may not benefit as much from intensification approaches. Additional baseline mutational data will be presented at the conference.

P11.58.A Case of a complex neurocutaneous syndrome characterized by extensive peripheral nerve sheath tumors and somatic ERBB2 mutation

Abstract Schwannomatosis is a rare genetic tumor predisposition syndrome characterized by the presence of multiple non-intradermal schwannomas and the definitive absence of vestibular nerve involvement. Though considered benign, the burden of tumors can cause significant morbidity in the form of motor dysfunction and refractory neuropathic pain. Treatment is focused on mitigating these symptoms, which includes resection of offending tumors when feasible and anti-angiogenesis therapy with bevacizumab. Options for targeted medical therapies are lacking. Schwannomatosis is molecularly distinct from the other neurofibromatoses and by definition lacks constitutional mutations in NF1 and NF2. Instead, constitutional mutations in SMARCB1 or LZTR1 are often the “first hit” in tumorigenesis. Activating mutations in ERBB2 (also designated HER2) have been identified as oncogenic drivers in peripheral nerve sheath tumors in patients who lack these typical constitutional tumor suppressor gene mutations. Here we report a case of extensive peripheral nerve sheath tumors in the setting of apparent somatic mosaicism of an ERBB2 mutation. A 48-year-old man with history of chronic back pain presented with diffuse enlarging soft tissues masses primarily involving the left neck and occiput. Examination revealed areas of cutaneous heterogeneous hyperpigmentation, decreased muscle bulk and dorsiflexor weakness in the bilateral lower extremities, and steppage gait. Neuroimaging showed extensive T2 hyperintense, mildly enhancing masses within the bilateral thoracic and lumbar spine neuroforamina, intercostal nerves, paraspinal musculature, and lumbosacral plexus. Tissue from two separate tumor sites was morphologically consistent with schwannoma with some features of neurofibroma. Immunohistochemistry revealed partial loss of staining for SMARCB1. Whole exome sequencing (WES) on blood and tumor tissue did not show pathogenic germline or somatic variants for NF1, NF2, SMARCB1, LZTR1, or an array of tumor predisposition syndromes. WES on tumor tissue from both sites however did reveal a somatic ERBB2 variant (p.D769Y), suggesting mosaicism. ERBB2 D769Y has previously been classified as an activating mutation that confers sensitivity to some small molecule receptor tyrosine kinase inhibitors. Patients with ERRB2-mutated peripheral nerve sheath tumors may have broader therapeutic options in the variety of available tyrosine kinase inhibitors studied in other cancers.

Association of ERBB2 Copy Number and Gene Coalterations With Trastuzumab Efficacy and Resistance in Human Epidermal Growth Factor Receptor 2-Positive Esophagogastric and Gastric Cancer.

PURPOSEERBB2 copy number (CN), measured using next-generation sequencing, is a predictive biomarker for trastuzumab efficacy in human epidermal growth factor receptor 2 (HER2)–positive advanced esophagogastric and gastric cancer (AGC). We aimed to investigate the association of ERBB2 amplification and gene coalterations with response and resistance to trastuzumab-combined chemotherapy.METHODSThe SCRUM-Japan GI-SCREEN was a comprehensive genomic profiling project of GI cancer tissues using Oncomine Cancer Research Panel and Oncomine Comprehensive Assay. From 885 patients with AGC who successfully underwent gene profiling, 74 with ERBB2 amplification (CN ≥ 4.0) and who received first-line trastuzumab-combined chemotherapy were selected, and ERBB2 CN and gene coalterations were assessed.RESULTSERBB2 CN did not differ in tumor response to trastuzumab-combined chemotherapy (one-way analysis of variance test, P = .37). Multivariate analysis using the Cox proportional hazard model revealed that ERBB2 CN (continuous log2-converted CN, hazard ratio, 0.76; 95% CI, 0.62 to 0.93; P < .01) and receptor/oncogene amplifications in the HER2 signaling pathway (hazard ratio, 2.5; 95% CI, 1.2 to 5.3; P = .01) were significant predictors for progression-free survival (PFS). ERBB2 variants coexisted in five patients (7%) and were missense mutations. Two patients with low variant allele frequencies (VAFs; 8%, 12%) showed high ERBB2 CN (55, 80) and durable response (≥ 20 months), whereas three patients with high VAFs (66%–90%) showed low ERBB2 CN (8-11) and no response with short PFS (1-10 months).CONCLUSIONERBB2 CN and gene coamplification in the HER2 signaling pathway were positive and negative predictors of PFS in trastuzumab-treated HER2-positive AGC patients, respectively. HER2-positive AGC patients with a high VAF of ERBB2 showed poor outcomes and may need HER2 tyrosine kinase inhibitors and trastuzumab deruxtecan.

Abstract 5014: The landscape of ERBB2 mutations in Chinese patients with solid tumors

Abstract Background: Receptor tyrosine kinase (ERBB2) is altered by mutations and amplifications in various cancer types. Meanwhile, FDA approved some drugs to use for variants in ERBB2. However, the landscape of ERBB2 mutations and copy number variants (CNV) in pan-cancer is unknown in Chinese. Methods: 599 gene-panel by next-generation sequencing (NGS) was performed on 2,570 patients with solid tumors including non-small cell lung cancer (NSCLC) (n = 660), esophagogastric (n = 89), colorectal (n = 966), breast (n = 156) and gastric cancer (n = 699). The mutations and CNV in ERBB2 were detected for characterize the landscape in Chinese patients with solid tumors. Results: The frequency of single nucleotide variants (SNV) and CNV in ERBB2 was 9% and 4.3% in pan-cancer and the rate of variants was 13.3%. The most frequency of CNV was occurred in breast cancer and the last was in non-small cell lung cancer (13.5% vs 1.1%). Nevertheless, the rate of SNV in different cancer species was almost the same as in pan-cancer (mean: 8.8%). The higher rate of variants in ERBB2 was breast cancer and the lower was NSCLC. The cancer types with the highest incidence of ERBB2 variants were the following: breast (22.4%), gastric adenocarcinoma (18.3%), esophagogastric cancer (11.2%), colorectal cancer (10.8%) and NSCLC (9.7%). Conclusion: Based on the results, the profile of variants in ERBB2 revealed more patients may profit from FDA-approval drugs in China. The patients with somatic mutations in ERBB2 are treated with trastuzumab deruxtecan and adotrastuzumab emtansine in NSCLC. The patients with amplification are treated with trastuzumab deruxtecan in most tumors, but margetuximab and chemotherapy can cure breast cancer, according to FDA-approval. Keywords: ERBB2 mutations, CNV, SNV, solid tumors Citation Format: Xiangyu Su, Hongmei Zhao. The landscape of ERBB2 mutations in Chinese patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5014.

Integrated approach to functional analysis of an ERBB2 variant of unknown significance detected by a cancer gene panel test

PurposeDealing with variants of unknown significance (VUS) is an important issue in the clinical application of NGS-based cancer gene panel tests. We detected a novel ERBB2 extracellular domain VUS, c.1157A > G p.(E401G), in a cancer gene panel test. Since the mechanisms of activation by ERBB2 extracellular domain (ECD) variants are not fully understood, we aimed to clarify those mechanisms and the biological functions of ERBB2 E401G.MethodsERBB2 E401G was selected as VUS for analysis because multiple software tools predicted its pathogenicity. We prepared ERBB2 expression vectors with the E401G variant as well as vectors with S310F and E321G, which are known to be activating mutations. On the basis of wild-type ERBB2 or mutant ERBB2 expression in cell lines without ERBB2 amplification or variants, we evaluated the phosphorylation of human epidermal growth factor receptor 2 and related proteins, and investigated with molecular dynamics (MD) simulation the mechanisms conferred by the variants. The biological effects of ERBB2 E401G were also investigated, both in vitro and in vivo.ResultsWe found that ERBB2 E401G enhances C-terminal phosphorylation in a way similar to S310F. MD simulation analysis revealed that these variants maintain the stability of the EGFR-HER2 heterodimer in a ligand-independent manner. Moreover, ERBB2 E401G-transduced cells showed an increased invasive capacity in vitro and an increased tumor growth capacity in vivo.ConclusionOur results provide important information on the activating mechanisms of ERBB2 extracellular domain (ECD) variants and illustrate a model workflow integrating wet and dry bench processes for the analysis of VUS detected with cancer gene panel tests.

Germline ERBB2/HER2 Coding Variants Are Associated with Increased Risk of Myeloproliferative Neoplasms.

Simple SummaryFamilial clustering of myeloproliferative neoplasms (MPN) is well known, with a 5- to 7-fold increased risk of MPN among first-degree relatives of MPN patients. However, the genetic susceptibility of this disease remains poorly understood. Exome sequencing of a familial MPN pedigree followed by a case-control analysis identified germline variants in the HER2/ERBB2 gene that associate with the MPN phenotype. ERBB2/HER2 is a novel susceptibility locus which contributes to cancer risk in combination with additional risk alleles.Familial cases of myeloproliferative neoplasms (MPN) are relatively common, yet few inherited risk factors have been identified. Exome sequencing of a kindred with a familial cancer syndrome characterized by both MPN and melanoma produced a germline variant in the ERBB2/HER2 gene that co-segregates with disease. To further investigate whether germline ERBB2 variants contribute to MPN predisposition, the frequency of ERBB2 variants was analyzed in 1604 cases that underwent evaluation for hematologic malignancy, including 236 cases of MPN. MPN cases had a higher frequency of rare germline ERBB2 coding variants compared to non-MPN hematologic malignancies (8.9% vs. 4.1%, OR 2.4, 95% CI: 1.4 to 4.0, p = 0.0028) as well as cases without a blood cancer diagnosis that served as an internal control (8.9% vs. 2.7%, OR 3.5, 95% CI: 1.4 to 8.3, p = 0.0053). This finding was validated via comparison to an independent control cohort of 1587 cases without selection for hematologic malignancy (8.9% in MPN cases vs. 5.2% in controls, p = 0.040). The most frequent variant identified, ERBB2 c.1960A > G; p.I654V, was present in MPN cases at more than twice its expected frequency. These data indicate that rare germline coding variants in ERBB2 are associated with an increased risk for development of MPN. The ERBB2 gene is a novel susceptibility locus which likely contributes to cancer risk in combination with additional risk alleles.

Germline ERBB2 Variants Associate with Myeloproliferative Neoplasms

Background: The myeloproliferative neoplasms (MPN) and myelodysplastic syndrome (MDS) are usually sporadic diseases, however, familial cases are well-described. Approximately 10% of MPN cases display familial clustering, and there is a 5-7 fold increased risk of developing an MPN among first degree relatives of MPN patients. While familial MDS appears to be less common, identification of multiple predisposition genes has led to the incorporation of this entity into the most recent WHO classification of myeloid neoplasms. Identification of predisposing germline mutations in the myeloid malignancies has led to a better understanding of the pathophysiology of these diseases and improved clinical care. However, many familial MDS and MPN cases exist in which the inherited genetic lesion is unknown. Our recent investigation of a multigenerational family with inherited MDS/AML marked by erythroid hyperplasia identified a missense variant in ERBB3 that co-segregated with the disease phenotype (1). In this study, we investigated a kindred with an autosomal dominant familial cancer syndrome characterized by both MPN and solid tumor malignancies.Methods: Whole exome sequencing was performed via the Baylor-Hopkins Center for Mendelian Genomics at Center for Inherited Disease Research at Johns Hopkins University. Saliva or peripheral blood samples were used as source DNA. Sequence analysis and variant calling/filtering was performed as previously described (1). Targeted sequencing of the ERBB genes was performed via MiSeq using an Illumina TruSeq Custom Amplicon (v1.5) designed to sequence the entire open reading frame of selected genes. Analysis was performed using Illumina BaseSpace TruSeq applications. Germline variants were identified via variant allele frequency and heterozygous, nonsynonymous variants with a minor allele frequency < 0.01 and a depth > 40x were prioritized.Results: The proband was diagnosed with a JAK2 V617F positive MPN at age 37. Subsequent personal cancer history included melanoma and renal carcinoma. Family history was significant for ovarian cancer and melanoma in her mother, MPN and melanoma in her maternal aunt, and bladder cancer in her maternal uncle (Figure 1A). Exome sequencing of the three family members affected with melanoma was performed in order to identify the predisposing inherited variant in this family. Sequence analysis produced 71 candidate rare, heterozygous, nonsynonymous single nucleotide variants (SNV), including one in the gene ERBB2 . Genotyping of 2 additional family members for the ERBB2 c.3182T>C;p.L1061P SNV established that it co-segregates with disease (Figure 1A). To further investigate the prevalence of rare germline ERBB2 and ERBB3 variants in both MPN and MDS, we performed targeted sequencing of these genes in two discovery cohorts: 1) 96 cases of MPN, of which 63 had a 1st or 2nd degree relative with a family history of MPN and 2) 19 cases of erythroid MDS/AML. Five out of 96 (5.2%) cases of MPN were found to have ERBB2 variants, while 3 of 96 (3.1%) had ERBB3 variants. Of the 19 erythroid MDS/AML cases, no ERBB2 variants were identified, while 1 had an ERBB3 variant. Further, one MPN case with a germline ERBB2 c.G3250T:p.D1084Y variant had a family history of breast cancer in her mother, who also carried this variant (Figure 1B). We analyzed a validation cohort of 630 cases of hematologic malignancies (approximately 20% MPN) sequenced as part of their clinical evaluation at the Sidney Kimmel Cancer Center. Similar to the findings in our discovery cohorts, rare germline ERBB2 variants were enriched in MPN, found in approximately 5% of MPN compared to only 1.8% of all other myeloid disorder diagnoses. In contrast, germline ERBB3 variants were present in 1.6% of MPN compared to 1.8% of the other myeloid disorders (Table).Conclusions: We have identified germline ERBB2 variants that co-segregate with disease in two kindreds with familial cancer syndromes manifesting as a mixed MPN and solid tumor phenotype. In addition, we show that germline ERBB2 and ERBB3 variants are present in patients with hematologic malignancies at a higher incidence than expected in the general population, and ERBB2 variants are associated with an MPN phenotype. Together, these data implicate these genes in the predisposition to myeloid malignancy as well as other familial cancer syndromes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Integrative statistical analyses of multiple liquid biopsy analytes in metastatic breast cancer

BackgroundSingle liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cell-free DNA (cfDNA).MethodsBlood was drawn from 26 hormone receptor-positive, HER2-negative MBC patients. CTC mRNA and EV mRNA were analyzed using a multi-marker qPCR. Plasma from CTC-depleted blood was utilized for cfDNA isolation. gDNA from CTCs was isolated from mRNA-depleted CTC lysates. CTC gDNA and cfDNA were analyzed by targeted sequencing. Hierarchical clustering was performed within each analyte, and its results were combined into a score termed Evaluation of multiple Liquid biopsy analytes In Metastatic breast cancer patients All from one blood sample (ELIMA.score), which calculates the contribution of each analyte to the overall survival prediction. Singular value decomposition (SVD), mutual information calculation, k-means clustering, and graph-theoretic analysis were conducted to elucidate the dependence between individual analytes.ResultsA combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA.score resulted in a highly significant correlation with overall survival, thereby bolstering evidence for the additive value of using multiple LBAs. Computation of mutual information indicated that none of the LBAs is independent of the others, but the ability of a single LBA to describe the others is rather limited—only CTC gDNA could partially describe the other three LBAs. SVD revealed that the strongest singular vectors originate from all four LBAs, but a majority originated from CTC gDNA. After k-means clustering of patients based on parameters of all four LBAs, the graph-theoretic analysis revealed CTC ERBB2 variants only in patients belonging to one particular cluster.ConclusionsThe additional benefits of using all four LBAs were objectively demonstrated in this pilot study, which also indicated a relative dominance of CTC gDNA over the other LBAs. Consequently, a multi-parametric liquid biopsy approach deconvolutes the genomic and transcriptomic complexity and should be considered in clinical practice.

Abstract PS6-60: A clustering and mutual information based analysis of the ELIMA study results: The additive value of multi-parametric liquid biopsies, including CTCs, EVs and cfDNA, in metastatic breast cancer

Abstract Background: To gain comprehensive insights into the aspects of genomic and transcriptomic complexity which could be beneficial for therapy management in metastatic breast cancer (MBC), we established the isolation and analysis of mRNA and gDNA from circulating tumor cells (CTCs), mRNA from extracellular vesicles (EVs), and cell-free DNA (cfDNA) from a minimized blood volume. Here, we aimed to assemble the results of all four analytes and elucidated the relevance of the diverse parameters within a multimodal data set. Methods: EDTA blood (2x 9 ml) was drawn from 26 MBC patients with hormone receptor-positive and HER2 negative primary tumors at the time of disease progression. CTCs and their mRNA were isolated using the AdnaTest EMT2/StemCell Select/Detect. Plasma of CTC-depleted blood was used for cfDNA isolation, while mRNA from EVs was isolated by exoRNeasy using the remaining blood. The mRNA purified from CTCs and EVs was analyzed by a multimarker qPCR panel. gDNA from CTCs was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/RNA Nano Kit prototype. CTC gDNA and cfDNA were analyzed with a customized QIAseq Targeted DNA Panel for Illumina with unique molecular indices. Consumables: QIAGEN, Germany. The statistical tools for evaluating the results included: Hierarchical clustering according to Ward’s method with Euclidean distance, singular value decomposition, mutual information calculation, and k-means clustering. Results: Isolation of mRNA and gDNA from CTCs, mRNA from EVs, and cfDNA was successfully established in a condensed workflow. 88% of the patients showed at least one variant in CTC gDNA or one overexpression signal in the CTC mRNA fraction. The mean number of variants/signals was also higher in CTC gDNA/mRNA when compared to cfDNA/EV mRNA. The analysis of individual analytes identified a similar number of patients (50%-73%) with actionable markers, but a multi-parametric evaluation of all four analytes identified actionable markers in 96% of the patients. After hierarchical clustering of the results of each individual analyte into four clusters, combining the two patient clusters with the worst overall survival resulted in prognostic value for CTC gDNA, cfDNA, and EV mRNA. Combination of the information above analyte borders showed additive value and resulted in a prognostic factor defined here as the ‘ELIMA score’. A calculation of the mutual information showed that CTC gDNA has the highest potential to describe the other three analytes. However, an Eigenvector analysis based on singular value decomposition revealed that the 10 most influential vectors contain parameters from all four analytes. This indicates that each of the analytes add valuable information not conveyed by the other analytes. K-means clustering was used to generate clusters based solely on CTC gDNA and based on all four analytes. A comparison of the clusters based on these two criteria showed that clustering based on all four analytes resulted in a division of patients according to their tumor histology type and ERBB2 variants in CTCs; thereby, underscoring the importance of taking all four analytes into consideration. Conclusion: We established a workflow for parallel isolation of multiple liquid biopsy analytes from a minimized blood volume. Though the mutual information calculation showed that CTC gDNA has a relatively greater ability to describe the other three analytes, further statistical analysis showed that each analyte carried information of additive value. Thus, a comprehensive picture of the genomic and transcriptomic complexity obtained by a multi-parametric liquid biopsy might enable easier identification of the most suitable therapy regimen for each individual patient in the future. Citation Format: Sabine Kasimir-Bauer, Vinay Suryaprakash, Markus Storbeck, Peter Hahn, Siegfried Hauch, Markus Sprenger-Haussels, Oliver Hoffmann, Rainer Kimmig, Corinna Keup. A clustering and mutual information based analysis of the ELIMA study results: The additive value of multi-parametric liquid biopsies, including CTCs, EVs and cfDNA, in metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-60.

Canonical ErbB-2 isoform and ErbB-2 variant c located in the nucleus drive triple negative breast cancer growth.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments.

Multimodal Targeted Deep Sequencing of Circulating Tumor Cells and Matched Cell-Free DNA Provides a More Comprehensive Tool to Identify Therapeutic Targets in Metastatic Breast Cancer Patients.

Cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for therapy management in oncology. We aimed to establish a multimodal liquid biopsy strategy that is usable with minimized blood volume to deconvolute the genomic complexity of metastatic breast cancer. CTCs were isolated from 10ml blood of 18 hormone receptor-positive and human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer patients. cfDNA was isolated from plasma generated after CTC depletion and targeted sequencing analyses were conducted. PIK3CA and ESR1 variants were less common in CTC gDNA, while ERBB2 variants were only detected in CTC gDNA. A total of 62% of all cfDNA variants were recovered in the matched CTC gDNA, while 72% of all variants were unique in either cfDNA (14 variants) or CTC gDNA (104 variants). The percentage of patients with no detectable cfDNA variants or CTC gDNA variants was 17%/11%, but a combined analysis identified variants in 94% of all patients. In univariate and multivariate regression models, ESR1 variants in cfDNA and CTC gDNA correlated significantly with survival. We suggest a coordinated analysis of both fractions in order to provide a comprehensive genomic footprint that may contribute to identifying the most suitable therapy for each individual.

Abstract P5-01-14: The combined blood analysis of cell-free DNA and genomic DNA of circulating tumor cells reveals additive value in hormone receptor-positive, HER2-negative metastatic breast cancer patients

Abstract Background: To gain comprehensive insights into the genomic complexity useful for therapy management in metastatic breast cancer (MBC), we aimed to isolate and analyze genomic DNA (gDNA) from circulating tumor cells (CTCs) and matched cell-free DNA (cfDNA) from a minimized blood volume. Methods: EDTA blood was drawn from 27 MBC patients with hormone receptor-positive and HER2 negative primary tumors at the time of disease progression. CTCs and CTC mRNA were isolated from 2 × 5 ml whole blood using the AdnaTest EMT2/StemCell Select/Detect. Plasma of CTC-depleted blood was used for cfDNA isolation. gDNA from CTCs was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/RNA Nano Kit prototype. CTC gDNA and cfDNA were analyzed with a customized QIAseq Targeted DNA Panel for Illumina with unique molecular indices (UMIs) analyzing AKT1, AR, BRCA1, BRCA2, EGFR, ERCC4, ERBB2, ERBB3, ESR1, FGFR1, KRAS, MUC16, PIK3CA, PIK3R1, PTEN, PTGFR and TGFB1. The library preparation protocol was slightly modified for the usage of CTC gDNA using the entire amount of the CTC gDNA eluate as input without pre-quantification followed by fragmentation and ligation with an increased number of adapters. All libraries were analyzed by paired-end sequencing on the Illumina NextSeq Sequencer with a NextSeq 550 System High-Output Kit, 2 × 150 bp reads with a mean coverage of 11000 x (CTC gDNA) and 8000 x (cfDNA). Consumables: QIAGEN, Germany. Results: Isolation of CTC gDNA and cfDNA was successfully established in a condensed workflow. The UMI coverage observed for cfDNA (2000 x) differed from the UMI coverage for CTC gDNA (500 x), resulting in dramatically increased lowest detectable allele frequency (AF) called with a confidence of 90% in CTC gDNA compared to cfDNA. On average, 6 CTC gDNA variants and 2 cfDNA variants were detected in each patient. Most variants were found in the MUC16 gene in both analytes. BRCA2 variants were the second most prevalent variants in CTC gDNA and cfDNA. PIK3CA and ESR1 variants were less common in CTC gDNA compared with cfDNA, while ERBB2 variants were only detected in CTC gDNA. 57% of all cfDNA variants (29/51) were recovered in the matched CTC gDNA, while 89% of all variants were unique in either CTC gDNA (125 variants) or cfDNA (22 variants). On average, the cfDNA variants with low AF (mean 15%) were not detected in CTC gDNA, while the cfDNA variants that were also found in CTC gDNA exhibited a mean AF of 44%. Similarly, the unique CTC gDNA variants had a mean AF of 23%, while the shared variants were prevalent with a mean AF of 47% in the CTC gDNA fraction. Comparing the patients, 11%/14% of the single CTC gDNA / cfDNA variants were detected in more than one patient, hence the majority of variants was patient-specific. The portion of patients without detectable cfDNA variants or CTC gDNA variants was 15% (4/27) / 11% (3/27), but combined analysis of CTC gDNA and cfDNA identified variants in 26 of all 27 MBC patients (96%). Conclusion: cfDNA and CTC gDNA showed additive variant information. Thus, it is advised to assess cfDNA and CTC gDNA variants to receive a comprehensive genomic picture that might enable to identify the most suitable therapy regimen in each individual patient in the future. Citation Format: Corinna Keup, Ann-Kathrin Bittner, Oliver Hoffmann, Mitra Tewes, Markus Storbeck, Peter Hahn, Siegfried Hauch, Markus Sprenger-Haussels, Rainer Kimmig, Sabine Kasimir-Bauer. The combined blood analysis of cell-free DNA and genomic DNA of circulating tumor cells reveals additive value in hormone receptor-positive, HER2-negative metastatic breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-14.

Abstract CT139: NCI Molecular Analysis for Therapy Choice (NCI-MATCH EAY131) arm B: Phase II study of afatinib in patients (pts) with HER2 (ERBB2) activating mutations

Abstract BACKGROUND: NCI-MATCH is a multi-cohort, signal finding trial that assigns pts with advanced cancers to targeted therapies based on central tumor genomic testing. Arm B evaluated afatinib, an ErbB family blocker, in pts with ERBB2 activating mutations. METHODS: Eligible pts had advanced refractory solid tumors, lymphoma, or multiple myeloma, tumor biopsies with selected ERBB2 single nucleotide variants or insertions/deletions, but no ERBB2 amplification detected by the NCI-MATCH next generation sequencing assay. Pts had performance status (PS) ≤1, left ventricular ejection fraction &amp;gt;50%, grade ≤1 diarrhea, and no prior HER2 therapy. Non-small cell lung cancers were excluded. Pts received afatinib 40 mg daily in 28 day cycles. Concomitant endocrine therapy was not allowed. Tumor assessments were every two cycles. The primary objective was overall response rate (ORR). Secondary objectives were: 6-month progression-free survival (PFS6), overall survival, toxicity and molecular correlates. Initial planned enrollment was 35 pts, but a subsequent amendment permitted accrual of up to 70 pts to provide additional information in rarer histologies. RESULTS: 59 pts were assigned and 40 enrolled (37 evaluable) to Arm B. Median age was 62 (range 29-83), 78% female, 32% PS0, and 58% had received &amp;gt;3 prior therapies. Tumor histologies: breast (n=12; 5 lobular/7 ductal, all ER+), colorectal (n=5), urothelial (n=4), biliary (n=3), cervix (n=2), small bowel (n=2) and other (n=9). The qualifying ERBB2 variants were L755S (n=6), V777L (n=6), V842I (n=6), S310F (n=6), D769Y (n=5), S310Y (n=4), V777_G778insGSP (n=2), and other (n=7). The ORR was 2.7% (n=1/37; 90% CI 0.14%-12.2%) and PFS6 was 11% (90% CI 5.0%-23.5%). A confirmed partial response (PR) occurred in a patient with adenocarcinoma of extra-mammary Paget disease of skin who progressed after cycle 6. Two unconfirmed PRs were observed (low grade serous gynecological tract and ER+/HER2- IHC breast ductal carcinoma). Of 12 evaluable breast cancer pts, there was 1 additional pt with lobular carcinoma (ER+/HER2-FISH) who had 51% reduction in target lesions but progressed due to a new lesion at cycle 6. The most common (&amp;gt;20%) treatment-related AEs were: diarrhea (68%), mucositis (43%), fatigue (40%), acneiform rash (30%) dehydration (27%), vomiting (27%), nausea (27%), anemia (27%) and anorexia (22%). Most AEs were grade ≤2. Four pts (11%) discontinued due to AEs. CONCLUSION: Although afatinib did not meet the pre-specified threshold for anti-tumor activity, the response in a rare tumor type is notable. The results in breast/gynecologic malignancies, along with the SUMMIT trial (Hyman Nature 2018), suggest differential activity of HER2 inhibitors in ERBB2 mutant cancers by tumor histology. The safety profile of afatinib was consistent with prior studies. Citation Format: Philippe L. Bedard, Shuli Li, Kari B. Wisinski, Eddy S. Yang, Sewanti A. Limaye, Edith P. Mitchell, James A. Zwiebel, Jeffrey Moscow, Robert J. Gray, Lisa M. McShane, Larry V. Rubenstein, David R. Patton, P Mickey Williams, Stanley R. Hamilton, Barbara A. Conley, Carlos L. Arteaga, Lyndsay N. Harris, Peter J. O'Dwyer, Alice P. Chen, Keith T. Flaherty. NCI Molecular Analysis for Therapy Choice (NCI-MATCH EAY131) arm B: Phase II study of afatinib in patients (pts) with HER2 (ERBB2) activating mutations [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 CT139.

Abstract 3267: Combined effect of multiple gene polymorphisms on cancer risk stratification

Abstract Cyclin D1 (CCND1) G870A polymorphism has been known to be a risk factor in multiple cancers. Cyclin D1 is a cell cycle regulator frequently mutated, amplified and overexpressed in a variety of tumors. Activation and over expression of CCND1 have been found in variety of tumors and alterations in CCND1 are thought to be involved in carcinogensis. CCND1 polymorphism A870G interferes with splicing and studies showed DNA damage in cells from subjects with ‘A’ allele to bypass G1/S check point of the cell cycle control mechanism more easily than damage in cells with G allele (wild type). A single base pair polymorphism (exon 4, G870A) in the CCND1 gene affects gene splicing. This identification and validation of contributing genetic factors such as single-nucleotide polymorphisms (SNPs) represents a critical step in the advance toward personalized medicine. Several gene expression correlation studies have been done but to our knowledge studies have not been reported on SNP’s of ERBB2 gene, ESR gene and CCND1 combined effect correlating with high risk. In this study, we examined the relationship between ERBB2 (codon 655), Estrogen receptor (ER Intron1 T&amp;lt;C) and cyclin D1 (Exon 4) polymorphic additive effect in cancers more specific to breast cancer. Genomic DNA was extracted from whole blood samples collected on study. The SNPs were tested by the PCR-restriction fragment length polymorphism (PCR-RFLP) method. Our results show individuals carrying CCND1 ‘A allele in combination with variant ERBB2/BsmAI uncut allele are significantly high (P&amp;lt;0.004) in cancer patients (N=232) as compared to controls. (N=222). Similarly individuals carrying CCND1 Variant ‘A ‘ allele in combination with ESR ‘C’ Allele showed high frequency with p&amp;lt;0.009. Both these combinations showed a strong and significant association with cancer but individually, ERBB2 variant did not show any significant association, ESR gene showed a borderline association and CCND1 showed strong association independent of these two gene polymorphisms. Earlier few studies have shown no association of ERBB2 mutation in agreement with the previous reports. In conclusion our data suggests that in complex diseases a single gene may not contribute to show its effect but in combination may show a profound effect in evaluating the risk. Citation Format: Radhika Gade Andavolu, Olivia Rodriguez, Anastasia Al Rubakovic, Catherine Stafford, Murthy V. Andavolu. Combined effect of multiple gene polymorphisms on cancer risk stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3267. doi:10.1158/1538-7445.AM2017-3267

Abstract LB-103: Landscape of somatic ERBB2 Mutations: Findings from AACR GENIE and comparison to ongoing ERBB2 mutant basket study

Abstract Background: AACR GENIE is an international data-sharing project that aggregates clinical-grade cancer genomic data. As a demonstration of utility, we evaluated the landscape of ERBB2 mutations in the first 18,486 patients included in this registry and compared it to the first 100 patients enrolled in an ongoing international Phase 2 SUMMIT ‘basket’ study of the pan-HER inhibitor neratinib in ERBB2 mutant solid tumors (NCT01953926). Results: ERBB2 mutations were identified in 2.8% (519/18,486) of patients in the GENIE cohort and observed at all participating centers. In total, there were 482 missense, 66 indels, 19 truncating mutations, and 14 structural variants. A total of 263 unique missense mutations were observed including 12 at previously identified hotspots which accounted for 69.2% of all missense mutations. 35 unique cancer types were represented. The tumor types with the highest proportion of ERBB2 mutations were bladder (12.8%, 82/641), breast (3.9%, 87/2230), colorectal (3.3%, 70/2102), and NSCLC (3%, 90/3006). Among patients with copy number data available (91%) 11% had concurrent ERBB2 amplification, most often in breast cancer. The most frequently observed alterations in ERBB2, adjusted for differing exon coverage between panels, was S310F/Y in 0.46% of the GENIE cohort (12.6% of samples with ERBB2 alterations), Y772_A775dup in 0.21% (6.9%), R678Q in 0.17% (4.5%), L755S in 0.16% (5.2%), V777L in 0.12% (3.8%), and V842I in 0.09% (3.1%). The distribution of specific ERBB2 variants differed significantly by tumor type with exon 20 insertions being most common in NSCLC (44.4%, 40/90), L755S (18.9%, 11/92) in breast, S310F/Y (26.9%, 28/104) in bladder, and V842I (13.9%, 10/72) in colorectal cancer. Structural variants included intragenic deletions (n=4) and fusions involving various partners including GRB7 (n=2), and one each of C1orf87, PPIL6, HEXIM2, THRA, ASIC2, BCA3, WIPF2. The frequencies of ERBB2 mutant cancer types observed in the GENIE cohort were generally comparable to those enrolled to the neratinib basket study including NSCLC (17 vs 22%, respectively), breast (16.4 vs 24%), bladder (15.5 vs 14%), colorectal (13.2 vs 17%), and endometrial (4.2 vs 6%). At the variant level, S310F/Y was less prevalent in GENIE compared to the neratinib study (12.6 vs 24%) while all other mutations were generally similar including L755S (5.2 vs 9%), R678Q (4.5 vs 2%), Y772_A775dup (6.9 vs 13%), V777L (3.8 vs 9%), and V842I (3.1 vs 6%). Conclusion: GENIE confirms that a diversity of ERBB2 mutations are prevalent across a variety of tumor types in patients with advanced cancer. The genomic landscape of ERBB2 mutations was largely similar in the population based GENIE cohort and the neratinib SUMMIT study, providing the first direct evidence that basket study enrollment accurately reflects the true landscape of the target alteration. Citation Format: Alison Schram, Helen H. Won, Fabrice Andre, Monica Arnedos, Funda Meric - Bernstam, Philippe L. Bedard, Kenna R. Shaw, Hugo Horlings, Christine Micheel, Ben Ho Park, Grace Mann, Alshad S. Lalani, Lillian Smyth, David B. Solit, Deborah Schrag, Mia A. Levy, Barrett J. Rollins, Mark Routbort, Charles L. Sawyers, Eva Lepisto, Michael F. Berger, David M. Hyman, on behalf of the AACR Project GENIE Consortium. Landscape of somatic ERBB2 Mutations: Findings from AACR GENIE and comparison to ongoing ERBB2 mutant basket study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-103. doi:10.1158/1538-7445.AM2017-LB-103

Abstract P2-03-10: A fit-for-purpose NGS system that reports ERBB2 (HER2) mutations and copy number variants for clinical trials research and drug development

Abstract Introduction: HER2-positive breast cancers comprise 20-30% by subtype and represent the second poorest prognosis. In addition to HER2 signaling activation via protein overexpression or gene amplification (measured by IHC or ISH), somatic mutations in the ERBB2 (HER2) gene have been identified in 2-4% of breast cancers and reported to drive HER2 pathway signaling and tumorigenesis. Irreversible HER2 tyrosine kinase inhibitors such as neratinib have been shown to potently inhibit breast tumors harboring somatic HER2 activating mutations and are under active clinical investigation. A targeted molecular characterization strategy is thus needed to support clinical trial research. Here, we present an integrated NGS system that identifies multiple classes of ERBB2aberrations, including single nucleotide variants, insertion/deletion mutations, and copy number variations (CNVs). Methods: Genomic DNA was isolated from FFPE human tumor tissues or FFPE-embedded engineered cell lines expressing ERBB2 mutations. Pre-analytical sample QC was performed by a novel qPCR assay that quantifies amplifiable DNA templates suitable for library preparation. NGS libraries were prepared using the QuantideX® NGS ERBB2 Mutations assay, with two-pool PCR-based enrichment to detect multiple types of ERBB2 genomic alterations. Libraries were sequenced (Illumina MiSeq®) and analyzed using QuantideX® Reporter software, directly incorporating the pre-analytical QC data from each DNA sample to improve variant call accuracy. Results: The QuantideX® NGS ERBB2 Mutations system targets 28% of ERBB2 coding bases covering exons 8 and 17 - 24, including codons 713-989 of the tyrosine kinase domain. The assay also detects focal, whole-arm and polysomy-induced HER2 amplification using sentinel flanking amplicons, and amplicons covering CEN17 and copy number-neutral chromosomal regions identified in the TCGA breast cancer cohort. The assay was analytically validated in a CLIA-laboratory which included an analysis of 6 mixtures and titrations of synthesized DNA bearing 31 ERBB2 mutations, 17 engineered cell lines and FFPE cell-line blocks, and 12 surgically-resected FFPE carcinomas. A multi-day/operator study revealed 100% sensitivity and PPV for detection of ERBB2 SNVs, indels and complex variants down to 5% mutation positive in wild-type DNA. The NGS system was also used to profile ERBB2 variants in 473 FFPE human tumor tissues, including 280 breast carcinomas. Mutation-positive samples were confirmed by an orthogonal NGS method. A subset of 46 FFPE tissue samples were assessed for CNVs by NGS and compared with matched droplet digital PCR (ddPCR) analysis. A correlation of R2 &amp;gt; 0.91 between NGS and ddPCR underscores the potential of the assay to reveal both ERBB2 mutations and copy number changes. Conclusions: The QuantideX® NGS ERBB2system integrates optimized wet- and dry-bench elements to achieve broad and accurate detection of ERBB2 mutations and copy number variants from challenging FFPE specimens. The technology has the potential to advance breast cancer clinical research and aid in the selection of patients for clinical trials, particularly those that assess HER2-directed therapies such as afatinib, lapatinib, and neratinib. Citation Format: Zhu H, Zeigler R, Bridger S, Larson JL, Kincaid A, Cardwell R, Haynes BC, Andruss BF, Latham GJ. A fit-for-purpose NGS system that reports ERBB2 (HER2) mutations and copy number variants for clinical trials research and drug development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-10.

Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.