ERBB2 Gene Research Articles

Genomic profiling using the UltraSEEK panel identifies discordancy between paired primary and breast cancer brain metastases and an association with brain metastasis-free survival

PurposeBrain metastases (BM) are an increasing clinical problem. This study aimed to assess paired primary breast cancers (BC) and BM for aberrations within TP53, PIK3CA, ESR1, ERBB2 and AKT utilising the MassARRAY® UltraSEEK® technology (Agena Bioscience, San Diego, USA).MethodsDNA isolated from 32 paired primary BCs and BMs was screened using the custom UltraSEEK® Breast Cancer Panel. Data acquisition and analysis was performed by the Agena Bioscience Typer software v4.0.26.74.ResultsMutations were identified in 91% primary BCs and 88% BM cases. TP53, AKT1, ESR1, PIK3CA and ERBB2 genes were mutated in 68.8%, 37.5%, 31.3%, 28.1% and 3.1% respectively of primary BCs and in 59.4%, 37.5%, 28.1%, 28.1% and 3.1% respectively of BMs. Differences in the mutations within the 5 genes between BC and paired BM were identified in 62.5% of paired cases. In primary BCs, ER-positive/HER2-negative cases harboured the most mutations (70%), followed by ER-positive/HER2-positive (15%) and triple-negatives (13.4%), whereas in BMs, the highest number of mutations was observed in triple-negative (52.5%), followed by ER-positive/HER2-negative (35.6%) and ER-negative/HER2-positive (12%). There was a significant association between the number of mutations in the primary BC and breast-to-brain metastasis-free survival (p = 0.0001) but not with overall survival (p = 0.056).ConclusionThese data demonstrate the discordancy between primary BC and BM, as well as the presence of clinically important, actionable mutations in BCBM. The UltraSEEK® Breast Cancer Panel provides a tool for BCBM that can be utilised to direct more tailored treatment decisions and for clinical studies investigating targeted agents.

Associations between brain abnormalities and common genetic variants for schizophrenia: a narrative review of structural and functional neuroimaging findings.

Schizophrenia is a common psychotic disease worldwide, and several genetic variants have been suggested to influence susceptibility to schizophrenia. However, the exact genetic and neural mechanisms underlying such relationships remain unclear. Neuroimaging endophenotypes have been considered to be desirable for finding genes influencing brain structure and function. Because of its high penetrance of genetic variants and wide applications in clinic practice, neuroimaging could be a great option in schizophrenia study. In this narrative review, we aim to provide a summary of current progress in neuroimaging findings to detect genetic variants that may influence the brain's structure and function, and thus improve understanding of how this interaction affects the onset of schizophrenia. Multiple common genetic variants for schizophrenia including the ZNF804A, DTNBP1, DAOA, AKT1, NTRK3, and ERBB4 genes are reviewed. In summary, the current investigations have reported effects of these genetic variants on both structural and functional brain abnormalities (e.g., altered grey matter volumes, white matter integrity and functional connectivity patterns) in schizophrenia within multiple brain regions, especially the frontal, temporal and hippocampal areas. These findings facilitate our understanding of schizophrenia and helping us with early diagnosis, prognosis prediction and medication. However, a deep insight into this field necessitate further investigations and there are a number of challenges to be settled, such as enlarging the sample size; solving the statistical, methodological, and technological limitations; performing not only cross-sectional but also longitudinal observations; focusing on the effect of copy number variants; figuring out the complexity of epistasis and gene-environment interaction; achieving clinical translations; and depict the effect of a polygenic risk score beyond those of single genetic markers.

Epigenetic Silencing of HER2 Expression during Epithelial-Mesenchymal Transition Leads to Trastuzumab Resistance in Breast Cancer.

HER2 receptor tyrosine kinase (encoded by the ERBB2 gene) is overexpressed in approximately 25% of all breast cancer tumors (HER2-positive breast cancers). Resistance to HER2-targeting therapies is partially due to the loss of HER2 expression in tumor cells during treatment. However, little is known about the exact mechanism of HER2 downregulation in HER2-positive tumor cells. Here, by analyzing publicly available genomic data we investigate the hypothesis that epithelial-mesenchymal transition (EMT) abrogates HER2 expression by epigenetic silencing of the ERBB2 gene as a mechanism of acquired resistance to HER2-targeted therapies. As result, HER2 expression was found to be positively and negatively correlated with the expression of epithelial and mesenchymal phenotype marker genes, respectively. The ERBB2 chromatin of HER2-high epithelial-like breast cancer cells and HER2-low mesenchymal-like cells were found to be open/active and closed/inactive, respectively. Decreased HER2 expression was correlated with increased EMT phenotype, inactivated chromatin and lower response to lapatinib. We also found that induction of EMT in the HER2-positive breast cancer cell line BT474 resulted in downregulated HER2 expression and reduced trastuzumab binding. Our results suggest that ERBB2 gene silencing by epigenetic regulation during EMT may be a mechanism of de novo resistance of HER2-positive breast cancer cells to trastuzumab and lapatinib.

Concentration-dependent transcriptome of zebrafish larvae for environmental bisphenol S assessment

The widespread use of bisphenol S (BPS) as an alternative to bisphenol A has captured attention due to its potential toxicity to aquatic organisms. In the present study, the zebrafish was used as a model to evaluate the toxicity of BPS and determine the underlying mechanisms. The environmental concentration-dependent (0, 0.1, 1, 10, 100, and 1000 μg/L BPS) transcriptome approach was employed in combination with toxicity assays to address the problem. Based on a weighted correlation network analysis, we speculated that excess reactive oxygen species (ROS) may initiate cellular events in BPS-exposed zebrafish, leading to multiple toxic effects. Furthermore, we used pathway enrichment analysis to identify key pathways (MAPK signalling pathway and metabolic pathways) that link the molecular mechanisms with different toxic effects. In addition, we performed protein-protein network and shortest path analyses to identify six hub genes (erbb2, rrm2, rps27a, his2h3c, cdk1, and mcm5) and their interactions. Moreover, we suggest that BPS may interact with erbb2 by molecular docking. Thus, the BPS-erbb2 interaction may activate the MAPK signalling and metabolic pathways, resulting in ROS production and then caused multiple toxic effects in zebrafish. This study provides information for characterising the mechanisms of BPS exposure in aquatic environments.

Mutations in circulating cell‐free tumour DNA: Predictors of survival in hepatocellular carcinoma

AbstractBackgroundHepatocellular carcinoma (HCC) incidence is increasing worldwide and prognostic biomarkers are urgently needed to guide treatment and reduce mortality. Circulating cell‐free DNA of tumour origin (ctDNA) is a novel, minimally invasive means of determining genetic alterations in cancer. We determined the utility of ctDNA as a prognostic biomarker of survival in HCC.MethodsPlasma cell‐free DNA and matched germline DNA were isolated from patients with HCC (n = 51) and cirrhosis (n = 10). Targeted, multiplex PCR ultra‐deep sequencing was performed using a liver cancer‐specific primer panel for genes ALB, AMPH, APC, ARID1A, ARID2, ATM, AXIN1, BAZ2B, BRAF, CSMD3, CTNNB1, DSE, ERBB2, HNF1A, IGFR2, IGSF10, KEAP1, MET, TP53, UBR3, USP25, ZIC3 and ZNF226. Associations between mutations in ctDNA and overall survival were analysed using Cox proportional hazards modelling.Results114 putative mutations (70 unique) in were detected in plasma ctDNA in 35 of 51 patients with HCC (69%). On univariable analysis, CSMD3 gene mutations were associated with shorter overall survival (Logrank HR 3.18, 95% CI 1.14‐8.86, P = .027). The median survival time was 15.5 months (IQR 7.77‐16.5 months) in patients with CSMD3 mutations compared with the median survival of 26.5 months (IQR 16.93‐46.07 months) in patients without CSMD3 mutations. Other factors associated with overall survival were advanced BCLC stage (HR 16.52, 95% CI 2.22‐122.94, P = .006) and Child‐Pugh Class (CPC HR 7.98, 95% CI 2.31‐27.61, P = .001). Cox proportional hazards modelling showed mutations in CSMD3 remained a significant independent risk for shorter overall survival in HCC when adjusted for age, BCLC stage and Child‐Pugh class (HR 4.91, 95% CI 1.60‐15.02, P = .005).ConclusionDetection of CSMD3 mutations in plasma ctDNA is associated with reduced overall survival in HCC patients, adjusted for potential confounding factors.

Association and epistatic analysis of white matter related genes across the continuum schizophrenia and autism spectrum disorders: The joint effect of NRG1-ErbB genes

Background Schizophrenia-spectrum disorders (SSD) and Autism spectrum disorders (ASD) are neurodevelopmental disorders that share clinical, cognitive, and genetic characteristics, as well as particular white matter (WM) abnormalities. In this study, we aimed to investigate the role of a set of oligodendrocyte/myelin-related (OMR) genes and their epistatic effect on the risk for SSD and ASD. Methods We examined 108 SNPs in a set of 22 OMR genes in 1749 subjects divided into three independent samples (187 SSD trios, 915 SSD cases/control, and 91 ASD trios). Genetic association and gene-gene interaction analyses were conducted with PLINK and MB-MDR, and permutation procedures were implemented in both. Results Some OMR genes showed an association trend with SSD, while after correction, the ones that remained significantly associated were MBP, ERBB3, and AKT1. Significant gene-gene interactions were found between (i) NRG1*MBP (perm p-value = 0.002) in the SSD trios sample, (ii) ERBB3*AKT1 (perm p-value = 0.001) in the SSD case-control sample, and (iii) ERBB3*QKI (perm p-value = 0.0006) in the ASD trios sample. Discussion Our results suggest the implication of OMR genes in the risk for both SSD and ASD and highlight the role of NRG1 and ERBB genes. These findings are in line with the previous evidence and may suggest pathophysiological mechanisms related to NRG1/ERBBs signalling in these disorders.

Abstract 2424: Screen for actionable ERBB3 mutations

Abstract Activating mutations and copy number variations in ERBB genes have been identified in several cancer types, and a number of cancer drugs targeting ERBB receptors have been approved for clinical use. These drugs include monoclonal antibodies (mAb) that selectively target either EGFR (ERBB1) or ERBB2 (HER2), and tyrosine kinase inhibitors (TKI) that block the kinase activity of all three kinase-competent ERBBs, i. e. EGFR, ERBB2, and ERBB4. ERBB3 is a unique member of the ERBB family in that it does not possess potent intrinsic kinase activity, and thus cannot be directly targeted with TKIs. However, it is still able to dimerize and activate the other three ERBB family members, and as a result, critically mediate oncogenic signaling as a member of heterodimeric complexes, such as the ERBB2/ERBB3 heterodimer. Thus, changes in ERBB3 structure and function may still affect responsiveness of cancer cells to ERBB TKIs. Moreover, several mAbs directly targeting ERBB3 in cancer are currently in preclinical and clinical development. To address whether ERBB3 mutations can affect the potential of ERBB3 to promote growth or affect sensitivity to ERBB inhibitors, we set up a screen for functional effects of all possible random ERBB3 missense or nonsense mutations in the context of ERBB2/ERBB3 heterodimers. To this end, the iSCREAM (in vitro screen of activating mutations) model of somatic evolution, recently developed in our laboratory, was chosen. This platform exploits randomly mutated cDNA libraries of the gene of interest created with error-prone PCR and allows the assessment of growth-advantage conferred by thousands of mutations in parallel. To set up the model for ERBB3 mutation screen, interleukin-3 (IL-3)-dependent Ba/F3 cells were engineered to express a homodimerization-incompetent ERBB2 V956R mutant together with ERBB3 constructs. The model was validated to serve as a readout for ERBB3's ability in activating ERBB2 kinase by demonstrating that cells expressing the two known transforming ERBB3 mutations, E928G and G284R, together with ERBB2 V956R survived and expanded in the absence of IL-3. However, control cells expressing ERBB2 V956R together with wild-type ERBB3 rapidly died in the absence of IL-3. The cell background with ERBB2 V956R expression will subsequently be used as a target for retroviral expression of cDNA library of randomly mutated ERBB constructs. After infection, the cell clones with activating ERBB3 mutations will be allowed to evolve and the activating mutations will be identified by ERBB3-targeted next generation sequencing. The goal is to identify activating ERBB3 mutations that are directly actionable or that provide predictive value for the use of drugs targeting ERBB signaling. Citation Format: Marika K. Koivu, Deepankar Chakroborty, Kari J. Kurppa, Klaus Elenius. Screen for actionable ERBB3 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2424.

Abstract 2729: Cell infiltration prediction of breast cancer tissues identifies basophils as potentially important immune cells in the tumor microenvironment

Abstract Background: Breast cancer (BC) is the most common and deadliest cancer in women worldwide. Hormone receptor (HR) positive tumors (luminal) are the most frequent while HER-2 overexpressing (HR-, HER2+) and triple negative (TNBC, HR-/HER2-) are most deadly. HR+ subtypes are more common in non-Hispanic White women while HER-2 and TNBC present either higher prevalence or risk of mortality in minority groups. Hispanic/Latinos (H/L) is the largest and fastest growing minority in the U.S.A. However, H/L women have been underrepresented in BC studies. We reported that luminal B is the most common BC subtype in a group of H/L and that ERBB2, GRB7 and MIEN1 genes have increased expression in women with higher Indigenous American ancestry. Immune infiltration of tumors has become a hallmark for the prediction of outcome. However, contrary to TNBC, luminal tumors have been considered low immunogenic. Immune infiltration of luminal B tumors in H/L has not yet been investigated. Our goal was to predict immune infiltration of luminal B tumors based on RNA-seq data and according to the levels of body mass index (BMI) and ancestral fractions (West African, European, Indigenous American). Methods: We performed RNA-seq in 70 luminal B tumors of H/L from Puerto Rico. Ancestry was estimated by genotyping on the Affymetrix U.K. Biobank array and global ancestry proportions determined with Admixture Software v1.3, using 1000 Genomes reference population for anchoring. BMI, age at diagnosis and tumor size were extracted from the Electronic Medical Records. We used Partek Flow for the RNA-seq analysis based on ancestry fraction and BMI. Prediction of cell populations was done in xCell. Correlation analysis was done in GraphPad Prism v7.05. Results: We found an inverse correlation between Indigenous American and γδ-T cell infiltration (R2= -0.3; p=0.03) while BMI was associated with increased infiltration of basophils (R2=0.3; p=0.026). Among women with high BMI, those with low European ancestry had increased levels of basophils infiltration R2=0.66; p=0.17) and those with high European ancestry had reduced infiltration of naïve CD8+ T cells (R2= -0.53; p=0.008) and reduced plasmacytoid dendritic cells (pDC, R2=-0.51; p=0.01). Conclusions: We found that luminal B tumors have infiltration of immune cells that may contribute to differences in the response to treatment and outcome. To the best of our knowledge, the role of infiltrating basophils into breast cancer tissues has not been described. However, circulating basophils and predicted infiltration of basophils into ovarian and colorectal cancer tissues have been correlated with survival. Our findings have to be confirmed in a larger set of samples and by additional methods, including immunohistochemistry. Citation Format: Jone Garai, Julie Dutil, Douglas W. Cress, Victoria Seewaldt, Jamie K. Teer, Laura Fejerman, Lucio Miele, Jovanny Zabaleta. Cell infiltration prediction of breast cancer tissues identifies basophils as potentially important immune cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2729.

Abstract 559: Next-generation sequencing of circulating tumor DNA in advanced lung cancer patients treated with the immune checkpoint inhibitor

Abstract Introduction: Advanced lung cancer is a highly lethal disease worldwide. Recently, immune checkpoint inhibitors (ICIs) have made a significant breakthrough in the management of advanced lung cancer. Even though only a proportion of patients respond to ICI, the duration of such responders due to immunological memory is remarkable and is longer than other agents in refractory disease. Circulating tumor DNA (ctDNA) exhibits the potential in identifying genomic alterations and monitoring ICI treatment in advanced lung cancer patients. Identification of these genomic alterations may help to triage patients who will achieve better clinical benefit with minimal toxicity in therapies with ICIs. Materials and Methods: ctDNA mutational changes were monitored in the longitudinal blood samples (n = 39) of seven stage IV lung adenocarcinoma patients between baseline and after ICI therapy (complete monitoring until disease progression or the end of follow-up). The mutation spectrum in these patients was detected by a next-generation sequencing panel consisting of 50 cancer-related genes. Results: Among the seven patients, 15 variants were identified. All patients harbored at least one mutation, with an average of 2.1 mutations per patient. The most frequently mutated genes were TP53 (42.9%) and EGFR (42.9%). Baseline measurement showed eight mutations (EGFR E746V, L747P, A763_Y764insFQEA; TP53 R158C, A159S; ATM V2439G; ERBB2 Y772_A755dup; KRAS Q61H) in five patients. Seven mutations (TP53 I254fs, I255del, A276P, R280S; EGFR I740_K745dup; FGFR2 G261fs; STK11 D162fs) were observed in four patients following ICI therapy, suggesting that these alterations might be acquired during treatment. Most importantly, all patients harbored at least one clinically targetable gene mutation, including ATM, EGFR, ERBB2, STK11, and KRAS. Among these genes, the oncogenic mutations of EGFR and ERBB2 are FDA-recognized biomarkers predictive of response to specific targeted drugs in non-small cell lung cancer. Conclusion: This study demonstrates that ctDNA monitoring is a useful method for molecular genotyping of advanced lung cancer patients. Genomic profiling of liquid biopsy may help to identify gene signatures for biomarkers predictive of response to treatment. The gene mutations identified in the individual patient of this study may help access the clinical benefit of therapy with ICIs or other drugs. The identification of druggable genes may potentially help to develop more effective treatment strategies. Considering the small percentage of patients receiving ICI therapy, this is a small-scale pilot study and a larger study is warranted. Citation Format: William C. Cho, Wen Hsiao, Kien Thiam Tan, Jacky Y. Li, Shu-Jen Chen. Next-generation sequencing of circulating tumor DNA in advanced lung cancer patients treated with the immune checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 559.

Abstract 1472: Novel EGFR WT sparing, HER2 selective inhibitors for the treatment of HER2 exon 20 insertion driven tumors address a clear unmet medical need

Abstract Activating mutations in ERBB2 receptors are tractable oncogenes in 2-3% of NSCLC patients for whom no approved targeted therapies are available. In this indication, oncogenic mutations in HER2 predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. We initiated a drug discovery program aiming at discovering novel HER2 selective inhibitors sparing EGFR WT activity. Focus was set on the most frequent HER2 mutation (ERBB2 A775 insYVMA), which is least sensitive to current compounds tested in clinical trials. Here, we report the identification and pharmacological characterization of novel selective HER2 exon 20 mutation TKIs that differ from currently tested TKIs such as poziotinib, TAK-788 or BDTX-189. We could demonstrate that selective inhibition of oncogenic HER2 signaling abrogates oncogenic signaling in in vitro models. Cell survival and proliferation was reduced, which translated into tumor regressions in preclinical CRISPR engineered xenotransplantation models of HER2 exon 20 mutants. The in vivo efficacy was confirmed in patient-derived tumor models. Our results suggest that HER2 exon 20 insertions can be effectively treated by a potent and highly selective HER2 inhibitor that spares EGFR wild type. These findings warrant the upcoming clinical testing in HER2 mutant NSCLC patients in order to effectively treat this aggressive type of cancer. Citation Format: Ralph A. Neumüller, Birgit Wilding, Dirk Scharn, Dietrich Böse, Valeria Santoro, Daniel Gerlach, Peter Ettmayer, Thomas Gerstberger, Julian Fuchs, Matthias Treu, Stephan Zahn, Anke Baum, Paolo Chetta, Mark Pearson, Darryl B. McConnell, Norbert Kraut, Flavio Solca. Novel EGFR WT sparing, HER2 selective inhibitors for the treatment of HER2 exon 20 insertion driven tumors address a clear unmet medical need [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1472.

Abstract 2313: The characteristics of ERBB2 exon 20 insertion in a large cohort of Chinese NSCLC patients

Abstract Background: ERBB2 mutations, especially exon 20 insertion (ERBB2ex20ins) had been extensively reported in NSCLC as classical driver factors. Previous researches indicate that NSCLC pts with ERBB2ex20ins are drug-resistant to afatinib, gefitinib and erlotinib. Up to now, approvals of targeted therapy of ERBB2ex20ins are still in process. Therefore, the objective of this study is to discover the features of ERBB2ex20ins in Chinese NSCLC patients, which could lead to more effective and personalized diagnosis and treatment. Methods: 20656 Chinese NSCLC cases who had undergone molecular profiling at a Clinical Laboratory Improvement Amendments (CLIA) certified genomics laboratory in China between January 2017 to May 2020 were screened for our study. Some patients' clinical characteristics and treatment history were retrieved from the database for further evaluation. Results: 11492 males and 9134 females were included in our cohort. ERBB2 gene mutation rate was 5.94% (1226/20656). Among them, 290 patients (1.4%), including 114 males and 176 females, harbored with ERBB2ex20ins, which include with p. Tyr772_Ala775dup (84.56%), p.Gly778_Pro780dup (10.74%), p.Ala775_Gly776insLeu(1.01%), p.Ala775_Gly776insVal(1.01%) and others(2.68%). Additionally, ERBB2ex20ins occurred more frequently in female patients (61% vs 39%, p<0.005, fisher exact test), and had a relatively young age (p<0.005, wilcoxon test) compared with others. Moreover co-variations of 290 pts are occurring with EGFR(4, 1.38%), KRAS(3, 1.03%), TP53(156, 53.79%), ERBB2 amp(21, 7.24%), MET amp (2, 0.69%), ALK amp (1, 0.34%) and RET fusion (1, 0.34%). Besides, a relatively lower TMB (p<0.005, wilcoxon test) is shown on the patients with ERBB2ex20ins. We followed up 5 patients with ERBB2ex20ins mutations, who were all insensitive to traditional radiotherapy and chemotherapy and had poor prognosis with short PFS (1.5-12.4 months) and short OS (3.9-13.7 months). Conclusion: Our results showed that the ERBB2ex20ins mutation tended to occur in younger, female patients. Additionally, ERBB2ex20ins mutated samples exhibited lower levels of TMB compared to wildtype tumors, which may not benefit from anti-PD1 treatment. We also found patients with ERBB2ex20ins mutation have been associated with short PFS and short OS. The detail mechanism of resistance and appropriate therapeutic strategies remain to be further study. Citation Format: Danhua Wang, Hongling Yuan, Honglin Zhu. The characteristics of ERBB2 exon 20 insertion in a large cohort of Chinese NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2313.

Identification of Atrial Fibrillation-Associated Genes ERBB2 and MYPN Using Genome-Wide Association and Transcriptome Expression Profile Data on Left-Right Atrial Appendages.

More reliable methods are needed to uncover novel biomarkers associated with atrial fibrillation (AF). Our objective is to identify significant network modules and newly AF-associated genes by integrative genetic analysis approaches. The single nucleotide polymorphisms with nominal relevance significance from the AF-associated genome-wide association study (GWAS) data were converted into the GWAS discovery set using ProxyGeneLD, followed by merging with significant network modules constructed by weighted gene coexpression network analysis (WGCNA) from one expression profile data set, composed of left and right atrial appendages (LAA and RAA). In LAA, two distinct network modules were identified (blue: p = 0.0076; yellow: p = 0.023). Five AF-associated biomarkers were identified (ERBB2, HERC4, MYH7, MYPN, and PBXIP1), combined with the GWAS test set. In RAA, three distinct network modules were identified and only one AF-associated gene LOXL1 was determined. Using human LAA tissues by real-time quantitative polymerase chain reaction, the differentially expressive results of ERBB2, MYH7, and MYPN were observed (p < 0.05). This study first demonstrated the feasibility of fusing GWAS with expression profile data by ProxyGeneLD and WGCNA to explore AF-associated genes. In particular, two newly identified genes ERBB2 and MYPN via this approach contribute to further understanding the occurrence and development of AF, thereby offering preliminary data for subsequent studies.

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.

Quantification of Human Epidermal Growth Factor Receptor 2 by Immunopeptide Enrichment and Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded and Frozen Breast Cancer Tissues

Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies. We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors. Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.

The Presence of Genomic Instability in Cerebrospinal Fluid in Patients with Meningeal Metastasis

PurposeThis study aimed to explore the genomic instability in cerebrospinal fluid (CSF) in patients with meningeal metastasis (MM).Material and MethodsWe collected the blood and CSF samples of 15 MM patients and one brain parenchymal metastasis (BPM) patient. A panel of 543 cancer-related genes was conducted to analyze the status of genomic instability in CSF and plasma cell-free DNA (cfDNA) of all patients. Subsequently, nine patients underwent low-depth whole-genome sequencing (WGS) analysis to verify the existence of genomic instability, followed by genomic scoring by the application of aneuploidy scores. Diagnosis-specific graded prognostic assessment (DS-GPA) score was utilized to assess the clinical status of MM patients.ResultsThere was significant difference in gene mutation between CSF cfDNA and plasma cfDNA in MM patients. Among them, 12 MM patients developed genomic instability in their CSF cfDNA, while the remaining 3 had stable genetic profile. Besides, BPM patients showed genomic stability in his CSF and paraffin-embedded tissue sections. No genomic instability was noticed in plasma cfDNA of all patients. Sensitive mutations on EGFR, ERBB2, ALK and KRAS genes and increased gene copy numbers of MET and ERBB2 were detected in 10 MM patients with genomic instability, as well as the EGFR gene mutation in one MM case with genomic stability. Additionally, MM patients with genomic instability had lower overall survival and higher aneuploidy scores and tumor mutation burden compared with those with genomic stability. Moreover, MM patients with higher DS-GPA scores benefited from better survival.ConclusionGenomic instability existed in the CSF cfDNA rather than plasma cfDNA of MM patients, which might be the underlying cause of the differences in MM.

Melatonin, Its Beneficial Effects on Embryogenesis from Mitigating Oxidative Stress to Regulating Gene Expression.

Embryogenesis is a complex multi-stage process regulated by various signaling molecules including pineal and extrapineal melatonin (MT). Extrapineal MT is found in the placenta and ovaries, where it carries out local hormonal regulation. MT is necessary for normal development of oocytes, fertilization and subsequent development of human, animal and avian embryos. This review discusses the role of MT as a regulator of preimplantation development of the embryo and its implantation into endometrial tissue, followed by histo-, morpho- and organogenesis. MT possesses pronounced antioxidant properties and helps to protect the embryo from oxidative stress by regulating the expression of the NFE2L2, SOD1, and GPX1 genes. MT activates the expression of the ErbB1, ErbB4, GJA1, POU5F1, and Nanog genes which are necessary for embryo implantation and blastocyst growth. MT induces the expression of vascular endothelial growth factor (VEGF) and its type 1 receptor (VEGF-R1) in the ovaries, activating angiogenesis. Given the increased difficulties in successful fertilization and embryogenesis with age, it is of note that MT slows down ovarian aging by increasing the transcription of sirtuins. MT administration to patients suffering from infertility demonstrates an increase in the effectiveness of in vitro fertilization. Thus, MT may be viewed as a key factor in embryogenesis regulation, including having utility in the management of infertility.

Comparison of Selection Signatures between Korean Native and Commercial Chickens Using 600K SNP Array Data.

Korean native chickens (KNCs) comprise an indigenous chicken breed of South Korea that was restored through a government project in the 1990s. The KNC population has not been developed well and has mostly been used to maintain purebred populations in the government research institution. We investigated the genetic features of the KNC population in a selection signal study for the efficient improvement of this breed. We used 600K single nucleotide polymorphism data sampled from 191 KNCs (NG, 38; NL, 29; NR, 52; NW, 39; and NY, 33) and 54 commercial chickens (Hy-line Brown, 10; Lohmann Brown, 10; Arbor Acres, 10; Cobb, 12; and Ross, 12). Haplotype phasing was performed using EAGLE software as the initial step for the primary data analysis. Pre-processed data were analyzed to detect selection signals using the ‘rehh’ package in R software. A few common signatures of selection were identified in KNCs. Most quantitative trait locus regions identified as candidate regions were associated with traits related to reproductive organs, eggshell characteristics, immunity, and organ development. Block patterns with high linkage disequilibrium values were observed for LPP, IGF11, LMNB2, ERBB4, GABRB2, NTM, APOO, PLOA1, CNTN1, NTSR1, DEF3, CELF1, and MEF2D genes, among regions with confirmed selection signals. NL and NW lines contained a considerable number of selective sweep regions related to broilers and layers, respectively. We recommend focusing on improving the egg and meat traits of KNC NL and NW lines, respectively, while improving multiple traits for the other lines.

Evolution and functional divergence of the ERBB receptor family.

Aim: The ERBB gene family plays an important rolein cell proliferation and differentiation, and aberrant activations could result in tumorigenesis, which makes this gene family an attractive drug target in the area of precision oncology. Materials & methods: Functional divergence analysis and conservation analysis were performed using ClustalW, MEGA7 and DIVERGE3 software. Results: One hundred and forty fivefunctional divergence residues sites, 94 totally conserved sites and averagely 345 conserved sites of individual gene member were obtained. Some have been reported to play rolein drug binding, tumorigenesis and drug resistance. Conclusion: Functional divergence residues with high posterior probabilities and conserved residues may possess certain functions, and aberrant alterations may confer drug resistance or contribute to tumorigenesis.

Evaluation of in silico tools for variant classification in clinically actionable NSCLC variants.

e13545 Background: Genetic variants beyond FDA-approved drug targets are often identified in NSCLC patients. To address this challenge, in silico variant classification tools are available to determine whether specific variants contribute to disease pathogenicity or remain benign. Although the performance of in silico tools has been analyzed in previous studies, it has not been analyzed for actionable targets of FDA-approved therapies for NSCLC. The aim of this study is to compare the performance of commonly used in silico tools in classifying the pathogenicity of actionable variants in NSCLC. Methods: We evaluated the performance of several in silico tools: PolyPhen-2, Align-GVGD, and MutationTaster2. A curated set of targetable NSCLC missense variants (n = 179) was used. The dataset consisted of variants in the BRAF, EGFR, ERBB2, KRAS, MET, ALK, and ROS1 genes based on their indications as molecular targets in the NCCN Guidelines for NSCLC. Pathogenic variants (n = 80) were curated based on available literature and annotations according to the NCCN Guidelines, OncoKB, My Cancer Genome, and AACR Project GENIE. Benign variants (n = 99) were curated from the dbSNP database with the inclusion criteria of a benign or likely benign ClinVar assertion. The overall accuracy, sensitivity, specificity, and Matthews correlation coefficient (MCC) of each in silico tool were determined. The performance of each in silico tool in predicting pathogenicity for subsets of sensitizing (n = 18) and resistant (n = 57) variants was also evaluated. Results: PolyPhen-2 HumVar demonstrated the highest overall accuracy (0.80), specificity (0.69), and MCC (0.63) of the in silico tools analyzed. PolyPhen-2 HumDiv (0.75) and MutationTaster2 (0.69) had similar overall accuracies while Align-GVGD (0.50) had the lowest overall accuracy. Align-GVGD also had the lowest MCC (0.08), with the other in silico tools ranging from 0.50 to 0.63. All the in silico tools achieved high sensitivities, with MutationTaster2 performing the highest (1.00) and Align-GVGD performing the lowest (0.86). The specificities were remarkably low (0.20-0.69) for all the in silico tools, with the lowest specificity demonstrated by Align-GVGD. The overall accuracies when classifying the subsets of sensitizing and resistant variants were generally high, ranging from 0.84 to 1.00. Conclusions: These results suggest that the performance of the evaluated in silico tools to predict the pathogenicity of clinically actionable NSCLC missense variants is not fully reliable. The tools analyzed in this study could be acceptable to rule out pathogenicity in variants given their higher sensitivities, but are limited when it comes to identifying pathogenicity in variants due to low specificities.