Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Primary Central Nervous System Lymphomas. A validation study of array-based Comparative Genomic Hybridization in Formalin-Fixed Paraffin-Embedded Tumor Specimens

ABSTRACT: Purpose: Only a limited number of genetic studies have been performed in primary central nervous system lymphomas (PCNSL), partly due to the rarity of the tumors and the very limited amount of available tissue. In this report, we present the first molecular characterization of copy-number abnormalities (CNA) of newly diagnosed PCNSL by array-based comparative genomic hybridization in formalin fixed paraffin embedded (FFPE) specimens and compare the results with matched frozen tumor specimens. Experimental design: We performed array-based comparative genomic hybridization (aCGH) in FFPE tissues from PCNSL. Results were compared with matched paired frozen tumors. Results: Our analysis confirmed the good to fair quality and reliability of the data generated from limited amounts of tumoral FFPE tissue. Overall, all PCNSL cases were characterized by highly complex karyotypes with a median of 23 copy-number abnormalities (CNA) per patient (range 17-47). Overall, 20 chromosomal regions were recurrently found in more than 40% of cases. Deletions of 6p21, 6q, 9p21.3 and gain of 12q12-q24.33 were the commonest CNA. Other minimal affected regions were defined, and novel recurrent CNAs affecting single genes were identified in 3q26.32 (TBL1XR1) and 8q12.1 (TOX). Conclusions: The results obtained are encouraging. Larger archival tissue collections can now be analyzed in order to complement the still fragmented knowledge we have of the genetic basis of the PCNSL. We included samples from 7 PCNSL patients. In 6 out of these 7 PCNSL patients (cases B – G), aCGH experiments were performed in DNA samples obtained from both snap-frozen and FFPE tissues for assay quality comparative purposes. In the remaining sample (case A), only frozen sample was analyzed.

ORGANISM(S): Homo sapiens

SUBMITTER: Esteban Braggio

PROVIDER: E-GEOD-28952 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lymphoplasmacytic lymphomas and marginal zone lymphomas of nodal, extra-nodal and splenic types account for 10% of non-Hodgkin lymphomas. They are similar at the cell differentiation level, sometimes making difficult to distinguish them from other indolent non-Hodgkin lymphomas. To better characterize their genetic basis, we performed array-based comparative genomic hybridization in 101 marginal zone lymphomas (46 MALT, 35 splenic and 20 nodal marginal zone lymphomas) and 13 lymphoplasmacytic lymphomas. Overall, 90.1% exhibited copy-number abnormalities. Lymphoplasmacytic lymphomas demonstrated the most complex karyotype (median=7 copy-number abnormalities), followed by MALT (4), nodal (3.5) and splenic marginal zone lymphomas (3). A comparative analysis exposed a group of copy-number abnormalities shared by several or all the entities with few disease-specific abnormalities. Gain of chromosomes 3, 12 and 18 and loss of 6q23-q24 (TNFAIP3) were identified in all entities. Losses of 13q14.3 (MIRN15A-MIRN16-1) and 17p13.3-p12 (TP53) were found in lymphoplasmacytic and splenic marginal zone lymphomas; loss of 11q21-q22 (ATM) in nodal, splenic marginal zone and lymphoplasmacytic lymphomas; loss of 7q32.1-q33 in MALT, splenic and lymphoplasmacytic lymphomas. Abnormalities affecting the NF-kB pathway were observed in 70% of MALT and lymphoplasmacytic lymphomas and 30% of splenic and nodal marginal zone lymphomas, suggesting distinct roles of this pathway in the pathogenesis/progression of these subtypes. Elucidation of the genetic alterations contributing to the pathogenesis of these lymphomas may guide to design specific therapeutic approaches. One hundred fourteen patients were included in this study: 46 MALT lymphomas (22 pulmonary, 11 salivary glands, 7 lacrimal glands and 6 gastrointestinal), 35 splenic marginal zone lymphomas, 20 nodal marginal zone lymphomas and 13 non-Waldenström’s Macroglobulinemia lymphoplasmacytic lymphomas. All cases were reviewed prior to study on paraffin sections with immunohistochemistry. Sections of each frozen tissue used for study were also reviewed by histological examination and immunohistochemistry before was submitted for the study.

Project description:In the study of tumor genetics, formalin-fixed paraffin-embedded (FFPE) tumors are the most readily available tissue samples. While DNA derived from FFPE tissue has been validated for array comparative genomic hybridization (aCGH) application, the suitability of such fragmented DNA for single-nucleotide polymorphism (SNP) array analysis has not been well examined. Furthermore, whole-genome amplification (WGA) has been used in the study of small precursor lesions to produce sufficient amount of DNA for aCGH analysis. It is unclear whether the same approach can be extended to SNP analysis. In this study, we examined the utility and limitations of genotyping platform performed on whole-genome amplified DNA from FFPE tumor samples for both copy number and SNP analyses. We analyzed the results obtained using DNA derived from matched FFPE and frozen tissue samples on Affymetrix 250K Nsp SNP array. Two widely used WGA methods, Qiagen (isothermal protocol) and Sigma (thermocycling protocol), were used to determine how WGA methods affect the results. We found that the use of DNA derived from FFPE tumors (without or with WGA) for high-resolution SNP array application can produce a significant amount of false positive and false negative findings. While some of these misinterpretations appear to cluster in genomic regions with high or low GC contents, the majority appears to occur randomly. Only large-scale chromosome LOH (>10Mb) can be reliably detected from FFPE tumor DNA samples (without or with WGA) but not smaller LOH or copy number alterations. Our findings here indicate a need for caution in SNP array data interpretation when using FFPE tumor-derived DNA, particularly with WGA. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved and FFPE mesenchymal tumor samples without or with WGA, as well as genomic snap-frozen non-neoplastic tissue DNA from 5 adult individuals to serve as reference DNA. WGA was performed using the REPLI-g® FFPE kit (Qiagen, Valencia, CA, USA) and GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA) in parallel in accordance with the manufacturers’ protocols. A two- to eight-hour individualized reaction time was used in the Qiagen platform for each sample. A gradient amount of initial DNA (10ng, 30ng, 60ng, 100ng and 150ng) was tested followed by gel electrophoresis and qualitative multiplex PCR assay to determine the quality of post-WGA products. At least four independent experiments were concurrently performed per template amplification. Four separateWGA reaction products were pooled for each sample for subsequent microarray analysis to minimize the amplification bias and allele dropout. One of the Affymetrix GeneChip® Human Mapping 500K Array Set (Nsp 250K SNP array) was used for genotyping analysis. Four gastrointestinal stromal tumors with known cytogenetic aberrations were included. Two cases were sucessfullly amplified and passed the quality tests. A total of 12 samples were compared between each other, including frozen tissue DNA (as reference), frozen tissue DNA with WGA (two platforms), FFPE tissue DNA, and FFPE tissue DNA with WGA (two platforms) from each case.

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Primary Central Nervous System Lymphomas. A validation study of array-based Comparative Genomic Hybridization in Formalin-Fixed Paraffin-Embedded Tumor Specimens

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