Influence of detection of pretreatment cytogenetic abnormalities on first complete remission and survival in adult acute lymphoblastic leukemia.

OBJECTIVE: Treatment of acute lymphoblastic leukemia (ALL) in adults focuses on the initial assessment of the prognostic relevant cytogenetic features as well as a response-guided therapy based on molecular data. We examined the importance of molecular-cytogenetic abnormalities for complete remission (CR) rates and the overall survival (OS) in adult ALLs. METHODS: Conventional cytogenetics and fluorescence in situ hybridization were performed on bone marrow cells from 33 newly-diagnosed ALL adults. Two karyotype categories [standard- risk group- normal karyotype, hyperdiplody and other structural aberrations, and high-risk group-t(11q23)/MLL, t(9;22)/bcr-abl, t(1;19), t(8;14), C-MYC and complex karyotype] and the biologically and clinically relevant ALL ploidy subgroups were prospectively defined. RESULTS: Chromosomal abnormalities were found in 52% of the cases with a high rate of poor-risk translocations - t(9;22), t(8q24), t(11q23), t(1;19). The total CR rate was 67% and the median time for achievement 2.33 months. Male sex, an age below 35 years and the absence of high risk translocations might have contributed to the high CR rates. Female patients, hyperdiplody, low white blood cells (WBC), and random cytogenetic aberrations had the longest OS. OS, 3- and 5-years survival periods were significantly shorter for poor-risk than standard risk group (p=.015, p=.001 and p=.005, respectively). CONCLUSION: This study emphasizes the lack of influence of cytogenetic aberrations on the CR and the time to achieve CR. However, our observations show that these aberrations are an independent prognostic factor in adult ALL - they allow predicting therapy resistance and the OS time after intense treatment.

Epidermal growth factor receptor gene copy number changes in lung cancer.

OBJECTIVE: The objective of this study was to assess the implication of copy number changes of epidermal growth factor receptor (EGFR) in lung cancer pathogenesis. MATERIALS AND METHODS: We used the highly reliable method of FISH, applied on tissue microarray (TMA), containing 306 lung tumors of different histological types, grades and tumor stage, in order to analyze the correlations between gene copy number changes and tumor phenotype. RESULTS: The frequency of EGFR copy number changes was 22.2%-2.8% amplifications and 19.4% gains. EGFR gains occurred more commonly in the squamous cell cancers (23.5%) than in adenocarcinomas (11.8%). Amplifications of EGFR were found only in the squamous cell cancers. Regarding cancer phenotype, there was a statistically significant correlation between EGFR copy number changes and histological grade (p = 0.001). No statistically significant relation could be observed with the metastatic spread of the tumors (lymphogenic and haematogenic) (p = 0.082 and p = 0.1, respectively). In our study EGFR could not be determined as a prognostic factor of survival (p = 0.6115). CONCLUSION: EGFR copy number changes could supplement the clinical significance of EGFR as a marker related to its pathogenesis and targeted therapy.

Gene-specific fluorescence in-situ hybridization analysis on tissue microarray to refine the region of chromosome 20q amplification in melanoma.

Several comparative genomic hybridization studies provide evidence for overrepresentation of the long arm of chromosome 20 in malignant melanoma. These studies also suggest that chromosome 20q contains genes that may contribute to melanoma pathogenesis. To refine the region of 20q amplification and to identify potential candidate genes involved in melanoma or even in melanoma progression from these regions, we combined fluorescence in-situ hybridization with MYBL2, ZNF217, CYP24 and STK6 specific probes (chromosomal region 20q13.1-q13.2) with high-throughput tissue microarray consisting of 280 primary melanomas and melanoma metastases. Low-level amplification ranging from 0.5 to 2.0% was detected for the tumor-related genes of interest. Higher frequencies of gain when compared with amplification were detected for MYBL2, ZNF217, CYP24 and STK6. Aneusomy of centromere 20 was observed in 29.9% of the analyzed tumors. A significantly higher frequency of ZNF217, CYP24 and STK6 total copy-number increase, as well as aneusomy of centromere 20, was found in the group of metastases when compared with the group of primary melanomas. Despite the technological advantage of fluorescence in-situ hybridization on tissue microarray, which allows refining regions of amplification, we were not able to recognize any of the MYBL2, ZNF217, CYP24 and STK6 genes as a particular relevant gene for melanoma tumorigenesis.

Identification of NQO1 and GSTs genotype frequencies in Bulgarian patients with Balkan endemic nephropathy.

BACKGROUND: Polymorphisms in NAD[P]H:quinone oxidoreductase (NQO1) and glutathione S-transferases (GSTs) have been reported to be associated with an increased risk for environmentally and/or occupationally induced renal and bladder cancers. Genetic factors related to chronic nephropathy and to urinary bladder or renal cancer development in Balkan endemic nephropathy (BEN) is unknown. In order to evaluate their possible role in BEN susceptibility, we determined the frequencies of NQO1 alleles *1, *2 and *3, as well as the GSTT1 and GSTM1 null genotypes in BEN patients and healthy subjects from a non-endemic region of Bulgaria. METHODS: The respective genotypes of 95 unrelated Bulgarian BEN patients and of 112 healthy individuals (control group) were determined by rapid cycle polymerase chain reaction (PCR) and detected with either SYBR green I fluorescent dye or melting curve analysis using allele specific probes. RESULTS: NQO1 genotyping showed a higher NQO1*2 allele frequency (23.68%) in BEN patients compared to controls (18.75%; p=0.219), while NQO1*3 allele frequencies were similar in both groups (2.63% in BEN patients vs. 2.23% in controls; p=0.791). The GSTT1 deficiencywas observed in 20% of BEN patients vs. 16.1% of controls (p=0.613). The GSTM1 null genotype was found in 45.3% of BEN patients vs. 51.8% of controls (p=0.674). There was no influence of NQO1 and GSTs genotypes found on BEN risk. CONCLUSIONS: Our results established that alleles NQO1*2 and NQO1*3, as well as lack of GSTT1 and GSTM1 did not influence the BEN risk. These findings provide novel information on the genetic heterogeneity in the healthy Bulgarian population.

High-throughput tissue microarray analysis of 11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinary bladder cancer.

Gene amplification is a common mechanism for oncogene overexpression. High-level amplifications at 11q13 have been repeatedly found in bladder cancer by comparative genomic hybridization (CGH) and other techniques. Putative candidate oncogenes located in this region are CCND1 (PRAD1, bcl-1), EMS1, FGF3 (Int-2), and FGF4 (hst1, hstf1). To evaluate the involvement of these genes in bladder cancer, a tissue microarray (TMA) containing 2317 samples was screened by fluorescence in situ hybridization (FISH). The frequency of gains and amplifications of all genes increased significantly from stage pTa to pT1-4 and from low to high grade. In addition, amplification was associated with patient survival and progression of pT1 tumours. Among 123 tumours with amplifications, 68.3% showed amplification of all four genes; 19.5% amplification of CCND1, FGF4, and FGF3; and 0.8% co-amplification of FGF4, FGF3, and EMS1. Amplification of CCND1 alone was found in 9% of the tumours, while EMS1 alone was amplified in 1.6% and FGF4 in 0.8%. Overall, the amplification frequency decreased with increasing genomic distance from CCND1, suggesting that, among the genes examined, CCND1 is the major target gene in the 11q13 amplicon in bladder cancer.

High-throughput tissue microarray analysis of erbB-2 gene amplification in urinary bladder cancer. A study of Bulgarian patients.

INTRODUCTION: Overexpression of the receptor Her-2 leads to increased proliferative response to epidermal growth factors which plays a key role in tumor development and growth. Increased oncoprotein level, in the majority of cases, is caused by erbB-2 gene amplification. To define the frequency of amplifications of erbB-2 in bladder cancer, and to determine their association with the tumor phenotype we utilized tissue microarray (TMA) approach. MATERIALS AND METHODS: We analyzed a TMA consisting of 159 transitional cell bladder carcinomas for erbB-2 gene amplification by dual-color FISH. RESULTS: The frequency of erbB-2 amplification was 5.3% of all successfully analyzed samples (4 of 75). It increased from minimally invasive (pT1) to muscle-invasive (pT2-4) tumors, as well as with advanced tumor grade (G1 to G2 to G3). All amplifications were cluster amplifications confirming the fact that erbB-2 amplification occurs intrachromosomally in bladder cancer. CONCLUSIONS: We concluded that despite its low incidence, erbB-2 amplification is of importance for the bladder tumor invasion and progression.