Pharmacoepigenetics of depression: no major influence of MAO-A DNA methylation on treatment response.

The monoamine oxidase A (MAO-A) gene has been suggested to be involved in the pathogenesis as well as the pharmacological treatment of major depressive disorder. In the present analysis, for the first time a pharmacoepigenetic approach was applied investigating the influence of DNA methylation patterns in the MAO-A regulatory and exon1/intron1 region on antidepressant treatment response. 94 patients of Caucasian descent with major depressive disorder (f = 61; DSM-IV) were analyzed for DNA methylation status at 43 MAO-A CpG sites via direct sequencing of sodium bisulfite treated DNA extracted from blood cells. Patients were also genotyped for the functional MAO-A VNTR. Clinical response to antidepressant treatment with escitalopram was assessed by intra-individual changes of HAM-D-21 scores after 6 weeks of treatment. Apart from two CpG sites, male subjects showed no or only very minor methylation. In female patients, lower methylation at two individual CpG sites in the MAO-A promoter region was nominally associated with impaired response to antidepressant treatment after 6 weeks (GRCh37/hg19: CpG 43.514.063, p = 0.04; CpG 43.514.684, p = 0.009), not, however, withstanding correction for multiple testing. MAO-A VNTR genotypes did not influence MAO-A methylation status. The present pilot data do not suggest a major influence of MAO-A DNA methylation on antidepressant treatment response. However, the presently observed trend towards CpG-specific MAO-A gene hypomethylation-possibly via increased gene expression and consecutively decreased serotonin and/or norepinephrine availability-to potentially drive impaired antidepressant treatment response in female patients might be worthwhile to be followed up in larger pharmacoepigenetic studies.

Serotonin transporter gene hypomethylation predicts impaired antidepressant treatment response.

Variation in the serotonin transporter gene (5-HTT; SERT; SLC6A4) has been suggested to pharmacogenetically drive interindividual differences in antidepressant treatment response. In the present analysis, a 'pharmaco-epigenetic' approach was applied by investigating the influence of DNA methylation patterns in the 5-HTT transcriptional control region on antidepressant treatment response. Ninety-four patients of Caucasian descent with major depressive disorder (MDD) (f = 61) were analysed for DNA methylation status at nine CpG sites in the 5-HTT transcriptional control region upstream of exon 1A via direct sequencing of sodium bisulfite treated DNA extracted from blood cells. Patients were also genotyped for the functional 5-HTTLPR/rs25531 polymorphisms. Clinical response to treatment with escitalopram was assessed by intra-individual changes of HAM-D-21 scores after 6 wk of treatment. Lower average 5-HTT methylation across all nine CpGs was found to be associated with impaired antidepressant treatment response after 6 wk (p = 0.005). This effect was particularly conferred by one individual 5-HTT CpG site (CpG2 (GRCh37 build, NC_000017.10 28.563.102; p = 0.002). 5-HTTLPR/rs25531 haplotype was neither associated with 5-HTT DNA methylation nor treatment response. This analysis suggests that DNA hypomethylation of the 5-HTT transcriptional control region - possibly via increased serotonin transporter expression and consecutively decreased serotonin availability - might impair antidepressant treatment response in Caucasian patients with MDD. This pharmaco-epigenetic approach could eventually aid in establishing epigenetic biomarkers of treatment response and thereby a more personalized treatment of MDD.

Monoamine oxidase A gene DNA hypomethylation - a risk factor for panic disorder?

The monoamine oxidase A (MAOA) gene has been suggested as a prime candidate in the pathogenesis of panic disorder. In the present study, DNA methylation patterns in the MAOA regulatory and exon 1/intron 1 region were investigated for association with panic disorder with particular attention to possible effects of gender and environmental factors. Sixty-five patients with panic disorder (44 females, 21 males) and 65 healthy controls were analysed for DNA methylation status at 42 MAOA CpG sites via direct sequencing of sodium bisulfate treated DNA extracted from blood cells. The occurrence of recent positive and negative life events was ascertained. Male subjects showed no or only very minor methylation with some evidence for relative hypomethylation at one CpG site in intron 1 in patients compared to controls. Female patients exhibited significantly lower methylation than healthy controls at 10 MAOA CpG sites in the promoter as well as in exon/intron 1, with significance surviving correction for multiple testing at four CpG sites (p≤0.001). Furthermore, in female subjects the occurrence of negative life events was associated with relatively decreased methylation, while positive life events were associated with increased methylation. The present pilot data suggest a potential role of MAOA gene hypomethylation in the pathogenesis of panic disorder particularly in female patients, possibly mediating a detrimental influence of negative life events. Future studies are warranted to replicate the present finding in independent samples, preferably in a longitudinal design.

Influence of whole arm loss of chromosome 16q on gene expression patterns in oestrogen receptor-positive, invasive breast cancer.

A whole chromosome arm loss of 16q belongs to the most frequent and earliest chromosomal alterations in invasive and in situ breast cancers of all common subtypes. Besides E-cadherin, several putative tumour suppressor genes residing on 16q in breast cancer have been investigated. However, the significance of these findings has remained unclear. Thus, other mechanisms leading to gene loss of function (eg haploinsufficiency, or distortion of multiple regulative subnetworks) remain to be tested as a hypothesis. To define the effect on gene expression of whole-arm loss of chromosome 16q in invasive breast cancer, we performed global gene expression analysis on a series of 18 genetically extensively characterized invasive ductal breast carcinomas and verified the results by quantitative real-time PCR (qRT-PCR). The distribution of the differential genes across the genome and their expression status was studied. A second approach by qRT-PCR in an independent series of 30 breast carcinomas helped to narrow down the observed effect. Whole-arm chromosome 16q losses, irrespective of other chromosomal changes, are associated with decreased expression of a number of candidate genes located on 16q (eg CDA08, CGI-128, SNTB2, NQO1, SF3B3, KIAA0174, ATBF1, GABARAPL2, KARS, GCSH, MBTPS1 and ZDHHC7) in breast carcinomas with a low degree of genetic instability. qRT-PCR provided evidence to suggest that the expression of these genes was reduced in a gene dosage-dependent manner. The differential expression of the candidate genes according to the chromosomal 16q-status vanished in genetically advanced breast cancer cases and changed ER status. These results corroborate previous reports about the importance of whole-arm loss of chromosome 16q in breast carcinogenesis and give evidence for the first time that haploinsufficiency, in the sense of a gene dosage effect, might be an important contributing factor in the early steps of breast carcinogenesis.

Norepinephrine and serotonin transporter genes: impact on treatment response in depression.

BACKGROUND/AIMS: The norepinephrine transporter (NET) and serotonin transporter (5-HTT) genes constitute promising candidate genes in major depression. Seven polymorphisms in the promoter, intronic and exonic region of the NET gene, as well as serotonin-transporter-linked promoter region (5-HTTLPR) and 5-HTT rs25531 polymorphisms were analyzed with respect to antidepressant treatment response with particular attention to gender effects and subtypes of melancholic or anxious depression. METHODS: 252 unrelated Caucasian patients (f = 142; m = 110) with major depression were genotyped for NET and 5-HTT polymorphisms. Genotype effects on Hamilton Depression Rating Scale score changes over 6 weeks of antidepressant treatment were analyzed using analysis of covariance with repeated measures. RESULTS: There was no effect of any of the 7 investigated NET, or the two 5-HTT polymorphisms, on the overall treatment response. An additional -/CT insertion/deletion (ins/del) polymorphism (rs58532686), however, was significantly associated with melancholic depression, with a better response in 12 patients carrying the deletion. Stratification for anxious versus nonanxious depression revealed a significantly detrimental effect of the less active 5-HTTLPR S allele (p = 0.007) and 5-HTTLPR/5-HTT rs25531 haplotypes on treatment response in patients with anxious depression. CONCLUSION: The present findings do not support a major impact of the NET and 5-HTT genes on antidepressant treatment response in major depression per se. However, there might be an impact of a -/CT ins/del polymorphism in the enhancer domain of the NET gene on treatment response in melancholic depression, which remains to be functionally investigated in future studies. The observed significant influence of the 5-HTT gene variation on antidepressant treatment in anxious depression points to anxious depression as a potential diagnostic entity of its own, requiring specific diagnostic and therapeutic attention.

Identification of acute myeloid leukaemia associated microRNA expression patterns.

MicroRNAs (miRNAs) play an important role in cellular differentiation and cancer pathogenesis. This study analysed the expression of 154 human miRNAs in acute myeloid leukaemia (AML) and control samples using a stem-loop real-time reverse transcription polymerase chain reaction approach. Global patterns of miRNA expression in AML, normal bone marrow (NBM) and CD34(+) progenitor cells allowed correct class predictions similar to whole genome microarray expression analyses that were performed at the same time. At single miRNA species level, MIRN23B was repressed in AML specimens compared to NBM and purified CD34(+) haematopoietic progenitor cells. In contrast, the MIRN221/MIRN222 cluster and MIRN34A were expressed at significantly higher levels in AML blasts. Patients with high MIRN221/MIRN222 expression showed low levels of KIT RNA and protein expression but the correlation between kit protein and KIT mRNA was significantly stronger than the correlation of either one with MIRN221/MIRN222. A global analysis between miRNA expression levels and mRNA expression of predicted target genes revealed only weak associations in the majority of miRNA species. Nonetheless, the presence of two or more miRNA binding sites within the mRNA was usually associated with a decrease in mRNA levels. Taken together, these findings provide evidence that specific miRNA expression patterns exist in AML.

Distinct amplification of an untranslated regulatory sequence in the egfr gene contributes to early steps in breast cancer development.

Overexpression of the epidermal growth factor receptor (egfr) gene is a common feature in breast cancer. We demonstrated recently that the expression of EGFR in breast cancer strongly correlates with the length of a CA simple sequence repeat within the first 2000 bases in intron 1 of the egfr gene [CA simple sequence repeat (CA-SSR) I; H. Buerger et al., Cancer Res., 60: 854-857, 2000]. Using a standardized semiautomated method of microsatellite analysis for loss of heterozygosity detection, we identified an allelic imbalance (AI) at the egfr locus in 55 of 163 primary breast cancer cases. Fine mapping of the chromosomal region at 7p12-15 around the egfr gene using 10 CA-SSR markers showed that mutations of egfr in breast cancer are frequently restricted to the first intron of egfr. Thereby, the simple sequence repeat CA-SSR I in intron 1 was affected in 84% of the patients with AI. Reverse transcription-PCR analysis of 23 breast cancer tissues with AI excluded the presence of in-frame deletions between exon 2 and exon 7. For additional characterization of the underlying phenomenon leading to the detection of an AI in microsatellite analysis, a quantitative 5'-nuclease assay for the first CA-SSR I in intron 1 was established. In breast cancer cases with AI the presence of amplifications of this sequence was shown. Kaplan-Meier analysis revealed a statistically significant worse prognosis for patients with AI in the cancer tissue at the egfr locus compared with patients without AI. Interestingly, 75% of the patients bearing AI of CA-SSR I in the tumor also showed AI at normal, nontumorous breast tissue. Our data strongly support the assumption that distinct amplifications in intronic sequences of the egfr gene, which enhance the basic transcription activity of the gene, represent one of the first steps in breast carcinogenesis. Furthermore, they point to the presence of prognosis-associated markers for breast cancer already in morphological normal breast tissue.

Modification of breast cancer risk in young women by a polymorphic sequence in the egfr gene.

The regulation of the epidermal growth factor receptor (egfr) gene in human cancer is not yet fully understood. Recent data on a polymorphic CA repeat located at the 5'-regulatory sequence in intron 1 of the egfr gene [egfr CA simple sequence repeat (SSR) I] point to a possible inheritance of cancer risk associated with the egfr gene. Furthermore, we have detected frequent allelic imbalances restricted to the egfr CA SSR I in breast cancer tissue and nontumorous breast tissue adjacent to invasive and in situ breast cancer representing amplifications. Therefore, we conducted a population-based case-control study to assess the relationship between the egfr polymorphism and breast cancer risk. Cases with a first primary breast cancer by age 50 years and age-matched population controls provided information on known and suspected risk factors. The allelic length of the egfr CA SSR was determined in 616 cases and 1072 population-sampled controls. Genotypes were categorized for analysis by allele length. Multivariate logistic regression was used to compare genotype distributions, accounting for other risk factors, and to investigate gene-environment interactions. We found a modifying effect, albeit no main effect, of the allelic length of the egfr polymorphism on breast cancer risk. The presence of two long alleles (>/==" BORDER="0">19 CA) was associated with a significantly elevated odds ratio (OR) of 10.4 [95% confidence interval (CI), 1.85-58.70] among women with a first-degree family history of breast cancer (P = 0.015 for interaction). The risk increase associated with high red meat consumption (OR, 10.68; 95% CI, 1.57-72.58) and the protective effect of high vegetable intake (OR, 0.07; 95% CI, 0.004-1.07) was also most pronounced among carriers of two long alleles (>/==" BORDER="0">19 CA). The length of the egfr CA SSR may increase the risk for familial breast cancers, and its effect could be modulated by dietary factors.

MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer.

Early-stage non-small cell lung cancer (NSCLC) can be cured by surgical resection, but a substantial fraction of patients ultimately dies due to distant metastasis. In this study, we used subtractive hybridization to identify gene expression differences in stage I NSCLC tumors that either did or did not metastasize in the course of disease. Individual clones (n=225) were sequenced and quantitative RT-PCR verified overexpression in metastasizing samples. Several of the identified genes (eIF4A1, thymosin beta4 and a novel transcript named MALAT-1) were demonstrated to be significantly associated with metastasis in NSCLC patients (n=70). The genes' association with metastasis was stage- and histology specific. The Kaplan-Meier analyses identified MALAT-1 and thymosin beta4 as prognostic parameters for patient survival in stage I NSCLC. The novel MALAT-1 transcript is a noncoding RNA of more than 8000 nt expressed from chromosome 11q13. It is highly expressed in lung, pancreas and other healthy organs as well as in NSCLC. MALAT-1 expressed sequences are conserved across several species indicating its potentially important function. Taken together, these data contribute to the identification of early-stage NSCLC patients that are at high risk to develop metastasis. The identification of MALAT-1 emphasizes the potential role of noncoding RNAs in human cancer.

High-throughput analysis of genome-wide receptor tyrosine kinase expression in human cancers identifies potential novel drug targets.

Novel high-throughput analyses in molecular biology allow sensitive and rapid identification of disease-related genes and drug targets. We have used quantitative real-time reverse transcription-PCR reactions (n = 23000) to analyze expression of all human receptor tyrosine kinases (n = 56) in malignant tumors (n = 313) of different origins and normal control samples (n = 58). The different tumor types expressed very different numbers of receptor tyrosine kinases: whereas brain tumors and testicular cancer expressed 50 receptor tyrosine kinases, acute myeloid leukemia (AML) samples expressed only 20 different ones. Specimens of similar tumor origin exhibited characteristic receptor tyrosine kinase expression patterns and were grouped together in hierarchical cluster analyses. When we focused on specific tumor entities, receptor tyrosine kinases were identified that were disease and/or stage specific. Leukemic blasts from AML bone marrow samples differed significantly in receptor tyrosine kinase expression compared with normal bone marrow and purified CD34+ cells. Among the differentially expressed receptor tyrosine kinases, we found FLT3, c-kit, CSF1 receptor, EPHB6, leukocyte tyrosine kinase, and ptk7 to be highly overexpressed in AML samples. Whereas expression changes of some of these were associated with altered differentiation patterns (e.g., CSF1 receptor), others, such as FLT3, were genuinely overexpressed in leukemic blasts. These data and the associated database (http://medweb.uni-muenster.de/institute/meda/research/) provide a comprehensive view of receptor tyrosine kinase expression in human cancer. This information can assist in the definition of novel drug targets.

Epigenetic signature of panic disorder: a role of glutamate decarboxylase 1 (GAD1) DNA hypomethylation?

Glutamate decarboxylases (GAD67/65; GAD1/GAD2) are crucially involved in gamma-aminobutyric acid (GABA) synthesis and thus were repeatedly suggested to play an important role in the pathogenesis of anxiety disorders. In the present study, DNA methylation patterns in the GAD1 and GAD2 promoter and GAD1 intron 2 regions were investigated for association with panic disorder, with particular attention to possible effects of environmental factors. Sixty-five patients with panic disorder (f=44, m=21) and 65 matched healthy controls were analyzed for DNA methylation status at 38 GAD1 promoter/intron2 and 10 GAD2 promoter CpG sites via direct sequencing of sodium bisulfate treated DNA extracted from blood cells. Recent positive and negative life events were ascertained. Patients and controls were genotyped for GAD1 rs3762556, rs3791878 and rs3762555, all of which are located in the analyzed promoter region. Patients with panic disorder exhibited significantly lower average GAD1 methylation than healthy controls (p<0.001), particularly at three CpG sites in the promoter as well as in intron 2. The occurrence of negative life events was correlated with relatively decreased average methylation mainly in the female subsample (p=0.01). GAD1 SNP rs3762555 conferred a significantly lower methylation at three GAD1 intron 2 CpG sites (p<0.001). No differential methylation was observed in the GAD2 gene. The present pilot data suggest a potentially compensatory role of GAD1 gene hypomethylation in panic disorder possibly mediating the influence of negative life events and depending on genetic variation. Future studies are warranted to replicate the present finding in independent samples, preferably in a longitudinal design.

Feasibility of azacitidine added to standard chemotherapy in older patients with acute myeloid leukemia--a randomised SAL pilot study.

INTRODUCTION: Older patients with acute myeloid leukemia (AML) experience short survival despite intensive chemotherapy. Azacitidine has promising activity in patients with low proliferating AML. The aim of this dose-finding part of this trial was to evaluate feasibility and safety of azacitidine combined with a cytarabine- and daunorubicin-based chemotherapy in older patients with AML. TRIAL DESIGN: Prospective, randomised, open, phase II trial with parallel group design and fixed sample size. PATIENTS AND METHODS: Patients aged 61 years or older, with untreated acute myeloid leukemia with a leukocyte count of <20,000/µl at the time of study entry and adequate organ function were eligible. Patients were randomised to receive azacitidine either 37.5 (dose level 1) or 75 mg/sqm (dose level 2) for five days before each cycle of induction (7+3 cytarabine plus daunorubicine) and consolidation (intermediate-dose cytarabine) therapy. Dose-limiting toxicity was the primary endpoint. RESULTS: Six patients each were randomised into each dose level and evaluable for analysis. No dose-limiting toxicity occurred in either dose level. Nine serious adverse events occurred in five patients (three in the 37.5 mg, two in the 75 mg arm) with two fatal outcomes. Two patients at the 37.5 mg/sqm dose level and four patients at the 75 mg/sqm level achieved a complete remission after induction therapy. Median overall survival was 266 days and median event-free survival 215 days after a median follow up of 616 days. CONCLUSIONS: The combination of azacitidine 75 mg/sqm with standard induction therapy is feasible in older patients with AML and was selected as an investigational arm in the randomised controlled part of this phase-II study, which is currently halted due to an increased cardiac toxicity observed in the experimental arm. TRIAL REGISTRATION: This trial is registered at clinical trials.gov (identifier: NCT00915252).

Low SMC1A protein expression predicts poor survival in acute myeloid leukemia.

Age is a strong adverse prognostic factor in acute myeloid leukemia. Little is known about the biology of acute myeloid leukemia in elderly patients. The aim of this study was to identify genes with age-dependent changes of expression in leukemic blasts and their relevance for the patient prognosis. Gene expression profiling was carried out by mRNA microarray analysis from blasts of 67 adult acute myeloid leukemia patients of different age (range, 17-80 years). Among the genes that correlated with age, PRPF4 and SMC1A were selected for protein expression studies on a tissue array containing bone marrow histologies of 135 patients with newly diagnosed AML of different ages. A significant correlation between mRNA expression levels and patient age was shown by 131 genes. Increasing age was associated with significantly decreased mRNA levels of SMC1A. On the protein level, expression of SMC1A was low or absent in 74 out of 116 acute myeloid leukemia specimens. Importantly, patients with low protein expression levels of SMC1A experienced significantly shortened event free (2.6 months versus 10.3 months, p=0.003) and overall survival (10.4 months versus 22.6 months, p=0.015). The SMC1A protein expression level remained a significant prognostic factor for event free survival (p=0.014) with a borderline significance for overall survival (p=0.066) in a multivariate analysis. SMC1A protein expression might play a role in the determination of the prognosis and might have possible implications in therapy decision in patients with acute myeloid leukemia.

Chromatin modifications induced by PML-RARalpha repress critical targets in leukemogenesis as analyzed by ChIP-Chip.

The translocation t(15;17) generates the chimeric PML-RARalpha transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARalpha transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)-chip experiments identified 372 direct genomic PML-RARalpha targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARalpha targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARalpha binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARalpha to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARalpha regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes.

The C/EBPdelta tumor suppressor is silenced by hypermethylation in acute myeloid leukemia.

Aberrant DNA methylation is the most frequent molecular alteration in acute myeloid leukemia (AML). To identify methylation-silenced genes in AML, we performed microarray analyses in U937 cells exposed to the demethylating agent 5-aza-deoxy-cytidine. Overall, 274 transcripts were significantly induced. Interestingly, C/EBPdelta expression was significantly induced (more than 10-fold) by demethylation whereas expression of all other C/EBP family members remained unchanged. The C/EBPdelta promoter was strongly methylated in different leukemic cell lines and showed signs of a repressed chromatin state. Analyses of the promoter regions of the entire C/EBP family (alpha, beta, gamma, delta, epsilon, zeta) in bone marrow samples from AML patients (n = 80) and controls (n = 15) by mass spectrometry revealed that C/EBPdelta is the most commonly hypermethylated C/EBP gene in AML. Hypermethylation occurred in more than 35% of AML patients at primary diagnosis. A significant correlation (P = .016) was observed between hypermethylation of the C/EBPdelta promoter and low expression of C/EBPdelta in AML patients. C/EBPdelta promoter activity was strongly repressed by methylation in vitro, and transcriptional repression partially depended on MeCP2 activity. C/EBPdelta exhibited growth-inhibitory properties in primary progenitor cells as well as in Flt3-ITD-transformed cells. Taken together, C/EBPdelta is a novel tumor suppressor gene in AML that is silenced by promoter methylation.

Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression.

Phyllodes tumors of the breast are rare biphasic tumors with the potential for invasion and metastatic spread. An important role of the epidermal growth factor receptor (EGFR) in phyllodes tumors has been proposed. However, detailed pathogenetic mechanisms remained unclear. We investigated 58 phyllodes tumors of the breast (40 benign, 10 borderline and eight malignant) by means of egfr fluorescence in situ hybridization (FISH) and gene dosage PCR for a regulatory sequence within intron 1 of egfr. Immunohistochemical staining was performed for EGFR, p16, p21, p27, p53, c-myc, Cyclin A, Cyclin D1, Cyclin E, c-kit and Ki67. Immunopositivity for EGFR was detected in 19% of phyllodes tumors (75% of all malignant tumors) in stromal tumor cells but not in the epithelial component. Whole-gene amplifications were seen by FISH in 15.8% (in stromal cells only) and intron 1 amplifications by gene dosage PCR in as much as 41.8% of all phyllodes tumors. Significant correlations were seen between tumor grade on the one hand and EGFR overexpression (P=0.001) and intron 1 amplifications (P<0.05) on the other. EGFR overexpression further correlated positively with immunohistochemical staining for p53, p16, Cyclin A, Cyclin E, Ki67 and c-kit. Presence of intron 1 amplifications correlated with p16 (P<0.01), p21 (P=0.009) and p53 immunoreactivity (P<0.001). Neither EGFR overexpression nor whole-gene amplification was observed in a control series of 167 fibroadenomas and only one of 43 (2.3%) exhibited intron 1 amplification in gene dosage PCR. In conclusion, our results show for the first time that activating mutations in and overexpression of egfr are associated with the progression in grade of phyllodes tumors of the breast. The observed association between intron 1 amplification and overexpression of EGFR provides further insight into regulation mechanisms of EGFR overexpression.

Characterization of a myeloid tyrosine phosphatase, Lyp, and its role in the Bcr-Abl signal transduction pathway.

The Bcr-Abl protein-tyrosine kinase is implicated in the development of chronic myeloid leukemia. The potential role of protein-tyrosine phosphatase in the regulation of Bcr-Abl signaling was explored. First, expression patterns of tyrosine phosphatases in leukemic cell lines were investigated using degenerate primers for reverse transcription-PCR followed by cloning and sequencing of the cDNA. Distinct patterns of distribution of phosphatase were found in erythroid and myeloid leukemic cell lines. Whereas some phosphatases were ubiquitously expressed, others were limited to specific cell types. Surprisingly, a previously cloned "lymphocyte-specific" phosphatase, Lyp, was frequently detected in a number of myeloid cell lines as well as normal granulocytes and monocytes. Lyp was localized to the cytosol, and overexpression of Lyp caused reduction in the phosphorylation levels of multiple proteins in KCL22 chronic myeloid leukemia blast cells including Cbl, Bcr-Abl, Erk1/2, and CrkL. Co-expression of Lyp and Bcr-Abl in Cos-7 cells resulted in decreased levels of Bcr-Abl, Grb2, and Myc. Overexpression of Lyp markedly suppressed anchorage-independent clonal growth of KCL22 cells. Taken together, the data suggest that Lyp may play an antagonistic role in signaling by the Bcr-Abl fusion protein.

Gene dosage PCR and fluorescence in situ hybridization reveal low frequency of egfr amplifications despite protein overexpression in invasive breast carcinoma.

The aim of this study was to assess the frequency of egfr whole gene and CA intron repeat amplification in invasive breast cancer as a mechanism for epidermal growth factor receptor (EGFR) protein overexpression. By means of tissue microarrays, protein overexpression and whole gene amplification were assessed in 222 cases of invasive breast cancer by immunohistochemistry and FISH, respectively. First intron CA repeat amplification was assessed by Taqman RT-PCR. With FISH and RT-PCR, 4.7 and 6.3% of cases showed whole gene and first intron CA repeat amplification, respectively. Amplification dosage varied between two- and four-fold in RT-PCR. By immunohistochemistry, 17.3% showed EGFR overexpression. There was a low correlation between the different methods. In all, 2.9% of cases showed both whole gene amplification and intron CA repeat amplification, and 90.3% of cases were negative for both. Nearly 20% of cases with immunohistochemical protein overexpression showed intron CA repeat amplification, and only 2.2% of cases that were negative on immunohistochemistry showed such amplification. In all, 13% of cases with protein overexpression showed amplification by FISH, and only 1.6% of cases that were negative on immunohistochemistry showed such amplification. Of the cases with EGFR overexpression, 4 (25%) showed either whole gene or intron CA repeat amplification. In conclusion, whole gene amplifications of egfr are rare in invasive breast cancer and explain protein overexpression in only about 12.5% of invasive breast cancer cases. First intron first CA repeat amplification is another important mechanism for EGFR protein overexpression, explaining protein overexpression in about 18.7% of cases. However, since about 75% of cases with EGFR protein overexpression lack either of these amplifications, other expression regulating mechanisms must be considered.

Allelic length of a CA dinucleotide repeat in the egfr gene correlates with the frequency of amplifications of this sequence--first results of an inter-ethnic breast cancer study.

Overexpression of the epidermal growth factor receptor (EGFR) is a common finding in invasive breast cancer and represents a potential target for new treatment options. However, little is known about the parameters that might indicate a potential clinical response for these anti-EGFR-based therapies. In order to gain further insights into the interplay between the length of a CA-SSR I repeat in intron 1 of egfr, copy numbers of this untranslated regulatory sequence, and protein expression, the present study investigated breast cancers from Germans and Japanese patients by microsatellite analysis, quantitative 5' nuclease assay by egfr enzyme-linked immunosorbent assay (ELISA), and comparative genomic hybridization (CGH). Japanese breast cancer patients displayed significantly longer alleles for the CA-SSR I repeat (p < 0.001), associated with significantly lower EGFR expression (mean 65 versus 36 fmol/mg membrane protein). Allelic imbalance (restricted to CA-SSR I) was observed in 55% of the informative Japanese breast cancers compared with only 34% of the German breast cancer reference group. Using a quantitative 5' nuclease assay for egfr, a significantly higher percentage of Japanese breast cancer patients revealed amplifications of the CA-SSR I repeat (p < 0.01). Japanese patients with these amplifications were characterized by a significantly higher EGFR content compared with the German breast cancer patients (p < 0.05). These data show, on the one hand, that the correlation of EGFR overexpression and an inherited CA repeat polymorphism within intron 1 of egfr is a general finding in breast cancer, as has been shown previously. On the other hand, the data demonstrate clearly for the first time an interaction between the length of a polymorphism in intron 1 of egfr as an inherited genetic factor and the frequency of egfr amplification, as an acquired genetic factor, both factors contributing to EGFR overexpression in breast cancer. This new knowledge about mechanisms of regulation of EGFR expression might serve as an additional basis for evaluating anti-EGFR-based therapies.