Tarja Niini

Abnormal expression of apoptosis-related genes in haematological malignancies: overexpression of MYC is poor prognostic sign in mantle cell lymphoma.

Summary. The expression of apoptosis‐related genes BCL2, BAX, BCL2L1, BCL2A1, MCL1, DAPK1 and MYC was studied by quantitative real‐time polymerase chain reaction on total RNA samples from patients with acute lymphoblastic leukaemia (ALL, n = 16), acute myeloid leukaemia (AML, n = 27), chronic myeloid leukaemia (CML, n = 12), mantle cell lymphoma (MCL, n = 19) and chronic lymphoid leukaemia (CLL, n = 32). BCL2, BAX, BCL2A1, MCL1, DAPK1 and MYC were overexpressed in all patient groups. BCL2L1 was underexpressed in CLL and CML, but not in AML, ALL and MCL. MCL1 levels were significantly higher in CD13 and CD33‐positive ALL, and in CD56‐positive AML samples. BCL2, BCL2L1, BCL2A1 and MCL1 were overexpressed and DAPK1 was underexpressed in CLL samples with a 11q23 deletion. MYC overexpression was significantly associated with shorter overall survival in MCL (P < 0·01). AML patients with a normal karyotype showed a higher frequency of BCL2A1 overexpression (P < 0·001) than those with an abnormal karyotype.

Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors

BackgroundEwing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas.MethodsArray comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5–192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest.ResultsCopy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, HDGF, confirmed that its expression was higher than in control samples. However, no association between HDGF expression and patient survival was observed.ConclusionWe conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.

Genomic Profiling of Chondrosarcoma: Chromosomal Patterns in Central and Peripheral Tumors

Purpose: Histologic grade is currently the best predictor of clinical course in chondrosarcoma patients. Grading suffers, however, from extensive interobserver variability and new objective markers are needed. Hence, we have investigated DNA copy numbers in chondrosarcomas with the purpose of identifying markers useful for prognosis and subclassification. Experimental Design: The overall pattern of genomic imbalances was assessed in a series of 67 chondrosarcomas using array comparative genomic hybridization. Statistical analyses were applied to evaluate the significance of alterations detected in subgroups based on clinical data, morphology, grade, tumor size, and karyotypic features. Also, the global gene expression profiles were obtained in a subset of the tumors. Results: Genomic imbalances, in most tumors affecting large regions of the genome, were found in 90% of the cases. Several apparently distinctive aberrations affecting conventional central and peripheral tumors, respectively, were identified. Although rare, recurrent amplifications were found at 8q24.21-q24.22 and 11q22.1-q22.3, and homozygous deletions of loci previously implicated in chondrosarcoma development affected the CDKN2A, EXT1, and EXT2 genes. The chromosomal imbalances in two distinct groups of predominantly near-haploid and near-triploid tumors, respectively, support the notion that polyploidization of an initially hyperhaploid/hypodiploid cell population is a common mechanism of chondrosarcoma progression. Increasing patient age as well as tumor grade were associated with adverse outcome, but no copy number imbalance affected metastasis development or tumor-associated death. Conclusion: Despite similarities in the overall genomic patterns, the present findings suggest that some regions are specifically altered in conventional central and peripheral tumors, respectively.

Expression of myeloid-specific genes in childhood acute lymphoblastic leukemia - A cDNA array study

Several specific cytogenetic changes are known to be associated with childhood acute lymphoblastic leukemia (ALL), and many of them are important prognostic factors for the disease. Little is known, however, about the changes in gene expression in ALL. Recently, the development of cDNA array technology has enabled the study of expression of hundreds to thousands of genes in a single experiment. We used the cDNA array method to study the gene expression profiles of 17 children with precursor-B ALL. Normal B cells from adenoids were used as reference material. We discuss the 25 genes that were most over-expressed compared to the reference. These included four genes that are normally expressed only in the myeloid lineages of the hematopoietic cells: RNASE2, GCSFR, PRTN3 and CLC. We also detected over-expression of S100A12, expressed in nerve cells but also in myeloid cells. In addition to the myeloid-specific genes, other over-expressed genes included AML1, LCP2 and FGF6. In conclusion, our study revealed novel information about gene expression in childhood ALL. The data obtained may contribute to further studies of the pathogenesis and prognosis of childhood ALL.

Array comparative genomic hybridization reveals frequent alterations of G1/S checkpoint genes in undifferentiated pleomorphic sarcoma of bone.

Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare tumor often difficult to differentiate from fibrosarcoma of bone (FSb), diagnostically. We applied array comparative genomic hybridization (array CGH) to screen for genes with potential importance in the tumor and compared the results with alterations seen in FSb. Twenty‐two fresh frozen tissue specimens from 20 patients (18 primary tumors and 4 local recurrences) with UPSb were studied. DNA was isolated and hybridized onto Agilent 244K CGH oligoarrays. The hybridization data were analyzed using Agilent DNA Analytics Software. The number of changes ranged from 2 to 168 (average = 66). Losses were most frequently seen at 8p, 9p, 10, 13q, and 18q, and gains at 4q, 5p, 6p, 7p, 8q, 12p, 14q, 17q, 19p, 20q, 22q, and X. Homozygous deletions of CDKN2A, RB1, TP53, and ING1 were seen in 8/20, 7/20, 3/20, and 2/20 cases, respectively. Hypermethylation of both p16INK4a and p14ARF was found in two cases with loss at CDKN2A. Inactivation either of CDKN2A, RB1, or TP53 was detected in 18/20 cases. One case showed high level gains of CDK4 and MDM2. Frequent gains were seen at MYC, PDGFRA, KIT, and KDR. Immunohistochemical positivity of KIT, PDGFRA, KDR, and PDGFRB was found in 8/14, 5/14, 4/14, and 4/14 cases, respectively. The regions most significantly discriminating between UPSb and FSb included RB1 and MYC. No homozygous deletions of RB1 were found in FSb. In conclusion, our analysis showed the disruption of G1/S checkpoint regulation to be crucial for the oncogenesis of UPSb.

An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma

BackgroundXenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers.MethodMicroarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods.ResultsThe most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers.ConclusionIn the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

Frequent deletion of CDKN2A and recurrent coamplification of KIT, PDGFRA, and KDR in fibrosarcoma of bone--an array comparative genomic hybridization study.

Very little is known about the genetics of fibrosarcoma (FS) of bone. We applied array comparative genomic hybridization (CGH) to identify genes and genomic regions with potential role in the pathogenesis of this tumor. Seventeen patients with FS of bone were included in the study. Array CGH analysis was carried out in 13 fresh frozen tissue specimens from 11 of these patients (nine primary tumors and four local recurrences). DNA was extracted and hybridizations were performed on Agilent 244K CGH oligoarrays. The data were analyzed using Agilent DNA Analytics Software. The number of changes per patient ranged from 0 to 132 (average = 43). Losses were most commonly detected at 6q, 8p, 9p, 10, 13q, and 20p. CDKN2A was homozygously deleted in 7/11 patients. Hypermethylation of both p16INK4a and p14ARF was found in 1/14 patients. An internal deletion of STARD13 was found in a region with common losses at 13q13.1. The most frequent gains were seen at 1q, 4q, 5p, 8q, 12p, 15q, 16q, 17q, 20q, 22q, and Xp. Single recurrent high level amplification was detected at 4q12, including KIT, PDGFRA, and KDR. No activating mutations were found in any of them. Immunohistochemistry revealed expression of PDGFRA and/or PDGFRB in 12/17 samples. Moreover, small regions of gains pinpointed genes of particular interest, such as IGF1R at 15q26.3 and CHD1L at 1q21.1. In conclusion, our analysis provided novel findings that can be exploited when searching for markers for diagnosis and prognosis, and targets of therapy in this tumor type.

Homozygous deletions of cadherin genes in chondrosarcoma-an array comparative genomic hybridization study.

Chondrosarcoma is a malignant bone tumor that is often resistant to chemotherapy and radiotherapy. We applied high resolution oligonucleotide array comparative genomic hybridization to 46 tumor specimens from 44 patients with chondrosarcoma and identified several genes with potential importance for the development of chondrosarcoma. Several homozygous deletions were detected. The tumor suppressor genes CDKN2A and MTAP were each homozygously deleted in four of the cases, and the RB1 gene was homozygously deleted in one. Two homozygous deletions of MTAP did not affect CDKN2A. Deletions were also found to affect genes of the cadherin family, including CDH4 and CDH7, each of which had a targeted homozygous loss in one case, and CDH19, which had a targeted homozygous loss in two cases. Loss of the EXT1 and EXT2 genes was uncommon; EXT1 was homozygously deleted in none and EXT2 in two of the cases, and large heterozygous losses including EXT1 and/or EXT2 were seen in three cases. Targeted gains and amplifications affected the MYC, E2F3, CDK6, PDGFRA, KIT, and PDGFD genes in one case each. The data indicate that chondrosarcomas develop through a combination of genomic imbalances that often affect the RB1 signaling pathway. The inactivation of cadherin genes may also be critical in the pathogenesis of the tumor.

Chapter 28 – Cytogenetic and molecular genetic alterations in bone tumors

The knowledge about the genetics of bone tumors has considerably expanded in the past two decades, including discovery of a number of tumor-specific genetic alterations. The studies have provided novel biomarkers for bone tumor diagnostics, as well as candidate targets for therapies. This review gives a comprehensive overview about genetic alterations in both benign and malignant bone tumors, covering nearly all bone tumor entities and lacking only a few rarest ones with extremely scarce data. In addition, the most important techniques used in the diagnostics and research of bone tumors are presented.