ERBB2 kinase domain mutation in a gastric cancer metastasis

ERBB2 is a member of the epidermal growth factor receptor (EGFR) family. Recent studies revealed that the kinase domain of the ERBB2 gene was mutated in human cancers, including gastric cancer. Despite the importance of cancer metastasis in the pathogenesis of cancers, data on the ERBB2 kinase domain mutation in cancer metastasis are lacking. In this study, to explore the possibility that ERBB2 mutation is involved in the metastasis mechanism, we analyzed the kinase domain of ERBB2 for the detection of somatic mutations in 58 gastric adenocarcinomas with lymph node metastasis. We found one ERBB2 mutation, which was detected in the lymph node metastasis, but not in the primary tumor of the same patient. The ERBB2 mutation was a missense mutation which substituted an amino acid in exon 21 (V832I). We simultaneously analyzed the somatic mutations of EGFR, K-RAS, PIK3CA and BRAF genes in the sample with the ERBB2 mutation, and found that this metastatic carcinoma did not harbor any of the mutations. Our data suggest that ERBB2 kinase domain mutation occasionally occurs in metastatic gastric carcinoma and might play a role in the metastatic process of some gastric carcinomas.     

Gene amplification, mutation, and protein expression of EGFR and mutations of ERBB2 in serous ovarian carcinoma

EGFR and erbB-2 are targets for specific cancer therapy. The purpose of this study was to examine the frequency and clinicopathological correlations of gene amplification, protein expression, and mutations of EGFR and ERBB2 in serous carcinoma, the most common and aggressive type of ovarian cancer. Tissue microarray constructed of 398 carcinomas was examined by chromogenic in situ hybridization (CISH) and by immunohistochemistry. Cases with amplification of EGFR by CISH were further analyzed by fluorescence in situ hybridization. One hundred ninety-eight samples were analyzed for mutations in exons 18, 19, or 21 of EGFR and in exon 20 of ERBB2 using denaturating high-performance liquid chromatography and direct sequencing. Amplification of EGFR was present in 12% (41/333), low-level gain in 43% (144/333), and protein overexpression in 17% (66/379) of the tumors. Both increased copy number and overexpression of EGFR were associated with high tumor grade, greater patient age, large residual tumor size, high proliferation index, aberrant p53, and poor patient outcome. Furthermore, increased copy number of EGFR was associated with increased copy number of ERBB2. No mutations were identified in EGFR, whereas one tumor had an insertion mutation in exon 20 of ERBB2. Both amplification and protein overexpression of EGFR occur in serous ovarian carcinoma, but EGFR copy number has a stronger prognostic value. This makes EGFR amplification a potentially useful criterion for selecting patients in clinical trials testing the effect of EGFR inhibitors in serous ovarian carcinoma.     

ERBB2 mutation frequency in lobular breast cancer with pleomorphic histology or high‐risk characteristics by molecular expression profiling

HER2‐positive breast cancer is defined by amplification or overexpression of the HER2/ERBB2 oncogene and accounts for about 15% of breast cancer cases. Somatic mutation of ERBB2 is an alternative mechanism, by which activation of HER2 signaling can occur. ERBB2 mutation has been associated with invasive lobular breast cancer (ILBC). This study investigates the frequency and phenotype of ILBC harboring mutated ERBB2. The ERBB2 mutation status was determined by next generation sequencing and/or pyrosequencing in n = 106 ILBCs, including n = 86 primary or locally recurrent tumors and n = 20 metastases from visceral organs, soft tissue, or skin. Immunohistochemical characteristics were determined using tissue microarrays. This series was enriched for ILBCs with pleomorphic histology and/or high‐risk expression profiles (Oncotype DX, recurrence score RS > 25). Nearly all specimens were E‐cadherin‐negative (99%), estrogen receptor (ER)‐positive (92%), and lacked ERBB2 overexpression (96%). ERBB2 mutations (p.V777L, p.L755S, p.S310F) were identified in 5/106 (5%) cases. ERBB2‐mutated cases included 2/86 (2%) primary tumors and 3/20 (15%) metastases (P = 0.045). ERBB2‐mutated cases were associated with loss of ER (2/7, 29%, P = 0.035) and histological grade 3 (4/34, 12%, P = 0.023), but not with solid growth (3/31, 10%, P = 0.148) or pleomorphic histology (2/27, 7%, P = 0.599). No ERBB2 mutation was detected in ILBCs with RS > 25 (0/22, 0%). In 10 patients with multiple matched specimens (n = 25), the ERBB2 mutational status was always concordant. In summary, a small subset of ILBCs harbors potentially actionable ERBB2 mutations. In ERBB2‐mutated ILBCs, no association with pleomorphic histology was found.     

Direct resequencing of the complete ERBB2 coding sequence reveals an absence of activating mutations in ERBB2 amplified breast cancer

Gene amplification is among the most common genetic abnormalities that cause cancer. One of the most clinically important gene amplifications in human cancer causes extensive reduplication of ERBB2. A variety of cancers also occasionally harbor somatic mutations in ERBB2. Gene amplification and activating mutations both have predictive value for clinical response to targeted inhibitors. Since the number of gene copies in an amplicon may exceed 100, and since amplicons may encompass multiple genes, high-resolution analysis of gene amplifications poses considerable technical challenges. We have overcome this obstacle by using emulsion-based resequencing to determine the sequence of many independently-amplified individual DNA molecules in parallel. We used this high throughput sequencing technology to analyze ERBB2 mutational status in five ERBB2 amplified cell lines (four breast, one ovarian) and two breast tumors. Genomic DNA was isolated and the 28 exons of ERBB2 were independently amplified. Amplicons were then pooled at equimolar ratios, subjected to emulsion PCR (emPCR) and finally to picotiter plate pyrosequencing. High-quality sequence data were obtained for all amplicons analyzed and no activating mutations within ERBB2 were identified. Although we did not find activating mutations within the multiple copies of ERBB2 in these samples, the results establish the utility of this technology as a feasible and cost-effective approach for high resolution analysis of amplified genes.     

Cooverexpression of ERBB1 and ERBB4 receptors predicts poor clinical outcome in pN+ oral squamous cell carcinoma with extranodal spread

Overexpression of members of the ErbB receptor family is common in oral squamous cell carcinomas (OSCC); however, their prognostic value for aggressive OSCC has been debated. Extranodal spread to cervical lymph nodes is the most significant prognostic indicator in OSCC. In the present study, we investigated the clinical significance of single versus paired overexpression of members of the ErbB receptor family in 82 OSCC patients with lymph nodes metastasis, with or without capsular rupture (CR) followed by at least 10 years. Immunohistochemistry analysis revealed a common overexpression of ErbB1 (P = 0.021), ErbB2 (P = 0.001), ErbB4 (P = 0.048), as well as MMP-2 (P = 0.043) in OSCC cases with CR+. Increased expression of ErbB1 was associated with MMP-2 in tumors with advanced clinical stages, including poorly differentiated (grade III) tumors (P < 0.050). Vascular embolization was associated with MMP-2 (P = 0.021) and MMP-13 (P = 0.010) overexpression. Survival analysis revealed a lower survival probability in tumors overexpressing ErbB1 (P = 0.038), ErbB4 (P = 0.043), and MMP-12 (P = 0.050). As well a strong association was observed in cases with high risk of recurrence and strong immunostaining for ErbB1 (P = 0.017), ErbB4 (P = 0.008), MMP-1 (P = 0.003), MMP-2 (P = 0.016), MMP-10 (P = 0.041), and MMP-13 (P = 0.005). Stratified multivariate survival analysis revealed a strong prognostic interdependence of ErbB1 and ErbB4 cooverexpression in predicting the worst overall and disease-free survivals (P = 0.0013 and P = 0.0004, respectively). Taken together, these results support a cooperation of ErbB1, ErbB4, and members of the MMP family in predicting OSCC invasion and poor clinical outcomes.     

MiR-148a inhibits angiogenesis by targeting ERBB3

MicroRNAs (miRNAs) play an important role in carcinogenesis in various solid cancers including breast can-cer. Down-regulation of microRNA-148a (miR-148a) has been reported in certain cancer types. However, the biological role of miR-148a and its related targets in breast cancer are unknown yet. In this study, we showed that the level of miR-148a was lower in MCF7 cells than that in MCF10A cells. V-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3) is a direct target of miR-148a in human breast cancer cells through direct binding of miR-148a to ERBB3 3’-UTR region. Overexpression of miR-148a in MCF7 cells inhibited ERBB3 expression, blocked the downstream pathway activation including activation of AKT, ERK1/2, and p70S6K1, and decreased HIF-1α expression. Furthermore, forced expression of miR-148a attenuated tumor angiogenesis in vivo. Our re-sults identify ERBB3 as a direct target of miR-148a, and provide direct evidence that miR-148a inhibits tumor an-giogenesis through ERBB3 and its downstream signaling molecules. This information would be helpful for target-ing the miR-148a/ERBB3 pathway for breast cancer prevention and treatment in the future.     

ERBB2, TBX2, RPS6KB1, and MYC alterations in breast tissues of BRCA1 and BRCA2 mutation carriers

Breast cancer risk is greatly increased in women who carry mutations in the BRCA1 or BRCA2 genes. Because breast cancer initiation is different between BRCA1/2 mutation carriers and women who do not carry mutations, it is possible that the mechanism of breast cancer progression is also different. Histopathologic and genetic studies have supported this hypothesis. To test this hypothesis further, we utilized a large cohort of women who underwent therapeutic mastectomy (TM) and contralateral prophylactic mastectomy (PM). From this cohort, we developed case groups of women with a family history of breast cancer with BRCA1/2 deleterious mutations, with unclassified variant alterations, and with no detected mutation and matched these cases with sporadic controls from the same TM and PM cohort. Fluorescence in situ hybridization was performed on paraffin sections by use of dual-color probes for ERBB2/CEP17, MYC/CEP8, TBX2/CEP17, and RPS6KB1/CEP17. All malignant and benign lesions, including putative precursor lesions, were studied. The invasive cancers from deleterious mutation carriers had a higher prevalence of duplication of MYC (P = 0.006) and TBX2 (P = 0.0008) compared to controls and a lower prevalence of ERBB2 amplification (P = 0.011). Coduplication of MYC and TBX2 was common in the in situ and invasive lesions from the deleterious mutation carriers. The odds ratio of having a BRCA1/2 mutation is 31.4 (95% CI = 1.7-569) when MYC and TBX2 are coduplicated but ERBB2 is normal. Unclassified variant carriers/no mutation detected and sporadic controls had a similar prevalence of alterations, suggesting that hereditary patients with no deleterious mutations follow a progression pathway similar to that of sporadic cases. With the exception of one atypical ductal hyperplasia lesion, no putative precursor lesion showed any detectable alteration of the probes tested. There was no significant intratumoral heterogeneity of genetic alterations. Our data confirm that a specific pattern of genomic instability characterizes BRCA1/2-related cancers and that this pattern has implications for the biology of these cancers. Moreover, our current and previous results emphasize the interaction between phenotype and genotype in BRCA1/2-related breast cancers and that a combination of morphologic features and alterations of ERBB2, MYC, and TBX2 may better define mechanisms of tumor progression, as well as determine which patients are more likely to carry BRCA1/2 mutations.     

ERBB2转基因小鼠乳腺癌模型在ERBB2阳性乳腺癌研究中的意义

ERBB2(erythroblastic leukemia viral oncogene homolog 2,成红细胞白血病病毒同源癌基因2)的表达与乳腺癌的发生与预后相关,并与病人的预后呈负相关。ERBB2转基因小鼠乳腺癌模型在ERBB2阳性乳腺癌的研究领域中意义重大:①明确了ERBB2与乳腺癌的发生有关;②用于ERBB2信号通路研究;③用于ERBB2诱发乳腺癌的机制研究;④用于ERBB2阳性乳腺癌治疗的研究。因此,本文对这些模型建立的意义做一概述。     

Heterogeneous amplification of ERBB2 in primary lesions is responsible for the discordant ERBB2 status of primary and metastatic lesions in gastric carcinoma

Kim M A, Lee H‐J, Yang H‐K, Bang Y‐J & Kim W H
(2011) Histopathology 59 , 822–831 Heterogeneous amplification of ERBB2 in primary lesions is responsible for the discordant ERBB2 status of primary and metastatic lesions in gastric carcinoma Aims: To determine the extent of HER2 homogeneity/heterogeneity in primary versus metastatic gastric carcinoma (GC). Materials and results: The human epidermal growth factor receptor 2 (HER2) status in primary and metastatic lesions was evaluated by immunohistochemistry (IHC) and fluorescence in‐situ hybridization (FISH). Four separate cohorts consisting of primary GC alone or primary GC paired with metastatic lesions were examined. In the FISH analysis of 325 primary GCs, eight cases (2.5%) showed amplification with a heterogeneous pattern, whereas 27 cases (8.3%) showed amplification with a homogeneous pattern, and in this cohort the discordant:concordant FISH ratio based on examination of three different areas in each primary lesion was 0.30:1. FISH testing using 250 paired primary and metastatic lesions revealed seven cases (2.8%) with discordant amplification. In metastatic disease positive conversion occurred in six cases (2.4%), whereas negative conversion happened in one case (0.4%). The discordant:concordant ratio of primary versus secondary lesions was 0.23:1. When the seven discordant cases were re‐evaluated using whole sections of primary GCs, six showed a heterogeneous pattern of amplification. Conclusions: These findings suggest that the discordant HER2 amplification observed in metastatic lesions is explained substantially by heterogeneity within primary tumours.     

Comparative multi-methodological measurement of ERBB2 status in breast cancer

The ERBB2 transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target in breast cancer. Accurate determination of ERBB2 status is a prerequisite for the establishment of prognostic significance and for the selection of ERBB2-overexpressing breast tumours for specific treatment. Unfortunately, there is no complete agreement on how this determination should be performed. This study has compared four methods of assessment of ERBB2 status. Two global, extraction-based methods using real-time quantitative PCR on DNA (Q-PCR) or RNA (RQ-PCR) and two non-global, tissue-based methods, ie fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were used. The 94 breast cancers tested were enriched in cases scoring 2+ using the IHC scoring system currently in use and for which the actual ERBB2 status remains ill defined. To determine the best parameters and reagents for assessment, two protocols for FISH and five anti-ERBB2 antibodies were used, and both FISH and IHC were carried out on the same tissue microarrays (TMAs). It is shown that the combination of the two tissue-based methods gives the best results. The use of either PCR-based method did not improve the results significantly. A new combined IHC score based on the association of two selected anti-ERBB2 antibodies (HercepTest trade mark and TAB250) and a dual scale for improved assessment of ERBB2 protein expression, particularly in 2+ cases, are proposed.     

Susceptibility loci affecting ERBB2/neu-induced mammary tumorigenesis in mice

Twenty percent of breast cancers exhibit amplification or overexpression of ERBB2/neu and a poor prognosis. As the susceptibility genes controlling ERBB2 tumorgenesis are unknown, in a genetic mapping project we crossed transgenic mice expressing the neu oncogene under control of MMTV promoter with recombinant congenic (RC) strains, which provided a high mapping power. RC strains differed considerably in tumor latency (P = 0.0002), suggesting a strong genetic control of tumor development. Linkage analysis in neu-transgene carrying F2 hybrids between the most susceptible and most resistant RC strain revealed three mammary tumor susceptibility (Mts) loci with main effects, Mts1 (chr. 4), Mts2 (chr. 10), Mts3 (chr. 19), and two interacting loci Mts4 (chr.6) and Mts5 (chr. 8), significantly affecting mammary tumor latency. Suggestive significance levels indicated control of tumor numbers by Mts1 alone and in interaction with Mts5, and by two additional interacting loci on chromosomes 1 and 8. These loci combined explain to a large extent the tumor latency and number in individual F2 mouse. We also identified a suggestive locus on chromosome 17 controls metastasis to the lung. The loci Mts1, Mts1b, and Mts3 are located in the Naad4-4,5 and Naad19-2 LOH-regions of neu-induced mammary tumors, corresponding to the frequent human breast cancer LOH-regions 1p34/1p36, and 10q25, respectively. These results expand the knowledge of ERBB2 tumorigenesis and point to a combined control of specific tumor phenotypes by germ-line polymorphisms and somatic alterations.     

Mutational and expressional analysis of ERBB3 gene in common solid cancers

ERBB3 is a member of EGFR family receptor tyrosine kinases, genetic alterations of which are common and therapeutically targeted in human cancers. Recently, somatic mutations of ERBB3 gene, including recurrent mutation in exon 3 altering Val104, were reported in gastric cancers (GC) and colorectal cancers (CRC), strongly suggesting its role in the development of GC and CRC. To examine whether the recurrent ERBB3 mutations of exon 3 occur in GC and CRC, and other malignancies as well, we analyzed the ERBB3 in 1677 cancer tissues by a single‐strand conformation polymorphism (SSCP) assay. We identified ERBB3 mutations altering the Val104 mutations in GC (0.5%) and CRC (2.2%). However, we did not find the ERBB3 mutations in the other cancers besides GC and CRC. We observed that an increased intensity of phosphorylated ERBB3 (pERBB3) in GC and CRC. Of note, all of the cancers with ERBB3 mutations displayed an increased intensity of pERBB3 immunostaining. Our data indicate that the recurrent ERBB3 mutations altering Val104 occur predominantly in GC and CRC. Also, the data suggest that ERBB3 is altered in GC and CRC by various ways, including somatic mutations and increased expression that might play roles in tumorigenesis.     

ERBB3 deficiency causes a multisystemic syndrome in human patient and zebrafish

The Erb-B2 receptor tyrosine kinase 3 (ERBB3) gene was first identified as a cause of lethal congenital contracture syndrome (OMIM 607598), while a recent study reported six additional patients carrying ERBB3 variants which exhibited distinct clinical features with evident intestinal dysmotility (OMIM 243180 ). The potential connection between these phenotypes remains unknown, and the ERBB3-related phenotype spectrum needs to be better characterized. Here, we described a patient presenting with a multisystemic syndrome including skip segment Hirschsprung disease, bilateral clubfoot deformity, and cardiac defect. Trio-whole exome sequencing revealed a novel compound heterozygous variant (c.1914-7C>G; c.2942_2945del) in the patient's ERBB3 gene. RT-PCR and in vitro minigene analysis demonstrated that variant c.1914-7C>G caused aberrant mRNA splicing. Both variants resulted in premature termination codon and complete loss of ERBB3 function. erbb3b knockdown in zebrafish simultaneously caused a reduction in enteric neurons in the distal intestine, craniofacial cartilage defects, and micrognathia, which phenotypically mimics ERBB3-related intestinal dysmotility and some features of lethal congenital contracture syndrome in human patients. These findings provide further patient and animal evidence supporting that ERBB3 deficiency causes a complex syndrome involving multiple systems with phenotypic variability among distinct individuals.     

No association between the ERBB3 gene and schizophrenia in a Japanese population

There is cumulative evidence that neuregulin 1 (NRG1) is a susceptibility gene for schizophrenia. Postmortem studies on brains from schizophrenia patients have revealed changes in the mRNA expression levels of v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), one of the NRG1 receptor genes. These observations suggest that NRG1-ERBB signaling is involved in the pathogenesis of schizophrenia. To assess whether the ERBB3 gene could be implicated in vulnerability to schizophrenia, we conducted a case-control (399 patients and 438 controls) association study in Japanese subjects. There were no significant association between the polymorphisms or haplotypes of ERBB3 and schizophrenia. The present study shows that ERBB3 does not play a major role in conferring susceptibility to schizophrenia in the Japanese population.