Functional 3'-UTR Variants Identify Regulatory Mechanisms Impacting Alcohol Use Disorder and Related Traits.

Although genome-wide association studies (GWAS) have identified loci associated with alcohol consumption and alcohol use disorder (AUD), they do not identify which variants are functional. To approach this, we evaluated the impact of variants in 3' untranslated regions (3'-UTRs) of genes in loci associated with substance use and neurological disorders using a massively parallel reporter assay (MPRA) in neuroblastoma and microglia cells. Functionally impactful variants explained a higher proportion of heritability of alcohol traits than non-functional variants. We identified genes whose 3'UTR activities are associated with AUD and alcohol consumption by combining variant effects from MPRA with GWAS results. We examined their effects by evaluating gene expression after CRISPR inhibition of neuronal cells and stratifying brain tissue samples by MPRA-derived 3'-UTR activity. A pathway analysis of differentially expressed genes identified inflammation response pathways. These analyses suggest that variation in response to inflammation contributes to the propensity to increase alcohol consumption.

Genetic Regulation of Human isomiR Biogenesis.

MicroRNAs play a critical role in regulating gene expression post-transcriptionally. Variations in mature microRNA sequences, known as isomiRs, arise from imprecise cleavage and nucleotide substitution or addition. These isomiRs can target different mRNAs or compete with their canonical counterparts, thereby expanding the scope of miRNA post-transcriptional regulation. Our study investigated the relationship between cis-acting single-nucleotide polymorphisms (SNPs) in precursor miRNA regions and isomiR composition, represented by the ratio of a specific 5'-isomiR subtype to all isomiRs identified for a particular mature miRNA. Significant associations between 95 SNP-isomiR pairs were identified. Of note, rs6505162 was significantly associated with both the 5'-extension of hsa-miR-423-3p and the 5'-trimming of hsa-miR-423-5p. Comparison of breast cancer and normal samples revealed that the expression of both isomiRs was significantly higher in tumors than in normal tissues. This study sheds light on the genetic regulation of isomiR maturation and advances our understanding of post-transcriptional regulation by microRNAs.

Intron-Retention Neoantigen Load Predicts Favorable Prognosis in Pancreatic Cancer.

PURPOSE: High tumor mutation burden (TMB) in many cancer types is associated with the production of tumor-specific neoantigens, a favorable outcome and response to immune checkpoint blockade (ICB) therapy. Besides mutation-derived neoantigens, aberrant intron retention also produces tumor neopeptides that could trigger an immune response. The relationship between intron-retention-derived tumor neoantigens (IR-neoAg) and clinical outcomes in pancreatic cancer remains uncertain. Here, we quantify IR-neoAg in pancreatic cancer and evaluate whether IR-neoAg load might serve as a biomarker for selecting patients who may benefit from ICB therapy. METHODS: We developed a computational approach to estimate patient-specific IR-neoAg load from transcriptome data available in The Cancer Genome Atlas pancreatic cancer cohort. Associations between IR-neoAg load and patient overall survival were evaluated using Kaplan-Meier estimates and Cox regression. Differential expression of immune checkpoint and HLA-I genes was evaluated in tumors with high IR-neoAg load. RESULTS: High IR-neoAg load predicted better overall survival in pancreatic cancer, although no association was found for TMB. IR-neoAg load remained a significant prognostic factor after adjusting for patient age, sex, tumor stage and grade, and TMB. Moreover, pancreatic tumors with both high IR-neoAg load and high HLA-I gene expression had similar gene expression profiles as other tumor types that showed response to anti-programmed cell death protein 1 therapy. CONCLUSION: IR-neoAg load is associated with favorable survival in pancreatic cancer. These findings provide strong evidence for considering IR-neoAgs when selecting patients who might benefit from ICB therapy.

Intron retention-induced neoantigen load correlates with unfavorable prognosis in multiple myeloma.

Neoantigen peptides arising from genetic alterations may serve as targets for personalized cancer vaccines and as positive predictors of response to immune checkpoint therapy. Mutations in genes regulating RNA splicing are common in hematological malignancies leading to dysregulated splicing and intron retention (IR). In this study, we investigated IR as a potential source of tumor neoantigens in multiple myeloma (MM) patients and the relationship of IR-induced neoantigens (IR-neoAg) with clinical outcomes. MM-specific IR events were identified in RNA-sequencing data from the Multiple Myeloma Research Foundation CoMMpass study after removing IR events that also occurred in normal plasma cells. We quantified the IR-neoAg load by assessing IR-induced novel peptides that were predicted to bind to major histocompatibility complex (MHC) molecules. We found that high IR-neoAg load was associated with poor overall survival in both newly diagnosed and relapsed MM patients. Further analyses revealed that poor outcome in MM patients with high IR-neoAg load was associated with high expression levels of T-cell co-inhibitory molecules and elevated interferon signaling activity. We also found that MM cells exhibiting high IR levels had lower MHC-II protein abundance and treatment of MM cells with a spliceosome inhibitor resulted in increased MHC-I protein abundance. Our findings suggest that IR-neoAg may represent a novel biomarker of MM patient clinical outcome and further that targeting RNA splicing may serve as a potential therapeutic strategy to prevent MM immune escape and promote response to checkpoint blockade.

Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients.

Single-cell RNA sequencing reveals gene expression differences between individual cells and also identifies different cell populations that are present in the bulk starting material. To obtain an accurate assessment of patient samples, single-cell suspensions need to be generated as soon as possible once the tissue or sample has been collected. However, this requirement poses logistical challenges for experimental designs involving multiple samples from the same subject since these samples would ideally be processed at the same time to minimize technical variation in data analysis. Although cryopreservation has been shown to largely preserve the transcriptome, it is unclear whether the freeze-thaw process might alter gene expression profiles in a cell-type specific manner or whether changes in cell-type proportions might also occur. To address these questions in the context of multiple myeloma clinical studies, we performed single-cell RNA sequencing (scRNA-seq) to compare fresh and frozen cells isolated from bone marrow aspirates of six multiple myeloma patients, analyzing both myeloma cells (CD138+) and cells constituting the microenvironment (CD138-). We found that cryopreservation using 90% fetal calf serum and 10% dimethyl sulfoxide resulted in highly consistent gene expression profiles when comparing fresh and frozen samples from the same patient for both CD138+ myeloma cells (R ≥ 0.96) and for CD138- cells (R ≥ 0.9). We also demonstrate that CD138- cell-type proportions showed minimal alterations, which were mainly related to small differences in immune cell subtype sensitivity to the freeze-thaw procedures. Therefore, when processing fresh multiple myeloma samples is not feasible, cryopreservation is a useful option in single-cell profiling studies.

Differential Splicing of Skipped Exons Predicts Drug Response in Cancer Cell Lines.

Alternative splicing of pre-mRNA transcripts is an important regulatory mechanism that increases the diversity of gene products in eukaryotes. Various studies have linked specific transcript isoforms to altered drug response in cancer; however, few algorithms have incorporated splicing information into drug response prediction. In this study, we evaluated whether basal-level splicing information could be used to predict drug sensitivity by constructing doxorubicin-sensitivity classification models with splicing and expression data. We detailed splicing differences between sensitive and resistant cell lines by implementing quasi-binomial generalized linear modeling (QBGLM) and found altered inclusion of 277 skipped exons. We additionally conducted RNA-binding protein (RBP) binding motif enrichment and differential expression analysis to characterize cis- and trans-acting elements that potentially influence doxorubicin response-mediating splicing alterations. Our results showed that a classification model built with skipped exon data exhibited strong predictive power. We discovered an association between differentially spliced events and epithelial-mesenchymal transition (EMT) and observed motif enrichment, as well as differential expression of RBFOX and ELAVL RBP family members. Our work demonstrates the potential of incorporating splicing data into drug response algorithms and the utility of a QBGLM approach for fast, scalable identification of relevant splicing differences between large groups of samples.

Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders.

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.

Integrative analysis of histopathological images and chromatin accessibility data for estrogen receptor-positive breast cancer.

BACKGROUND: Existing studies have demonstrated that the integrative analysis of histopathological images and genomic data can be used to better understand the onset and progression of many diseases, as well as identify new diagnostic and prognostic biomarkers. However, since the development of pathological phenotypes are influenced by a variety of complex biological processes, complete understanding of the underlying gene regulatory mechanisms for the cell and tissue morphology is still a challenge. In this study, we explored the relationship between the chromatin accessibility changes and the epithelial tissue proportion in histopathological images of estrogen receptor (ER) positive breast cancer. METHODS: An established whole slide image processing pipeline based on deep learning was used to perform global segmentation of epithelial and stromal tissues. We then used canonical correlation analysis to detect the epithelial tissue proportion-associated regulatory regions. By integrating ATAC-seq data with matched RNA-seq data, we found the potential target genes that associated with these regulatory regions. Then we used these genes to perform the following pathway and survival analysis. RESULTS: Using canonical correlation analysis, we detected 436 potential regulatory regions that exhibited significant correlation between quantitative chromatin accessibility changes and the epithelial tissue proportion in tumors from 54 patients (FDR < 0.05). We then found that these 436 regulatory regions were associated with 74 potential target genes. After functional enrichment analysis, we observed that these potential target genes were enriched in cancer-associated pathways. We further demonstrated that using the gene expression signals and the epithelial tissue proportion extracted from this integration framework could stratify patient prognoses more accurately, outperforming predictions based on only omics or image features. CONCLUSION: This integrative analysis is a useful strategy for identifying potential regulatory regions in the human genome that are associated with tumor tissue quantification. This study will enable efficient prioritization of genomic regulatory regions identified by ATAC-seq data for further studies to validate their causal regulatory function. Ultimately, identifying epithelial tissue proportion-associated regulatory regions will further our understanding of the underlying molecular mechanisms of disease and inform the development of potential therapeutic targets.

regSNPs-ASB: A Computational Framework for Identifying Allele-Specific Transcription Factor Binding From ATAC-seq Data.

Expression quantitative trait loci (eQTL) analysis is useful for identifying genetic variants correlated with gene expression, however, it cannot distinguish between causal and nearby non-functional variants. Because the majority of disease-associated SNPs are located in regulatory regions, they can impact allele-specific binding (ASB) of transcription factors and result in differential expression of the target gene alleles. In this study, our aim was to identify functional single-nucleotide polymorphisms (SNPs) that alter transcriptional regulation and thus, potentially impact cellular function. Here, we present regSNPs-ASB, a generalized linear model-based approach to identify regulatory SNPs that are located in transcription factor binding sites. The input for this model includes ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing) raw read counts from heterozygous loci, where differential transposase-cleavage patterns between two alleles indicate preferential transcription factor binding to one of the alleles. Using regSNPs-ASB, we identified 53 regulatory SNPs in human MCF-7 breast cancer cells and 125 regulatory SNPs in human mesenchymal stem cells (MSC). By integrating the regSNPs-ASB output with RNA-seq experimental data and publicly available chromatin interaction data from MCF-7 cells, we found that these 53 regulatory SNPs were associated with 74 potential target genes and that 32 (43%) of these genes showed significant allele-specific expression. By comparing all of the MCF-7 and MSC regulatory SNPs to the eQTLs in the Genome-Tissue Expression (GTEx) Project database, we found that 30% (16/53) of the regulatory SNPs in MCF-7 and 43% (52/122) of the regulatory SNPs in MSC were also in eQTL regions. The enrichment of regulatory SNPs in eQTLs indicated that many of them are likely responsible for allelic differences in gene expression (chi-square test, p-value < 0.01). In summary, we conclude that regSNPs-ASB is a useful tool for identifying causal variants from ATAC-seq data. This new computational tool will enable efficient prioritization of genetic variants identified as eQTL for further studies to validate their causal regulatory function. Ultimately, identifying causal genetic variants will further our understanding of the underlying molecular mechanisms of disease and the eventual development of potential therapeutic targets.

Highly robust model of transcription regulator activity predicts breast cancer overall survival.

BACKGROUND: While several multigene signatures are available for predicting breast cancer prognosis, particularly in early stage disease, effective molecular indicators are needed, especially for triple-negative carcinomas, to improve treatments and predict diagnostic outcomes. The objective of this study was to identify transcriptional regulatory networks to better understand mechanisms giving rise to breast cancer development and to incorporate this information into a model for predicting clinical outcomes. METHODS: Gene expression profiles from 1097 breast cancer patients were retrieved from The Cancer Genome Atlas (TCGA). Breast cancer-specific transcription regulatory information was identified by considering the binding site information from ENCODE and the top co-expressed targets in TCGA using a nonlinear approach. We then used this information to predict breast cancer patient survival outcome. RESULT: We built a multiple regulator-based prediction model for breast cancer. This model was validated in more than 5000 breast cancer patients from the Gene Expression Omnibus (GEO) databases. We demonstrated our regulator model was significantly associated with clinical stage and that cell cycle and DNA replication related pathways were significantly enriched in high regulator risk patients. CONCLUSION: Our findings demonstrate that transcriptional regulator activities can predict patient survival. This finding provides additional biological insights into the mechanisms of breast cancer progression.

Lipopolysaccharides Improve Mesenchymal Stem Cell-Mediated Cardioprotection by MyD88 and stat3 Signaling in a Mouse Model of Cardiac Ischemia/Reperfusion Injury.

Bone marrow-derived mesenchymal stem cells (MSCs) improve cardiac function after ischemia/reperfusion injury, in part, due to the release of cytoprotective paracrine factors. Toll-like receptor 4 (TLR4) is expressed in MSCs and regulates the expression of cytoprotective factors, cytokines, and chemokines. Lipopolysaccharide (LPS) stimulation of TLR4 activates two distinct signaling pathways that are either MyD88 dependent or MyD88 independent/TIR-domain-containing adapter-inducing interferon-ß (TRIF) dependent. While it was reported previously that LPS treatment improved MSC-mediated cardioprotection, the mechanism underlying such improved effect remains unknown. To study the role of MyD88 signaling in MSC cardioprotective activity, wild type (WT) and MyD88-/- MSCs were treated with LPS (200 ng/mL) for 24 h. WT and MyD88-/- MSCs with or without LPS pretreatment were infused into the coronary circulation of isolated mouse hearts (Langendorff model) and then subjected to ischemia (25 min) and reperfusion (50 min). Saline served as a negative control. Both untreated and LPS-pretreated WT MSCs significantly improved postischemic recovery of myocardial function of isolated mouse hearts, as evidenced by improved left ventricular developed pressure and ventricular contractility assessment (ie, the rate of left ventricle pressure change over time, ± dp/dt). LPS-pretreated WT MSCs conferred better cardiac function recovery than untreated MSCs; however, such effect of LPS was abolished when using MyD88-/- MSCs. In addition, LPS stimulated stat3 activity in WT MSCs, but not MyD88-/- MSCs. stat3 small interfering RNA abolished the effect of LPS in improving the cardioprotection of WT MSCs. In conclusion, this study demonstrates that LPS improves MSC-mediated cardioprotection by MyD88-dependent activation of stat3.

Estrogen-Dependent Upregulation of Adcyap1r1 Expression in Nucleus Accumbens Is Associated With Genetic Predisposition of Sex-Specific QTL for Alcohol Consumption on Rat Chromosome 4.

Humans show sex differences related to alcohol use disorders (AUD). Animal model research has the potential to provide important insight into how sex differences affect alcohol consumption, particularly because female animals frequently drink more than males. In previous work, inbred strains of the selectively bred alcohol-preferring (P) and non-preferring (NP) rat lines revealed a highly significant quantitative trait locus (QTL) on rat chromosome 4, with a logarithm of the odds score of 9.2 for alcohol consumption. Recently, interval-specific congenic strains (ISCS) were developed by backcrossing the congenic P.NP line to inbred P (iP) rats to further refine the chromosome 4 QTL region. Two ISCS sub-strains, ISCS-A and ISCS-B, were obtained with a narrowed QTL, where the smallest region of overlap consisted of 8.9 Mb in ISCS-B. Interestingly, we found that females from both ISCS lines consumed significantly less alcohol than female iP controls (p < 0.05), while no differences in alcohol consumption were observed between male ISCS and iP controls. RNA-sequencing was performed on the nucleus accumbens of alcohol-naïve female ISCS-B and iP rats, which revealed differentially expressed genes (DEG) with greater than 2-fold change and that were functionally relevant to behavior. These DEGs included down-regulation of Oxt, Asb4, Gabre, Gabrq, Chat, Slc5a7, Slc18a8, Slc10a4, and Ngfr, and up-regulation of Ttr, Msln, Mpzl2, Wnt6, Slc17a7, Aldh1a2, and Gstm2. Pathway analysis identified significant alterations in gene networks controlling nervous system development and function, as well as cell signaling, GABA and serotonin receptor signaling and G-protein coupled receptor signaling. In addition, ß-estradiol was identified as the most significant upstream regulator. The expression levels of estrogen-responsive genes that mapped to the QTL interval and have been previously associated with alcohol consumption were measured using RT-qPCR. We found that expression of the Adcyap1r1 gene, encoding the pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor, was upregulated in female ISCS-B compared to female iP controls, while no differences were exhibited in males. In addition, sequence variants in the Adcyap1r1 promoter region showed a differential response to estrogen stimulation in vitro. These findings demonstrate that rat chromosome 4 QTL contains genetic variants that respond to estrogen and are associated with female alcohol consumption.

Cytogenetic features of human trophoblast cell lines SWAN-71 and 3A-subE.

Immortalization of primary cells with telomerase is thought to maintain normal phenotypic properties and avoid chromosomal abnormalities and other cancer-associated changes that occur following simian virus 40 tumor antigen (SV40 Tag) induced immortalization. However, we report that the human telomerase reverse transcriptase (hTERT)-immortalized SWAN-71 trophoblast cell line has a near pentaploid 103∼119,XXXX[cp20] karyotype. Additionally, DNA typing analysis indicated that SWAN-71 cells have acquired microsatellite instability. In comparison, the post-crisis SV40-transformed trophoblast cell line 3A-subE was hypertriploid 69∼81,XX[cp20]. Both cell lines contained multiple specific clonal rearrangements. These findings emphasize the need to monitor for genetic instability in hTERT-immortalized cells.

Analytic variability in immunohistochemistry biomarker studies.

BACKGROUND: Despite the widespread use of immunohistochemistry (IHC), there are no standardization guidelines that control for antibody probe variability. Here we describe the effect of variable antibody reagents in the assessment of cancer-related biomarkers by IHC. METHODS: Estrogen receptor (ER), epidermal growth factor receptor (EGFR) 1, and human epidermal growth factor receptor 3 (HER3) were evaluated by quantitative immunofluorescence. Correlations between ER clones 1D5, SP1, F10, and ER60c, and EGFR monoclonal 31G7, 2-18C9, H11, and 15F8, and polyclonal 2232 antibodies were assessed in 642 breast cancer patients. HER3 was measured by RTJ1, RTJ2, SGP1, M7297, RB-9211, and C-17 antibodies in 42 lung cancer patients. Survival analysis was done with the use of multiple cutoff points to reveal any prognostic classification. RESULTS: All ER antibodies were tightly correlated (Pearson's r(2) = 0.94-0.96; P < 0.0001) and western blotting confirmed their specificity in MCF-7 and BT474 cells. All EGFR antibodies but 2232 yielded specific results in western blotting; however, only 31G7 and 2-18C9 were strongly associated (Pearson's r(2) = 0.61; P < 0.0001). HER3 staining was nonspecific and nonreproducible. High EGFR-expressing patients had a worse prognosis when EGFR was measured with H11 or 31G7 (log rank P = 0.015 and P = 0.06). There was no statistically significant correlation between survival and EGFR detected by 2-18C9, 15F8, or polyclonal 2232 antibodies. CONCLUSIONS: Antibody validation is a critical analytic factor that regulates IHC readings in biomarker studies. Evaluation of IHC proficiency and quality control are key components toward IHC standardization. IMPACT: This work highlights the importance of IHC standardization and could result in the improvement of clinically relevant IHC protocols.

New platform for controlled and sustained delivery of the EGF receptor tyrosine kinase inhibitor AG1478 using poly(lactic-co-glycolic acid) microspheres.

Inhibition of the epidermal growth factor receptor (EGFR) reduces tumour growth and metastases and promotes axon regeneration in the central nervous system. Current EGFR inhibition strategies include the administration of reversible small-molecule tyrosine kinase inhibitors (TKIs). However, to be effective in vivo sustained delivery is required. This study explored the feasibility of encapsulating the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) in poly(lactic-co-glycolic acid) (PLGA) microspheres using three different emulsion methods: solid-in-oil-in-water, oil-in-water and oil-in-water with co-solvent. Addition of a co-solvent increased loading and release of AG1478 and significantly (p < 0.001) decreased microsphere size. Co-solvent addition also prolonged AG1478 release from 6 months to over 9 months. Once released AG1478 remained bioactive and inhibited EGFR in immortalized rat fibroblasts and EGFR-amplified human carcinoma cells. These results demonstrate that AG1478 can be encapsulated in PLGA with sustained release and retain bioactivity; thereby providing a new platform for controlled administration of EGFR TKIs.

Characterization and expression of novel 60-kDa and 110-kDa EGFR isoforms in human placenta.

The epidermal growth factor receptor (EGFR) and related family members (ERBB2, ERBB3, and ERBB4) previously have been shown to play pivotal roles in the development of female reproductive tissues, in blastocyst implantation, and in placental differentiation. We have cloned and sequenced several naturally occurring alternative transcripts of the human and mouse EGFR genes, which encode novel receptor isoforms containing varying portions of the extracellular ligand-binding domain, but lacking the transmembrane and cytoplasmic domain sequences. The human 1.8-kb and 3-kb alternative EGFR transcripts encode secreted 60-kDa and cell surface-associated 110-kDa EGFR isoforms, respectively. We have developed quantitative ribonuclease protection assays to study the expression of these alternative transcripts in human tissues. Similar to the full-length EGFR mRNAs, the highest expression level of these alternative transcripts occurs in placenta. We speculate that both of these EGFR isoforms may be important regulators of EGF-mediated cell growth and differentiation in human placenta.

Generation and characterization of polyclonal antibodies specific for human p110 sEGFR.

The EGFR/ERBB family of receptor tyrosine kinases mediates intracellular signal transduction pathways important in the regulation of cell growth, differentiation, and transformation. We previously have reported the cloning and expression of a 3 kb alternative EGFR transcript which encodes a 110 kDa form of the receptor (p110 sEGFR). This receptor isoform is identical to the extracellular region of the full-length 170 kDa EGFR through amino acid 603; in addition, p110 sEGFR contains 78 unique carboxy-terminal amino acids. Here, we report the generation and characterization of polyclonal antisera specific for the unique carboxy-terminal sequence of p110 sEGFR. Polyclonal antisera were generated by immunizing rabbits with synthetic peptides corresponding to peptides contained within the unique carboxy-terminal sequence of p110 sEGFR. Immunoglobulin fractions from antisera which tested positive for immune reactivity to these peptides by ELISA were affinity-purified by protein G and peptide-based chromatography. This affinity-purified immunoglobulin fraction specifically recognizes p110 sEGFR by ELISA, immunoprecipitation, immunoblot analysis, and immunocytochemical methods. No cross-reactivity with full-length p170 EGFR is observed using any of these detection methods. These new polyclonal antibodies will be useful in determining the expression, localization, and function of p110 sEGFR, and importantly will allow us to distinguish between the expression of this receptor isoform and p170 EGFR.

A chromosomal region 7p11.2 transcript map: its development and application to the study of EGFR amplicons in glioblastoma.

Cumulative information available about the organization of amplified chromosomal regions in human tumors suggests that the amplification repeat units, or amplicons, can be of a simple or complex nature. For the former, amplified regions generally retain their native chromosomal configuration and involve a single amplification target sequence. For complex amplicons, amplified DNAs usually undergo substantial reorganization relative to the normal chromosomal regions from which they evolve, and the regions subject to amplification may contain multiple target sequences. Previous efforts to characterize the 7p11.2 epidermal growth factor receptor ) amplicon in glioblastoma have relied primarily on the use of markers positioned by linkage analysis and/or radiation hybrid mapping, both of which are known to have the potential for being inaccurate when attempting to order loci over relatively short (<1 Mb) chromosomal regions. Due to the limited resolution of genetic maps that have been established through the use of these approaches, we have constructed a 2-Mb bacterial and P1-derived artificial chromosome (BAC-PAC) contig for the EGFR region and have applied markers positioned on its associated physical map to the analysis of 7p11.2 amplifications in a series of glioblastomas. Our data indicate that EGFR is the sole amplification target within the mapped region, although there are several additional 7p11.2 genes that can be coamplified and overexpressed with EGFR. Furthermore, these results are consistent with EGFR amplicons retaining the same organization as the native chromosome 7p11.2 region from which they are derived.