Spatiotemporal 7q11.23 protein network analysis implicates the role of DNA repair pathway during human brain development.

Recurrent deletions and duplications of chromosome 7q11.23 copy number variants (CNVs) are associated with several psychiatric disorders. Although phenotypic abnormalities have been observed in patients, causal genes responsible for CNV-associated diagnoses and traits are still poorly understood. Furthermore, the targeted human brain regions, developmental stages, protein networks, and signaling pathways, influenced by this CNV remain unclear. Previous works showed GTF2I involved in Williams-Beuren syndrome, but pathways affected by GTF2I are indistinct. We first constructed dynamic spatiotemporal networks of 7q11.23 genes by combining data from the brain developmental transcriptome with physical interactions of 7q11.23 proteins. Topological changes were observed in protein-protein interaction (PPI) networks throughout different stages of brain development. Early and late fetal periods of development in the cortex, striatum, hippocampus, and amygdale were observed as the vital periods and regions for 7q11.23 CNV proteins. CNV proteins and their partners are significantly enriched in DNA repair pathway. As a driver gene, GTF2I interacted with PRKDC and BRCA1 to involve in DNA repair pathway. The physical interaction between GTF2I with PRKDC was confirmed experimentally by the liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified that early and late fetal periods are crucial for 7q11.23 genes to affect brain development. Our results implicate that 7q11.23 CNV genes converge on the DNA repair pathway to contribute to the pathogenesis of psychiatric diseases.

Multiple desmoplastic Spitz nevi with BRAF fusions in a patient with ring chromosome 7 syndrome.

Patients with non-supernumerary ring chromosome 7 syndrome have an increased incidence of hemangiomas, café-au-lait spots, and melanocytic nevi. The mechanism for the increased incidence of these benign neoplasms is unknown. We present the case of a 22-year-old man with ring chromosome 7 and multiple melanocytic nevi. Two nevi, one on the right ear and the other on the right knee, were biopsied and diagnosed as desmoplastic Spitz nevi. Upon targeted next-generation DNA sequencing, both harbored BRAF fusions. Copy number alterations and fluorescence in situ hybridization (FISH) for BRAF suggested that the fusions arose on the ring chromosome 7. Hence, one reason for increased numbers of nevi in patients with non-supernumerary ring chromosome 7 syndrome may be increased likelihood of BRAF fusions, due to the instability of the ring chromosome.

Multiorgan failure with abnormal receptor metabolism in mice mimicking Samd9/9L syndromes.

Autosomal dominant sterile α motif domain containing 9 (Samd9) and Samd9L (Samd9/9L) syndromes are a large subgroup of currently established inherited bone marrow failure syndromes that includes myelodysplasia, infection, growth restriction, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE), ataxia pancytopenia, and familial monosomy 7 syndromes. Samd9/9L genes are located in tandem on chromosome 7 and have been known to be the genes responsible for myeloid malignancies associated with monosomy 7. Additionally, as IFN-inducible genes, Samd9/9L are crucial for protection against viruses. Samd9/9L syndromes are caused by gain-of-function mutations and develop into infantile myelodysplastic syndromes associated with monosomy 7 (MDS/-7) at extraordinarily high frequencies. We generated mice expressing Samd9LD764N, which mimic MIRAGE syndrome, presenting with growth retardation, a short life, bone marrow failure, and multiorgan degeneration. In hematopoietic cells, Samd9LD764N downregulates the endocytosis of transferrin and c-Kit, resulting in a rare cause of anemia and a low bone marrow reconstitutive potential that ultimately causes MDS/-7. In contrast, in nonhematopoietic cells we tested, Samd9LD764N upregulated the endocytosis of EGFR by Ship2 phosphatase translocation to the cytomembrane and activated lysosomes, resulting in the reduced expression of surface receptors and signaling. Thus, Samd9/9L is a downstream regulator of IFN that controls receptor metabolism, with constitutive activation leading to multiorgan dysfunction.

Germline predisposition in myeloid neoplasms: Unique genetic and clinical features of GATA2 deficiency and SAMD9/SAMD9L syndromes.

Increasing awareness about germline predisposition and the widespread application of unbiased whole exome sequencing contributed to the discovery of new clinical entities with high risk for the development of haematopoietic malignancies. The revised 2016 WHO classification introduced a novel category of "myeloid neoplasms with germline predisposition" with GATA2, CEBPA, DDX41, RUNX1, ANKRD26 and ETV6 genes expanding the spectrum of hereditary myeloid neoplasms (MN). Since then, more germline causes of MN were identified, including SAMD9, SAMD9L, and ERCC6L2. This review describes the genetic and clinical spectrum of predisposition to MN. The main focus lies in delineation of phenotypes, genetics and management of GATA2 deficiency and the novel SAMD9/SAMD9L-related disorders. Combined, GATA2 and SAMD9/SAMD9L (SAMD9/9L) syndromes are recognized as most frequent causes of primary paediatric myelodysplastic syndromes, particularly in setting of monosomy 7. To date, ~550 cases with germline GATA2 mutations, and ~130 patients with SAMD9/9L mutations had been reported in literature. GATA2 deficiency is a highly penetrant disorder with a progressive course that often rapidly necessitates bone marrow transplantation. In contrast, SAMD9/9L disorders show incomplete penetrance with various clinical outcomes ranging from spontaneous haematological remission observed in young children to malignant progression.

Engineering of targeted megabase-scale deletions in human induced pluripotent stem cells.

Recurrent chromosomal deletions spanning several megabases are often found in hematological malignancies. The ability to engineer deletions in model systems to functionally study their effects on the phenotype would enable, first, determination of whether a given deletion is pathogenic or neutral and, second, identification of the critical genes. Incomplete synteny makes modeling of deletions of megabase scale challenging or impossible in the mouse or other model organisms. Furthermore, despite the breakthroughs in targeted nuclease technologies in recent years, engineering of megabase-scale deletions remains challenging and has not been achieved in normal diploid human cells. Large deletions of the long arm of chromosome 7 (chr7q) occur frequently in myelodysplastic syndrome (MDS) and are associated with poor prognosis. We previously found that we can model chr7q deletions in human induced pluripotent stem cells (iPSCs) using a modified Cre-loxP strategy. However, this strategy did not afford control over the length and boundaries of the engineered deletions, which were initiated through random chromosome breaks. Here we developed strategies enabling the generation of defined and precise chromosomal deletions of up to 22 Mb, using two different strategies: "classic" Cre-loxP recombination and CRISPR/Cas9-mediated DNA cleavage. As proof of principle, we illustrate that phenotypic characterization of the hematopoiesis derived from these iPSCs upon in vitro differentiation allows further definition of the critical region of chr7q whose hemizygosity impairs hematopoietic differentiation potential. The strategies we present here can be broadly applicable to engineering of diverse chromosomal deletions in human cells.

Association of chromosome 7 aneuploidy measured by fluorescence in situ hybridization assay with muscular invasion in bladder cancer.

BACKGROUND: The preoperative prediction of muscular invasion status is important for adequately treating bladder cancer (BC) but nevertheless, there are some existing dilemmas in the current preoperative diagnostic accuracy of BC with muscular invasion. Here, we investigated the potential association between the fluorescence in situ hybridization (FISH) assay and muscular invasion among patients with BC. A cytogenetic-clinical nomogram for the individualized preoperative differentiation of muscle-invasive BC (MIBC) from non-muscle-invasive BC (NMIBC) is also proposed. METHODS: All eligible BC patients were preoperatively tested using a FISH assay, which included 4 sites (chromosome-specific centromeric probe [CSP] 3, 7, and 17, and gene locus-specific probe [GLP]-p16 locus). The correlation between the FISH assay and BC muscular invasion was evaluated using the Chi-square tests. In the training set, univariate and multivariate logistic regression analyses were used to develop a cytogenetic-clinical nomogram for preoperative muscular invasion prediction. Then, we assessed the performance of the nomogram in the training set with respect to its discriminatory accuracy and calibration for predicting muscular invasion, and clinical usefulness, which were then validated in the validation set. Moreover, model comparison was set to evaluate the discrimination and clinical usefulness between the nomogram and the individual variables incorporated in the nomogram. RESULTS: Muscular invasion was more prevalent in BC patients with positive CSP3, CSP7 and CSP17 status (OR [95% CI], 2.724 [1.555 to 4.774], P < 0.001; 3.406 [1.912 to 6.068], P < 0.001 and 2.483 [1.436 to 4.292], P = 0.001, respectively). Radiology-determined tumor size, radiology-determined clinical tumor stage and CSP7 status were identified as independent risk factors of BC muscular invasion by the multivariate regression analysis in the training set. Then, a cytogenetic-clinical nomogram incorporating these three independent risk factors was constructed and was observed to have satisfactory discrimination in the training (AUC 0.784; 95% CI: 0.715 to 0.853) and validation (AUC 0.743; 95% CI: 0.635 to 0.850) set. The decision curve analysis (DCA) indicated the clinical usefulness of our nomogram. In models comparison, using the receiver operator characteristic (ROC) analyses, the nomogram showed higher discriminatory accuracy than any variables incorporated in the nomogram alone and the DCAs also identified the nomogram as possessing the highest net benefits at wide range of threshold probabilities. CONCLUSION: CSP7 status was identified as an independent factor for predicting muscular invasion in BC patients and was successfully incorporated in a clinical nomogram combining the results of the FISH assay with clinical risk factors.

Impact of clinical features, cytogenetics, genetic mutations, and methylation dynamics of CDKN2B and DLC-1 promoters on treatment response to azacitidine.

Azacitidine (AZA) is a DNA hypomethylation agent administered in myeloid neoplasms; however, there is still a lack of established predictors of response. We studied 113 patients with myelodysplastic syndromes (n = 85) or acute myeloid leukemia (n = 28) who received AZA to assess the predictive value on response of clinical features, cytogenetics, and molecular markers. Overall, 46 patients (41%) responded to AZA. Platelet doubling after the first AZA cycle was associated with a better response (68% vs. 32% responders, P = 0.041). Co-occurrence of chromosome 7 abnormalities and 17p deletion was associated with a worse response (P = 0.039). Pre-treatment genetic mutations were detected in 98 patients (87%) and methylation of CDKN2B and DLC-1 promoters were detected in 50 (44%) and 37 patients (33%), respectively. Patients with SF3B1 mutations showed a better response to AZA (68% vs. 35% responders, P = 0.008). In contrast, subjects with mutations in transcription factors (RUNX1, SETBP1, NPM1) showed a worse response (20% vs. 47% responders, P = 0.014). DLC-1 methylation pre-treatment was associated with poor clinical features and its reduction post-treatment resulted in a better response to AZA in MDS patients (P = 0.037). In conclusion, we have identified several predictors of response to AZA that could help select the best candidates for this treatment.

Imprinting methylation in SNRPN and MEST1 in adult blood predicts cognitive ability.

Genomic imprinting is important for normal brain development and aberrant imprinting has been associated with impaired cognition. We studied the imprinting status in selected imprints (H19, IGF2, SNRPN, PEG3, MEST1, NESPAS, KvDMR, IG-DMR and ZAC1) by pyrosequencing in blood samples from longitudinal cohorts born in 1936 (n = 485) and 1921 (n = 223), and anterior hippocampus, posterior hippocampus, periventricular white matter, and thalamus from brains donated to the Aberdeen Brain Bank (n = 4). MEST1 imprint methylation was related to childhood cognitive ability score (-0.416 95% CI -0.792,-0.041; p = 0.030), with the strongest effect evident in males (-0.929 95% CI -1.531,-0.326; p = 0.003). SNRPN imprint methylation was also related to childhood cognitive ability (+0.335 95%CI 0.008,0.663; p = 0.045). A significant association was also observed for SNRPN methylation and adult crystallised cognitive ability (+0.262 95%CI 0.007,0.517; p = 0.044). Further testing of significant findings in a second cohort from the same region, but born in 1921, resulted in similar effect sizes and greater significance when the cohorts were combined (MEST1; -0.371 95% CI -0.677,-0.065; p = 0.017; SNRPN; +0.361 95% CI 0.079,0.643; p = 0.012). For SNRPN and MEST1 and four other imprints the methylation levels in blood and in the five brain regions were similar. Methylation of the paternally expressed, maternally methylated genes SNRPN and MEST1 in adult blood was associated with cognitive ability in childhood. This is consistent with the known importance of the SNRPN containing 15q11-q13 and the MEST1 containing 7q31-34 regions in cognitive function. These findings, and their sex specific nature in MEST1, point to new mechanisms through which complex phenotypes such as cognitive ability may be inherited. These mechanisms are potentially relevant to both the heritable and non-heritable components of cognitive ability. The process of epigenetic imprinting-within SNRPN and MEST1 in particular-and the factors that influence it, are worthy of further study in relation to the determinants of cognitive ability.

Characterization of inv(3) cell line OCI-AML-20 with stroma-dependent CD34 expression.

Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.

Monosomy 7 in Pediatric Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) in children and adolescents are a rare heterogeneous group of clonal stem cell disorders. Complete or partial loss of chromosome 7 constitutes the most common cytogenetic abnormality encountered in any type of childhood MDS, is associated with more advanced disease, and usually requires a timely allogeneic stem cell transplantation. This article provides insights into the current understanding of the genotype, phenotype, and clonal evolution patterns in pediatric MDS associated with loss of chromosome 7.

Therapy-related acute lymphoblastic leukemia has distinct clinical and cytogenetic features compared to de novo acute lymphoblastic leukemia, but outcomes are comparable in transplanted patients.

Therapy-related acute lymphoblastic leukemia remains poorly defined due to a lack of large data sets recognizing the defining characteristics of this entity. We reviewed all consecutive cases of adult acute lymphoblastic leukemia treated at our institution between 2000 and 2017 and identified therapy-related cases - defined as acute lymphoblastic leukemia preceded by prior exposure to cytotoxic chemotherapy and/or radiation. Of 1022 patients with acute lymphoblastic leukemia, 93 (9.1%) were classified as therapy-related. The median latency for therapy-related acute lymphoblastic leukemia onset was 6.8 years from original diagnosis, and this was shorter for patients carrying the MLL gene rearrangement compared to those with other cytogenetics. When compared to de novo acute lymphoblastic leukemia, therapy-related patients were older (P<0.01), more often female (P<0.01), and had more MLL gene rearrangement (P<0.0001) and chromosomes 5/7 aberrations (P=0.02). Although therapy-related acute lymphoblastic leukemia was associated with inferior 2-year overall survival compared to de novo cases (46.0% vs 68.1%, P=0.001), prior exposure to cytotoxic therapy (therapy-related) did not independently impact survival in multivariate analysis (HR=1.32; 95% CI: 0.97-1.80, P=0.08). There was no survival difference (2-year = 53.4% vs 58.9%, P=0.68) between the two groups in patients who received allogenic hematopoietic cell transplantation. In conclusion, therapy-related acute lymphoblastic leukemia represents a significant proportion of adult acute lymphoblastic leukemia diagnoses, and a subset of cases carry clinical and cytogenetic abnormalities similar to therapy-related myeloid neoplasms. Although survival of therapy-related acute lymphoblastic leukemia was inferior to de novo cases, allogeneic hematopoietic cell transplantation outcomes were comparable for the two entities.

Chromosome mis-segregation and cytokinesis failure in trisomic human cells.

Cancer cells display aneuploid karyotypes and typically mis-segregate chromosomes at high rates, a phenotype referred to as chromosomal instability (CIN). To test the effects of aneuploidy on chromosome segregation and other mitotic phenotypes we used the colorectal cancer cell line DLD1 (2n = 46) and two variants with trisomy 7 or 13 (DLD1+7 and DLD1+13), as well as euploid and trisomy 13 amniocytes (AF and AF+13). We found that trisomic cells displayed higher rates of chromosome mis-segregation compared to their euploid counterparts. Furthermore, cells with trisomy 13 displayed a distinctive cytokinesis failure phenotype. We showed that up-regulation of SPG20 expression, brought about by trisomy 13 in DLD1+13 and AF+13 cells, is sufficient for the cytokinesis failure phenotype. Overall, our study shows that aneuploidy can induce chromosome mis-segregation. Moreover, we identified a trisomy 13-specific mitotic phenotype that is driven by up-regulation of a gene encoded on the aneuploid chromosome.

[CHARACTERISTIC OF ENDOMETRIAL MESENCHYMAL STEM CELLS IN CULTURE OBTAINED FROM PATIENT WITH ADENOMYOSIS].

Adenomyosis is form of endometriosis, common diseases of female reproductive system, which can lead to infertility in women. in this study we are obtained and characterized cell line endometrial mesenchymal stem cells from a patient with adenomyosis, and compare obtained cells with the cell line of healthy donor. Aim of this study was to assesses the extent of differences between cells from donor with adenomyosis and cells from healthy donor. Was established that compared lines had morphology like fibroblasts, were differentiated in adipocytes, were expressed mesenchymal markers and didn't expressed haematopoietic markers. Cytogenetic analysis of differentially stained metaphase chromosomes on G-banding (passage 6-7) showed that healthy donor's cells had predominantly normal karyotype. The cellular line from a patient with diagnosis of "adenomyosis" had a lot of cells with changes in karyotype's structure. These changes were related with aneuploidy of cellular population and the presence non-random chromosomal breaks, often in chromosomes 7 and 11. Analysis of this data allows the cells from adenomyosis characterized physiological stability in culture and karyotypic instability with non-random involvement certain chromosomal set. The cellular line obtained from donor with adenomyosis showed signs destabilization of he genome, typical for cell transformation. Division of adenomyosis cells to the 26th passage is stopped and these cells entered into a phase of replicative aging. Based on this, we can conclude that founded karyotype's hanges do not lead to transformation and immortalization of cells in vitro.

Telomere attrition and candidate gene mutations preceding monosomy 7 in aplastic anemia.

The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.

Genetic profile of T-cell acute lymphoblastic leukemias with MYC translocations.

MYC translocations represent a genetic subtype of T-lineage acute lymphoblastic leukemia (T-ALL), which occurs at an incidence of ∼6%, assessed within a cohort of 196 T-ALL patients (64 adults and 132 children). The translocations were of 2 types; those rearranged with the T-cell receptor loci and those with other partners. MYC translocations were significantly associated with the TAL/LMO subtype of T-ALL (P = .018) and trisomies 6 (P < .001) and 7 (P < .001). Within the TAL/LMO subtype, gene expression profiling identified 148 differentially expressed genes between patients with and without MYC translocations; specifically, 77 were upregulated and 71 downregulated in those with MYC translocations. The poor prognostic marker, CD44, was among the upregulated genes. MYC translocations occurred as secondary abnormalities, present in subclones in one-half of the cases. Longitudinal studies indicated an association with induction failure and relapse.

Whole exome sequencing identifies novel recurrently mutated genes in patients with splenic marginal zone lymphoma.

The pathogenesis of splenic marginal zone lymphoma (SMZL) remains largely unknown. Recent high-throughput sequencing studies have identified recurrent mutations in key pathways, most notably NOTCH2 mutations in >25% of patients. These studies are based on small, heterogeneous discovery cohorts, and therefore only captured a fraction of the lesions present in the SMZL genome. To identify further novel pathogenic mutations within related biochemical pathways, we applied whole exome sequencing (WES) and copy number (CN) analysis to a biologically and clinically homogeneous cohort of seven SMZL patients with 7q abnormalities and IGHV1-2*04 gene usage. We identified 173 somatic non-silent variants, affecting 160 distinct genes. In additional to providing independent validation of the presence of mutation in several previously reported genes (NOTCH2, TNFAIP3, MAP3K14, MLL2 and SPEN), our study defined eight additional recurrently mutated genes in SMZL; these genes are CREBBP, CBFA2T3, AMOTL1, FAT4, FBXO11, PLA2G4D, TRRAP and USH2A. By integrating our WES and CN data we identified three mutated putative candidate genes targeted by 7q deletions (CUL1, EZH2 and FLNC), with FLNC positioned within the well-characterized 7q minimally deleted region. Taken together, this work expands the reported directory of recurrently mutated cancer genes in this disease, thereby expanding our understanding of SMZL pathogenesis. Ultimately, this work will help to establish a stratified approach to care including the possibility of targeted therapy.

Chromosome 2p gain in monoclonal B-cell lymphocytosis and in early stage chronic lymphocytic leukemia.

Recent studies have described chromosome 2p gain as a recurrent lesion in chronic lymphocytic leukemia (CLL). We investigated the 2p gain and its relationship with common prognostic biomarkers in a prospective series of 69 clinical monoclonal B-cell lymphocytosis (cMBL) and 218 early stage (Binet A) CLL patients. The 2p gain was detected by FISH in 17 patients (6%, 16 CLL, and 1 cMBL) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e., del(11)(q23) and del(17)(p13) (P = 0.002), were significantly more frequent in 2p gain cases, as well as unmutated status of IGHV (P < 1 × 10(-4) ) and CD38 (P < 1 × 10(-4) ) and ZAP-70 positive expression (P = 0.003). Furthermore, 2p gain patients had significantly higher utilization of stereotyped B-cell receptors compared with 2p negative patients (P = 0.009), and the incidence of stereotyped subset #1 in 2p gain patients was significantly higher than that found in the remaining CLLs (P = 0.031). Transcriptional profiling analysis identified several genes significantly upregulated in 2p gain CLLs, most of which mapped to 2p. Among these, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Thus, 2p gain can be present since the early stages of the disease, particularly in those cases characterized by other poor prognosis markers. The finding of genes upregulated in the cells with 2p gain provides new insights to define the pathogenic role of this lesion.

Chromosome 7-centric analysis of proteomics data from a panel of human colon carcinoma cell lines.

In this manuscript, we describe a shotgun proteomics approach for a comprehensive proteomic analysis of samples including total lysates, membrane, secretome, and exosome fractions from a panel of colorectal cancer cell lines. We will present an analysis of our proteomics data in two alternative formats. First we will discuss a traditional analysis of our data, in which we identify a number of cancer-associated proteins using various proteomic data analysis tools. In a second approach, we use a chromosome format to organize the proteomic data on chromosome 7, allowing the identification of clusters of cancer-associated genes with boundaries defined by physical proximity on different chromosomes.

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma.

BACKGROUND: The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival. METHODS: The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining. RESULTS: Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient's survival, while there was no connection with pathological stage. CONCLUSION: In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.