Microarray-based Genomic Profiling Research Articles

Superior Identification of Prognostic Relevant Copy Number Abnormalities By SNP-Based Genomic Arrays As Compared to Interphase FISH in Multiple Myeloma

Multiple myeloma (MM) is a neoplasm that exhibits a broad heterogeneity in both biological behavior and clinical presentation. Specific copy number abnormalities (CNAs) such as hyperdiploidy, 1p loss, 1q gain, 13q loss and 17p loss (including the TP53 gene), and IGH translocations, such as t(4;14)(p16;q32) and t(14;16)(q32;q23), provide important information regarding prognosis and treatment response.Interphase fluorescence in situ hybridization (FISH) on enriched plasma cells, currently used in clinical diagnostics of MM, is a targeted test aimed at specific genomic loci. However, it is laborious and provides only genetic information of the probe targets. Microarray-based genomic profiling is a high-resolution tool that enables genome-wide analyses for copy number alterations (CNA), including focal CNA (<5 Mb) and regions of copy neutral loss of heterozogosity (CNLOH) that cannot be identified by FISH. A limitation of SNP-based array is its inability to identify balanced translocations.The aim of this study was to compare FISH with SNP-based genomic arrays with respect to the detection yield for prognostic relevant genetic copy number abnormalities in enriched plasma cell samples from MM patients. In addition we have set up a diagnostic work flow in which on one sample of enriched plasma cells interphase FISH for (balanced) IGH translocations as well as SNP-based array for identification of CNA can be performed.SNP-based genomic array profiling and FISH were performed in 37 MM patients. After enrichment of CD138 plasma cells half of each sample was treated with 0.075M KCl and, subsequently, fixed with 3:1 methanol/acetic acid and transferred to a microscopic slide for subsequent FISH. From the remaining part of the CD138-enriched plasma cell fraction DNA was extracted to perform SNP-based genomic array. Interphase FISH was performed according to standard methods using the following probes D5S23/D5S721/CEP9/CEP15, LSI13 (13q14), LSI TP53 (17p13.1) (all from Abbott Molecular, USA), and CDKN2C/CKS1B (from Cytocell, UK). 200 nuclei were analyzed by two different investigators and the detection limit was set at 20% as proposed by the EMN (Ross et al 2012; Haematologica 97:1272-1277). SNP-based array was performed using the CytoScan HD array platform (Affymetrix, USA), using the interpretation criteria as proposed by Schoumans (Schoumans et al 2016; Genes Chromosomes Cancer 55:480-491). Data regarding FISH and SNP-based array were obtained in a fully blinded fashion.All prognostic relevant CNA as observed by FISH were also identified when only SNP-based genomic arrays would have been performed, including 4 cases with loss of 1p, 19 cases with gain of 1q, 14 cases with loss of 13q, 4 cases with loss of 17p, and 20 cases with a hyperdiploid karyotype. However, SNP-based arrays identified 20 additional prognostic relevant abnormalities which were not observed by interphase FISH for several reasons. Due to a higher detection limit of the applied SNP-based array platform, 2 cases with loss of 17p (abnormality present in 15-20% of the cells) and 1 case with loss of 13q and a hyperdiploid karyotype (present in 15% of the cells) were observed by SNP-array only. Four cases showed a 1p21 or 1p16 loss, which were not observed by FISH since these deleted regions were outside the 1p32 probe target region. In 3 cases tumor-associated regions of CNLOH were observed involving the regions 13q and 17p. Finally, in 4 cases in which FISH was suggestive for a hyperdiploid karyotype, the SNP-based array information regarding whole genome analysis and allele frequencies demonstrated that these 4 cases appeared to have a doubled up DNA content in their plasma cells, and therefore the losses of 1p, 13q and 17p were not observed by interphase FISH.In conclusion, we demonstrate that SNP-based arrays are superior in the identification of prognostic relevant CNA in MM. SNP-based array do identify all CNA as observed by FISH, and in addition, identifies additional prognostic relevant abnormalities, such as loss of 1p, 13q, and 17p, that escaped the detection by FISH. The prognostic relevance of the CNLOH and the loss of 1p21 and 1p16 regions requires further evaluation in prospective clinical trials. DisclosuresZweegman:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding.

Microarray-based genomic profiling and in situ hybridization on fibrotic bone marrow biopsies for the identification of numerical chromosomal abnormalities in myelodysplastic syndrome.

BackgroundMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematological malignancies. In MDS patients with a fibrotic bone marrow the aspiration of cells often fails (dry-tap), which hampers standard karyotyping. Obtaining genetic data from these fibrotic marrows is therefore challenging, and up till now in situ hybridization applied to bone marrow biopsies is the only option. The microarray-based genomic profiling technology has already proven its value for bone marrow aspirates and peripheral blood samples, but has never been applied to the technically challenging bone marrow biopsies. We describe an approach for microarray-based genomic profiling on bone marrow biopsies and demonstrate its ability to obtain clinically relevant cytogenetic aberrations. In addition the data were compared with those obtained by in situ hybridization and karyotyping.ResultsWe have evaluated the success rate of microarray-based genomic profiling by studying twenty-one bone marrow biopsies (7 fibrotic MDS, 12 non-fibrotic MDS and 2 reactive), by microarray-based genomic profiling and in situ hybridization (12 of 21 cases). The data obtained with these techniques were compared with conventional karyotyping data on corresponding bone marrow aspirates. Of the 15 copy number aberrations that were detected by in situ hybridization, 13 were concordant with microarray-based genomic profiling and karyotyping, whereas two hybridizations were misinterpreted. In 20 of 21 patients, the data obtained by microarray-based genomic profiling and karyotyping were identical or differences could be explained by the presence of marker chromosomes, complex karyotypes, clonal heterogeneity or disease progression.ConclusionsWe demonstrate that genome wide microarray-based genomic profiling performed on bone marrow biopsies has a similar success rate compared to in situ hybridization, and prevents misinterpretation of chromosomal losses as observed by FISH. In addition, equal to even higher resolutions were obtained with genomic profiling compared to conventional karyotyping. Our findings indicate that microarray-based profiling, even on bone marrow biopsies, is a valid approach for the identification of genetic abnormalities. This is a valuable substitution in cases of fibrotic MDS lacking cytogenetic results.

Molecular basis of chronic lymphocytic leukemia diagnosis and prognosis.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults and is characterized by a clonal accumulation of mature apoptosis-resistant neoplastic cells. It is also a heterogeneous disease with a variable clinical outcome. Here, we present a review of currently known (epi)genetic alterations that are related to the etiology, progression and chemo-refractoriness of CLL. Relevant literature was identified through a PubMed search (1994-2014) of English-language papers using the terms CLL, signaling pathway, cytogenetic abnormality, somatic mutation, epigenetic alteration and micro-RNA. CLL is characterized by the presence of gross chromosomal abnormalities, epigenetic alterations, micro-RNA expression alterations, immunoglobulin heavy chain gene mutations and other genetic lesions. The expression of unmutated immunoglobulin heavy chain variable region (IGHV) genes, ZAP-70 and CD38 proteins, the occurrence of chromosomal abnormalities such as 17p and 11q deletions and mutations of the NOTCH1, SF3B1 and BIRC3 genes have been associated with a poor prognosis. In addition, mutations in tumor suppressor genes, such as TP53 and ATM, have been associated with refractoriness to conventional chemotherapeutic agents. Micro-RNA expression alterations and aberrant methylation patterns in genes that are specifically deregulated in CLL, including the BCL-2, TCL1 and ZAP-70 genes, have also been encountered and linked to distinct clinical parameters. Specific chromosomal abnormalities and gene mutations may serve as diagnostic and prognostic indicators for disease progression and survival. The identification of these anomalies by state-of-the-art molecular (cyto)genetic techniques such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), single nucleotide polymorphism (SNP) microarray-based genomic profiling and next-generation sequencing (NGS) can be of paramount help for the clinical management of these patients, including optimal treatment design. The efficacy of novel therapeutics should to be tested according to the presence of these molecular lesions in CLL patients.

Identification of prognostic relevant chromosomal abnormalities in chronic lymphocytic leukemia using microarray-based genomic profiling

BackgroundCharacteristic genomic abnormalities in patients with B cell chronic lymphocytic leukemia (CLL) have been shown to provide important prognostic information. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA), currently used in clinical diagnostics of CLL, are targeted tests aimed at specific genomic loci. Microarray-based genomic profiling is a new high-resolution tool that enables genome-wide analyses. The aim of this study was to compare two recently launched genomic microarray platforms, i.e., the CytoScan HD Array (Affymetrix) and the HumanOmniExpress Array (Illumina), with FISH and MLPA to ascertain whether these latter tests can be replaced by either one of the microarray platforms in a clinical diagnostic setting.ResultMicroarray-based genomic profiling and FISH were performed in all 28 CLL patients. For an unbiased comparison of the performance of both microarray platforms 9 patients were evaluated on both platforms, resulting in the identification of exactly identical genomic aberrations. To evaluate the detection limit of the microarray platforms we included 7 patients in which the genomic abnormalities were present in a relatively low percentage of the cells (range 5-28%) as previously determined by FISH. We found that both microarray platforms allowed the detection of copy number abnormalities present in as few as 16% of the cells. In addition, we found that microarray-based genomic profiling allowed the identification of genomic abnormalities that could not be detected by FISH and/or MLPA, including a focal TP53 loss and copy neutral losses of heterozygosity of chromosome 17p.ConclusionFrom our results we conclude that although the microarray platforms exhibit a somewhat lower limit of detection compared to FISH, they still allow the detection of copy number abnormalities present in as few as 16% of the cells. By applying similar interpretation criteria, the results obtained from both platforms were comparable. In addition, we conclude that both microarray platforms allow the identification of additional potential prognostic relevant abnormalities such as focal TP53 deletions and copy neutral losses of heterozygosity of chromosome 17p, which would have remained undetected by FISH or MLPA. The prognostic relevance of these novel genomic alterations requires further evaluation in prospective clinical trials.

Identification Of Chromosomal Abnormalities By Microarray-Based Genomic Profiling As Compared To Karyotyping In Patients With Low and Intermediate-1 Risk Myelodysplastic Syndromes Within a Prospective Clinical Trial: A Study On Behalf Of The HOVON89 Study Group

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic disorders, characterized by ineffective hematopoiesis resulting in cytopenias and a highly variable clinical course. Cytogenetics are routinely used as one of the diagnostic, prognostic and therapeutic markers in the clinical management of MDS (Greenberg et al. Blood 120:2454,2012). Although karyotyping is generally considered as the gold standard in the cytogenetic characterization of MDS, 40-60% of the patients exhibit a normal karyotype. Intrinsically, the resolution of karyotyping is limited by its capacity to detect only those copy number changes that are microscopically visible (5-10 Mb in size). In contrast, microarray-based genomic profiling analyses allow a genome-wide detection of copy number alterations (CNAs), down to 100 kb in size, and regions of copy neutral loss of heterozygosity (CNLOH). Such analyses also overcome some of the other major limitations of karyotyping such as low success rate due to inadequate metaphase yield and/or poor banding quality. We have compared karyotyping and fluorescence in situ hybridization (FISH) with microarray-based genomic profiling with respect to the detection yield for genetic abnormalities in bone marrow samples from lower risk MDS patients.We used the HOVON89 study-cohort, a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low/intermediate-1 risk MDS; www.trialregister.nl; NTR1825; EudraCT nr.: 2008-002195-10. Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). Data regarding cytogenetics, FISH and microarray were obtained in a fully blinded fashion for 68 MDS patients. For microarray-based genomic profiling we used the recently launched high resolution CytoScan HD Array (Affymetrix) platform. The following interpretation criteria were applied: (i) the threshold for CNAs was set at >5 Mb, (ii) inclusion of <5 Mb CNAs segments that coincide with known cancer genes as reported on www.sanger.ac.uk and (iii) the threshold for CNLOH was set at >10 Mb and to telomere. Karyotyping and interphase FISH were performed using standard cytogenetic methods. In all 4 patients where karyotyping revealed no metaphases interphase FISH was performed.Thirty-six of the 68 (53%) MDS patients had an abnormal microarray profile. Of interest, in 13 of these 36 patients no abnormalities were observed by karyotyping and/or FISH. All these abnormalities observed by microarray only, involved focal <5 Mb CNAs (containing e.g. the TET2, RUNX1 and DNMT3A genes) and regions of CNLOH (coinciding with 1p, 2p, 4q, 7q, 11p, 11q, 12q, 14q and 18q), which are all out of the scope of karyotyping and FISH. All CNAs identified by karyotyping and FISH were also observed by microarray-based genomic profiling, including a case with loss of 5q in 5% of the cells and loss of 7q in 9% of the cells as observed by interphase FISH and another case with loss of 5q in 3 of 20 the analyzed metaphases by karyotyping. These observations demonstrate the high sensitivity of the CytoScan HD array platform for the identification of CNAs in (small) subclones. As expected balanced translocations such as t(3;3)(q21;q26) and t(2;14)(q37;q22) present in 2 of the patients in our cohort were not identified by microarray-based genomic profiling. This study will be extended to all patients to be included in the HOVON89 trial.In conclusion, we demonstrate that in the present cohort of 68 patients, microarray-based genomic profiling allows the identification of almost all copy number abnormalities also observed by karyotyping and FISH. In addition, we show that microarray-based genomic profiling allows the detection of potential prognostic relevant abnormalities (focal CNAs and CNLOH) which would have remained undetected by karyotyping and FISH. The predictive and/or prognostic value of these novel CNAs and CNLOH will be evaluated within the ongoing prospective clinical HOVON89 trial in lower risk MDS. Disclosures:No relevant conflicts of interest to declare.

Validation Of a Multi-Diagnostic Approach Including Flow Cytometry To Identify Specific Risk Categories Within Low and Intermediate-1 Risk Myelodysplastic Syndromes Within a Prospective Clinical Trial: A Study On Behalf Of The HOVON89 Study Group

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of hematopoietic stem cell disorders, characterized by ineffective hematopoiesis resulting in cytopenias and variable risk of acute myeloid leukemia (AML). To make an accurate distinction between specific risk categories in MDS, especially in low and intermediate risk MDS, a multi-diagnostic approach is recommended. To verify the efficacy of multiple diagnostic tools in MDS we used the HOVON89 study-cohort (a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low-intermediate-1 risk MDS; trial registered at www.trialregister.nl as NTR1825; EudraCT nr.: 2008-002195-10.Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). We collect data on cytomorphology (CM), conventional cytogenetics (CCG), fluorescence in situ hybridization (FISH) and microarray-based genomic profiling. In addition, we performed flow cytometric (FC) analysis according to European LeukemiaNet guidelines (Van de Loosdrecht et al., Haematologica 2009 and Leukemia 2012). Current CM results (N=98) identified: 8 refractory anemia (RA); 16 refractory anemia with ringed sideroblasts (RARS); 43 refractory cytopenia with multilineage dysplasia with/without ringed sideroblasts (RCMD/RCMD-RS); 16 refractory anemia with excess blast-1 (RAEB-1), 5 chronic myelomonocytic leukemia-1 (CMML-1) and 10 patients with isolated del(5q). CCG analysis (N=101) indicated 2 very good risk, 84 good risk, 13 intermediate risk and 2 poor risk patients according to the IPSS-R risk categories (Greenberg et al., Blood 2012). In addition, interphase FISH analysis (N=72) was normal in 15 patients, in 6 patients the del(5q) was confirmed. From 68 MDS patients data from both CCG and microarray were available. Microarray-based genomic profiling identified genomic abnormalities such as copy neutral loss of heterozygosity and small (<5 Mb) copy number alterations coinciding with a cancer gene in 13 patients with normal CCG. As expected in one patient the balanced translocation t(3;3)(q21;q26) was not identified by microarray-based genomic profiling.FC analysis (N=82) evaluated aberrancies with regard to count, marker expression level and lineage infidelity marker expression on myeloid progenitors, B cell progenitors, maturing myeloid/monocytic and erythroid cells. Current, validated FC-scoring systems identified 60/81 (Della Porta et al., Haematologica 2012) and 61/81 (Wells et al., Blood 2003) patients with MDS. Both scoring systems do not evaluate dyserytropoiesis and dysmegakaropoiesis, thereby possibly not recognizing RA, RARS or RCMD patients with only dyserytropoiesis and dysmegakaropoiesis. We integrated both scoring systems into one score (Van de Loosdrecht et al., Leukemia 2012, and JNCCN 2013). Patients were divided into 3 categories: not likely MDS (A), signs of dysmyelopoiesis (B) and fitting MDS (C). This score diagnosed 69/78 patients as probably or likely MDS by FC (B or C, figure 1). Remarkably, patients scored as ‘category A' only displayed dyserytropoiesis and/or dysmegakaryopoiesis by CM. FC identified dyserytropoiesis in these patients, however, a consensus erytroid flow score is not yet validated. In addition, FC identified different risk categories within the patient group with no genetic abnormalities (based on CCG, FISH and microarray-based genomic profiling; data not shown). [Display omitted] In conclusion, this is the first prospective study in low/int-1 risk MDS that validates FC as a valuable diagnostic tool in MDS (sensitivity of FC in this cohort: 88%). FC only failed to recognize some patients with only dyserytropoiesis and dysmegakaryopoiesis by CM, not evaluated by the current scoring system. Thirteen patients with unilineage dysplasia by CM had multilineage dysplasia by FC. We postulate that RA/RARS patients with multi-lineage dysplasia by FC may have clinical features of RCMD patients and therefore a higher risk on transformation to AML. Clinical follow-up data are expected within 1.5 year.In near future, a multi-diagnostic approach may i) identify risk categories within well defined IPSS-R subgroups, ii) predict risk on transformation and iii) select patients who might benefit from new emerging drugs for low-int-1 risk MDS. Disclosures:No relevant conflicts of interest to declare.

Performance of a prognostic genomic signature for early-stage NSCLC in matched fresh frozen and RNA-stabilized tissue.

7532 Background: Recent clinical studies have demonstrated the benefit of adjuvant chemotherapy (ACT) in some early-stage non-small cell lung cancer (NSCLC) patients. A 15-gene signature, developed using fresh frozen (FF) tissue, has been shown to be an independent prognostic marker that identifies high risk patients who may benefit from ACT. Use of this signature in tissue preserved in an RNA stabilization reagent is desired for easier access to tumor tissue in the clinical setting. Methods: Matched FF and RNAlater-preserved (RNAL) tissues were obtained from 43 NSCLC patients. Patients provided written consent for the collection of tumor tissue at the time of surgery under an IRB approved protocol. Each tissue sample was split into 2 pieces, creating biological replicates for each tissue format. For each patient, RNA was extracted from 4 tissue pieces (2 FF, 2 RNAL), followed by microarray-based genomic profiling (Affymetrix U133 Plus 2.0). The 15-gene signature was applied to each profile, generating a numerical risk score and a risk category (high, low) using methods previously established (Zhu 2010 J Clin Oncol). The level of agreement was evaluated within biological replicates of each tissue format, as well as between the averaged biological replicates of matched FF and RNAL tissues. Results: The concordance in risk category between averaged biological replicates of matched FF and RNAL tissues was 84%, with a Pearson correlation of 0.74. This level of agreement is comparable to the inherent reproducibility of the assay observed within biological replicates of FF tissue, which demonstrated concordance of 79% and Pearson correlation of 0.83. In addition, a statistical in silico simulation was used to demonstrate that if the risk scores in this study had spanned the full dynamic range of the assay while maintaining the same level of inherent reproducibility observed in the current study, the level of concordance would be 89% with a Pearson correlation of 0.93. Conclusions: The level of agreement between matched FF and RNAL tissues is not inferior to that seen within FF biological replicates. Therefore, the 15-gene signature maintains its performance when used in RNAlater-preserved NSCLC tissues.

Microarray-Based Genomic Profiling Facilitates Genetic Subgrouping and Detects Critical Submicroscopic Aberrations Associated with Prognosis in Pediatric Acute Lymphoblastic Leukemia (ALL)

Abstract 1444 Objective:Risk-adapted therapeutic categories in acute lymphoblastic leukemia (ALL) take into account several key parameters, including cytogenetics. Because accurate conventional chromosome (CC) studies in ALL are hampered by low mitotic indexes and poor chromosome morphology, fluorescence in situ hybridization (FISH) and other molecular methods such as RT-PCR are currently used to complement karyotyping. We evaluated the contribution of oligo/SNP microarrays for providing additive genetic information in ALL that is not obtained by karyotype studies. Methods:Specimens from 24 children and young adults (12 M;12 F), including 3 patients with Down syndrome (DS), were processed for pre-B ALL cytogenetics work-up plus SNP/Oligo microarray. The median age was 4 y (range, 2–21 y); 23 patients had a pre-B-cell immunophenotype. Unstimulated CC (n=23) and pre-B ALL FISH studies (n=20) were performed using standard protocols. Genomic DNA was extracted from the residual bone marrow samples and processed for genome-wide copy number analyses on the Cytoscan HD oligo/SNP microarray (Affymetrix). Results:CC detected abnormalities that allowed prognostic subgrouping in 19 (83%) of 23 patients tested; the 24th patient was not tested with CC but showed an ETV6-RUNX1 fusion on FISH. Microarray genomic profiling allowed genetic subgrouping in the 4 cases with suboptimal or non-informative CC results. Overall, microarray detected a median of 5 additional copy number aberrations (CNAs) per patient (range, 1–27), including 18 additional CNAs in a T-cell ALL patient with only deletion 9p detected by CC and FISH. The 4 most common deletions detected by array involved CDKN2A (n=10, including 4 biallelic deletions) and ETV6, SESN1/6q16.1, and IKZF1 (6 cases each); sporadic deletions involved genes affecting B-cell development, cell cycle progression, DNA repair, and tumor progression were also seen. Five of the 7 patients with ETV6-RUNX1 translocation also showed deletions or disruptions at or near these 2 loci, suggesting the presence of the “cryptic” t(12;21). No balanced translocations were detected. Clonal diversity was easily detectable by microarray; however, a case with 64 chromosomes and a case with both 2n and 4n clones were difficult to interpret. At least 1 extended area of copy neutral loss-of heterozygosity (>5 Mb) was seen in 8/24 (33%) cases, including a 17q region that encompassed IKZF3; however, in most cases the significance of these CN-LOH changes was not clear. Significant “high risk” prognostic alterations identified by array but not detected by CC included 3 CRLF2-rearragements (found in 2 of the 3 DS patients) and disruption of the IKZF1 locus (6 patients). IKZF1 deletions were detected in a 5-y-old DS-ALL patient with CRLF2-P2PY8, a 20-y-old DS-ALL patient with high hyperdiploidy, an 18-y-old patients with IGH-CRLF2 confirmed by FISH (CC failed), another 18-y-old patient with a normal karyotype, and 1 patient each with iAMP(21) and dic(9;20) ALL. Conclusion:Submicroscopic IKZF1 deletions have been associated with drug resistance and a high risk of treatment failure in ALL, signifying critically important prognostic information needed for clinical management. Accordingly, OligoSNP arrays provide a comprehensive approach for accurately identifying clinically significant abnormalities in ALL that may be missed by routine chromosome study and targeted FISH panels alone. Array testing is a highly sensitive complementary molecular cytogenetic assay that should be offered to newly-diagnosed ALL patients, especially when CC is non-informative, to facilitate genetic subgrouping and define tumor markers that may help monitor a patient's clinical course. Disclosures:No relevant conflicts of interest to declare.

High detection rate of clinically relevant genomic abnormalities in plasma cells enriched from patients with multiple myeloma

Multiple myeloma is a heterogeneous disease, which is characterized by the occurrence of specific genomic abnormalities that are both of diagnostic and prognostic relevance. Since the detection of these abnormalities through molecular-genetic techniques is hampered by the overall low percentage of plasma cells present in primary bone marrow aspirates, we assessed the efficacy of these techniques in enriched plasma cell fractions from 61 multiple myeloma patients. Using interphase FISH, genomic abnormalities could be detected in 96% of the enriched samples as compared to 61% in the cultured whole bone marrow samples. We also found that microarray-based genomic profiling of enriched plasma samples facilitates the detection of additional, possibly clinically relevant, genomic abnormalities. We conclude that the genomic delineation of enriched plasma cells from multiple myeloma patients results in a significantly increased detection rate of clinically relevant genomic abnormalities. In order to facilitate molecular-genetic data interpretation, we recommend morphological assessment of plasma cell purity after enrichment.

Microarray‐based genomic profiling as a diagnostic tool in acute lymphoblastic leukemia

In acute lymphoblastic leukemia (ALL) specific genomic abnormalities provide important clinical information. In most routine clinical diagnostic laboratories conventional karyotyping, in conjunction with targeted screens using e.g., fluorescence in situ hybridization (FISH), is currently considered as the gold standard to detect such aberrations. Conventional karyotyping, however, is limited in its resolution and yield, thus hampering the genetic diagnosis of ALL. We explored whether microarray-based genomic profiling would be feasible as an alternative strategy in a routine clinical diagnostic setting. To this end, we compared conventional karyotypes with microarray-deduced copy number aberration (CNA) karyotypes in 60 ALL cases. Microarray-based genomic profiling resulted in a CNA detection rate of 90%, whereas for conventional karyotyping this was 61%. In addition, many small (< 5 Mb) genetic lesions were encountered, frequently harboring clinically relevant ALL-related genes such as CDKN2A/B, ETV6, PAX5, and IKZF1. From our data we conclude that microarray-based genomic profiling serves as a robust tool in the genetic diagnosis of ALL, outreaching conventional karyotyping in CNA detection both in terms of sensitivity and specificity. We also propose a practical workflow for a comprehensive and objective interpretation of CNAs obtained through microarray-based genomic profiling, thereby facilitating its application in a routine clinical diagnostic setting.

Microarray-Based Genomic DNA Profiling Technologies in Clinical Molecular Diagnostics

Microarray-based genomic DNA profiling (MGDP) technologies are rapidly moving from translational research to clinical diagnostics and have revolutionized medical practices. Such technologies have shown great advantages in detecting genomic imbalances associated with genomic disorders and single-gene diseases. We discuss the development and applications of the major array platforms that are being used in both academic and commercial laboratories. Although no standardized platform is expected to emerge soon, comprehensive oligonucleotide microarray platforms-both comparative genomic hybridization arrays and genotyping hybrid arrays-are rapidly becoming the methods of choice for their demonstrated analytical validity in detecting genomic imbalances, for their flexibility in incorporating customized designs and updates, and for the advantage of being easily manufactured. Copy number variants (CNVs), the form of genomic deletions/duplications detected through MGDP, are a common etiology for a variety of clinical phenotypes. The widespread distribution of CNVs poses great challenges in interpretation. A broad survey of CNVs in the healthy population, combined with the data accumulated from the patient population in clinical laboratories, will provide a better understanding of the nature of CNVs and enhance the power of identifying genetic risk factors for medical conditions. MGDP technologies for molecular diagnostics are still at an early stage but are rapidly evolving. We are in the process of extensive clinical validation and utility evaluation of different array designs and technical platforms. CNVs of currently unknown importance will be a rich source of novel discoveries.

Microarray-based comparative genomic profiling of reference strains and selected Canadian field isolates of Actinobacillus pleuropneumoniae

BackgroundActinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines.ResultsUsing hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 A. pleuropneumoniae L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements.ConclusionAlthough A. pleuropneumoniae strains are essentially clonal, M-CGH analysis of the reference strains of the fifteen serovars and representative field isolates revealed several classes of genes that were divergent or absent. Not surprisingly, these included genes associated with capsule biosynthesis as the capsule is associated with sero-specificity. Several of the conserved genes were identified as candidates for vaccine development, and we conclude that M-CGH is a valuable tool for reverse vaccinology.

Simultaneously detection of genomic and expression alterations in prostate cancer using cDNA microarray

Prostate cancer is a common disease among men but the knowledge of the prostate carcinogenesis is still limited. cDNA microarray-based comparative genomic hybridization (CGH) and expression profiling were performed to screen the genomic and the expression changes in prostate cancer respectively. The two data were integrated to study the influence of genomic aberrations on gene expression and seek for the genes with their expression affected by the genomic aberrations. Real-time PCR was performed to evaluate the array data. Array-based CGH detected gains at 2q, 3p/q, 5q, 6q, 8q, 9p, 10p/q, 11q, 12p, 14q, and 19p/q and losses at 1p, 2p, 4q, 6p/q, 7p, 11p/q, 12q, 17p/q, 19p/q, and Xp/q in more than 20% prostate tumors and narrowed these aberrations. For example, the gain of 8q was mapped to five minimal regions. Novel aberrations were also identified, such as loss at Xq21.33-q22.2. Expression profiling discovered the significant biological processes involved in the prostate carcinogenesis, such as exogenous antigen presentation via MHC class II and protein ubiquitination. Integration analysis revealed a weak positive correlation between genomic copy number and gene expression level. Fifty-three genes showed their expression directly affected by the genomic aberrations possibly, including more than one member of Ras superfamily and major histocompatibility complex (MHC). These genes are involved in multiple biological processes. Integration of the CGH and expression data provided more information than separate analysis. Although the direct influence of genomic aberrations on gene expression seems weak, the influence can be extended by indirect regulation through a few directly affected genes. Because the influence can be persistent, the genes directly affected by the genomic aberrations may play key roles in the prostate carcinogenesis and are worth further analysis.

Identification of genes with correlated patterns of variations in DNA copy number and gene expression level in gastric cancer

To identify DNA copy number changes that had a direct influence on mRNA expression in gastric cancer, cDNA microarray-based comparative genomic hybridization (aCGH) and gene expression profiling were performed using 17 K cDNA microarrays. A set of 158 genes showing Pearson correlation coefficients over 0.6 between DNA copy number changes and mRNA expression level variations was selected. In an independent gene expression profiling of 60 tissue samples, the 158 genes were able to distinguish most of the normal and tumor tissues in an unsupervised hierarchical clustering, suggesting that the differential expression patterns displayed by this specific group of genes are most likely based on the gene copy number changes. Furthermore, 43 statistically significant ( P < 0.01) genes were selected that correctly distinguished all of the tissue samples. The copy number changes detected by aCGH can be verified by fluorescence in situ hybridization and real-time polymerase chain reaction. The selected genes include those that were previously identified as being tumor suppressors or deleted in various tumors, including GATA binding protein 4 (GATA4), monoamine oxidase A (MAOA), cyclin C (CCNC), and oncogenes including malignant fibrous histiocytoma amplified sequence 1 (MFHAS1/MASL1), high mobility group AT-hook 2 (HMGA2), PPAR binding protein (PPARBP), growth factor receptor-bound protein 7 (GRB7), and TBC1 (tre-2, BUB2, cdc16) domain family, member 1 (TBC1D1).