Detection Of Gene Copy Number Research Articles

Abstract 551: Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer

Abstract Loss of expression of the PTEN gene is associated with poorer prognosis in many solid tumors. While cfDNA-based liquid biopsy testing are gradually adopted as the routine testing method in the clinical setting, most of them are unable to reliably detect copy number loss and/or are limited reporting copy number amplification events only. Method: We have developed a capture-based CLIA-certified NGS assay, PredicineCARE, to robustly identify tumor-derived aberrations including SNVs, Indels, fusions, copy number amplifications and deletions from blood samples. Analytical validation of the assay was performed with commercial standard reference materials and contrived cell lines. Copy number loss of PTEN was also confirmed by Immunohistochemistry (IHC) assay in 15 clinical samples. Clinical utility of PTEN copy number loss was further established by its significant association with poor outcome in clinical patients with metastatic castration-resistant prostate cancer (mCRPC). Results: Analytical validation of the PredicineCARE assay demonstrated its super sensitivity and robustness of detecting homozygous copy number loss at approximately 10% tumor fraction. Both the sensitivity and Positive Prediction Value reached 100% when overall gene copy number is below 1.75 copies. When comparing to tissue IHC results reviewed by two independent pathologists, PredicineCARE liquid biopsy assay revealed 86.7% concordance with IHC in detecting PTEN loss (80% sensitivity, 100% specificity, and 100% PPV). The copy number loss was further confirmed by low-pass whole-genome sequencing of cfDNA samples. When the assay was applied to profile mCRPC patients, copy number loss of PTEN was detected at the prevalence rate of 19.2% (10/52) and 35.9% (23/64) respectively. Notably, patients harboring PTEN copy number loss showed significantly worse overall survival in the two cohorts independently (log-rank P = 0.0001 and 0.0004 respectively). Conclusion: We have developed PredicineCARE for clinical use, a highly sensitive cfDNA-based assay to detect gene copy number variants in addition to gene mutations and fusions. Its ability to detect gene copy number loss in low fraction of tumor contents as a valuable molecular biomarker holds great potential for clinical applications and patient selections in personalized precision oncology, particularly for investigational drugs targeting the AKT-PI3K-PTEN pathway. Citation Format: Xiaoxi Dong, Minghua Zhang, Tiantian Zheng, Lu Tan, Feng Xie, Amy Xiaohong Wang, Pan Du, Shidong Jia, Chris Lu, Jianjun Yu. Clinical utility of cell-free DNA based PTEN copy number loss in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 551.

Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

BackgroundCopy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs.MethodsA multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.ResultsThere were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated.ConclusionsThe developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

An investigation into gene copy number determination in transgenic yeast; The importance of selecting a reliable real-time PCR standard

Nowadays, Pichia pastoris is a well-known yeast for the production of recombinant proteins. The yield of protein production tightly depends on the copy number of the gene of interest into the host chromosome. Real-time PCR has been used as a high throughput method for molecular detection of gene copy number. In light of determining an absolute gene copy number, the reliability of the qPCR quantification standard is a major issue and it can be a potential source of errors in the final results. Since the literature on this issue is inconclusive, we set out to find a reliable quantification method that allows comparing results in different laboratories. We generated standard curves for two genomic loci (5′UTR AOX1 and ARG4) and for plasmid DNA carrying hGM-CSF coding sequence. These data was used to calculate the integrated hGM-CSFcDNA copy number in a recombinant P. pastoris clone. In our expriments the 5′UTR AOX1 gene showed a more accurate quantification standard, based on more efficient amplification and better reproducibility. The results obtained in this study showed that the differences in terms of structure and length between circular plasmid and linear gDNA could be the source of significant differences in the pattern of DNA amplification.

A model for the impact of FFPE section thickness on gene copy number measurement by FISH

Fluorescent in situ hybridization (FISH) assays to detect gene amplification such as HER2 or MET in tumors are used for prognosis evaluation and selection of targeted therapies. Although FISH guidelines recommended 4~6 μm FFPE sections, many laboratories use 2~3 μm sections, which is a common practice for H&E staining and immunohistochemistry. A former study concluded that section thickness did not affect FISH results. We found, however, that thinner FFPE sections may lead to false negative results for gene amplification. A mathematic model was constructed and cell-line based controls with known gene copy number were prepared, and the model had a reasonable fit with the experimental data. The model revealed that even when counting the apparently full-sized nuclear images, many of them have partial volumes, which leads to under-estimation of gene copy number. Therefore, improperly thinner sections are prone to give false negative results, and thicker sections give a better approximation to the true value. The discrepancy between this and the former study was discussed. In summary, the model applies generally to FISH/ISH detection of gene copy number, and section thickness is an important parameter to control for precision medicine research, assay development, clinical trials and daily practice in pathology laboratory.

Recommendations for the clinical interpretation and reporting of copy number gains using gene panel NGS analysis in routine diagnostics

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity.

Relation between mycoplasma infection and recurrent spontaneous abortion.

This study explores the possible relation between cervical infections with Ureaplasma urealyticum (UU) and Mycoplasma hominis (MH) and the occurrence of recurrent spontaneous abortion (RSA). 132 patients with RSA (observation group) and 96 normal pregnancy volunteers undergoing planned abortions (control group) were selected successively and enrolled in the investigation. Cervical secretion samples were obtained for each subject. Bacterial cultures were started to detect UU, MH and other bacterial infections, and fluorescence quantitative PCR was used to detect gene copy number in chorion and decidual tissues. Additionally, ELISA (enzyme-linked immunosorbent assay) was used to detect anticardiolipin antibody (ACA) to rate positivity of Immunoglobulin M (IgM) and IgG in secretions, and Western blot was applied to quantify the expression levels of Interleukin (IL)-6, tumor necrosis factor-α (TNF-α), prostacyclin (PGI2) and bax/bcl-2. Our results showed the UU, MH, and overall bacterial infection rate of chorionic and decidual tissues, and the gene copy number of UU, MH were higher in the observation group than those in the control group (p<0.05). Moreover, the ACA-IgM and IgG positive rates in secretions of the observation group were significantly higher than those in the control group (p<0.05). Finally, the expression levels of IL-6, TNF-α, PGI2, and bax/bcl-2 were higher than those in the control group as well (p<0.05). Our results support the notion that RSA might be associated with UU and MH infection, could influence the occurrence of other bacterial infections and could stimulate ACA expression, inflammatory response, thrombogenesis, and factors associated with cell apoptosis, increasing the risk for an abortion during pregnancy.

Use of quantitative PCR technique for determining gene copy number in the genome of Beauveria bassiana transformant

This study describes the use of quantitative PCR (qPCR) to determine gene copy number in Beauveria bassiana genome. This procedure has been improved by generating a reference strain to calibrate PCR amplification efficiency and using a γ-tubulin gene as internal control. The reference strain was constructed by introducing one copy of phosphinothricin resistance gene and two copies of green fluorescent protein gene into the wild-type strain. Finally, this improved procedure was validated via determining the gene copy number in the previously characterized transformants, and displays a great potential in rapid detection of gene copy number in the genome of transformant.

High Resolution Copy Number Variation Data in the NCI-60 Cancer Cell Lines from Whole Genome Microarrays Accessible through CellMiner

Array-based comparative genomic hybridization (aCGH) is a powerful technique for detecting gene copy number variation. It is generally considered to be robust and convenient since it measures DNA rather than RNA. In the current study, we combine copy number estimates from four different platforms (Agilent 44 K, NimbleGen 385 K, Affymetrix 500 K and Illumina Human1Mv1_C) to compute a reliable, high-resolution, easy to understand output for the measure of copy number changes in the 60 cancer cells of the NCI-DTP (the NCI-60). We then relate the results to gene expression. We explain how to access that database using our CellMiner web-tool and provide an example of the ease of comparison with transcript expression, whole exome sequencing, microRNA expression and response to 20,000 drugs and other chemical compounds. We then demonstrate how the data can be analyzed integratively with transcript expression data for the whole genome (26,065 genes). Comparison of copy number and expression levels shows an overall medium high correlation (median r = 0.247), with significantly higher correlations (median r = 0.408) for the known tumor suppressor genes. That observation is consistent with the hypothesis that gene loss is an important mechanism for tumor suppressor inactivation. An integrated analysis of concurrent DNA copy number and gene expression change is presented. Limiting attention to focal DNA gains or losses, we identify and reveal novel candidate tumor suppressors with matching alterations in transcript level.

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines

We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

32-OR

Aim Killer cell immunoglobulin-like receptor (KIR) complex is polygenic and polymorphic. Our PCR-SSP Multiplex detects the presence of 14 KIR genes in 4 PCR reactions, but genotype and Centromeric/telomeric (Cen/Tel) haplotype assignments are performed manually, which can be time-consuming when analyzing large data sets. The aim of this study was to validate a new real-time (RT)-PCR KIR genotyping Kit (LinkSeq KIR) using 40 samples. Methods RT-PCR is a sensitive method, does not include post-PCR analysis steps and can be easily automated. LinkSeq KIR Kit with SYBR green uses a PCR-based method and melt curve analysis to determine KIR specificity. A separate and unique control well determined DNA quality. SureTyper software assigns KIR genotypes, as well as KIR Cen/Tel haplotypes. Results KIR genotypes using the LinkSeq method agreed with previous typing results, except one 2DS2 gene and one 2DP1 gene were initially reported as negative. Upon review, software threshold settings were ignoring somewhat weaker positive signals. After adjusting software analysis settings, all samples were concordant. We were not able to detect several framework KIR genes for one sample: 2DL4, 3DP1 and 3DL1 and 3DS1 corresponding to genotype #194, ID from allelefrequencies.net which was observed in one person from South Africa. The LinkSeq method also confirmed this unusual genotype and haplotypes were assigned as: CenB/B, TelB/B. Conclusions In summary, the Linkage RT-PCR KIR genotyping kit is specific and sensitive, the amount of DNA is comparable with our multiplexing method, and post-PCR analysis is completely automated. This is the main advantage of using RT-PCR for large studies. Suretyper also assigns haplotypes based on KIR location (Cooley et al 2010). Our recommendation was to adopt the RT-PCR test to detect gene copy-number, which is the key to assigning KIR haplotypes and distinguishing homozygotes from hemizygotes.

A molecular framework for risk assessment of a virus-tolerant transgenic pepper line

The Cucumber mosaic virus coat protein (CMV-CP) gene-transgenic pepper lines exhibit high tolerance to Cucumber mosaic virus (CMV) strains. In this study, E7, one of the CMVP0-CP transgenic chili pepper events selected by screening was further characterized. Southern blotting and inverse PCR analysis revealed that the E7 event contains a single copy of the inserted gene cassette whose flanking sequences appear to be noncoding and intergenic. We searched for pepper-specific DNA sequence candidates as an endogenous reference gene for GM-pepper detection. We found that only one copy of CaSIG4 and lipocalin genes are present in the pepper genome and their sequences were determined to be pepper-specific. The characterization of the genomic sequences flanking the transgene, as well as the availability of the pepper-specific single copy CaSIG4 and lipocalin genes as endogenous reference genes, enabled the design of E7-event-specific PCR-based quantitative detection methods. The CMV-CP protein levels in the CMV-inoculated wild-type pepper tissues were approximately 60 times higher than those in the uninoculated and CMV-inoculated E7 pepper tissues. These results suggested that the amount of CMV-CP expressed in transgenic pepper tissue was negligible relative to the amount of CMV-CP in the virus-infected wild-type pepper consumed by human beings. This work may prove useful for risk assessment studies of transgenic pepper lines. Furthermore, the characterized single copy genes, lipocalin and CaSIG4, may be used to develop a method to detect gene copy number variations in the pepper genome.

An assay of gene copy number and its application based on heteroduplex products

The aim of this study was to set up a simple and efficient method for detecting gene copy number, based on heteroduplex products from single-tube PCR/DHPLC. Single-nucleotide polymorphisms (SNPs) on the α-globin gene and chromosome 21 were used as examples. And the formula for quantitative calculation of gene copy number was deduced—based on the peak heights of homoduplexes and heteroduplexes on the DHPLC pattern. 27 samples (14 normal DNA and 13 cases of trisomy-21) were assessed with this method, and 160 samples (48 normal DNA and 112 α-thalassemia samples) were assessed with this method combined with a duplex PCR/DHPLC. Results for 184 of 187 cases were concordant with the known genotypes; three cases of trisomy-21 could not be detected because the target SNPs were homozygous. In conclusion, quantitative assessment of heteroduplex products from single-tube PCR/DHPLC is simple and rapid, and can be used to detect α-thalassemia gene deletions (α − 3.7 , α − 4.2 ) and trisomy-21.

Incidence and Clinicopathological Relevance of Gene Copy Number Abnormalities Detected by a Single Quantitative PCR Assay in Mantle Cell Lymphomas.

The genetic hallmark of mantle cell lymphoma (MCL) is the t(11;14)(q13;q32). Fluorescence in situ hybridisation (FISH) and microarray based comparative genomic hybridisation (array CGH) experiments have demonstrated that additional genetic clonal alterations occur in the majority of MCL and may have prognostic or pathological relevance. We previously developed and validated by CGH or FISH an inexpensive and sensitive genomic PCR assay (Multiplex PCR of Short Fluorescent Fragments, QMPSF) to detect gene copy number abnormalities in diffuse large B-cell lymphoma and chronic lymphocytic leukemia (Haematologica 2008,93:543-Leukemia, 2007,21:1460). In the aim to determine the incidence and clinical relevance of recurrent additional genomic number abnormalities, we specifically designed a single QMPSF assay dedicated for MCL and correlated results with pathological and clinical data. For this purpose, a series of 42 newly diagnosed MCL cases with available frozen-and paraffin-embedded lymph node tissues and clinical features were selected [median age=67y; median MCL international prognostic index (MIPI)=6.1; low risk 21%, intermediate risk 33%, high risk 45%; 3-year overall survival (OS) rate=38%]. The assay was designed according to the most frequent gene copy number abnormalities reported in MCL, allowing simultaneous analysis of 8 relevant genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, and MDM2) and 2 reference genes (SEM4F and CECR1). DNA copy number gains of MYC, CDK2, CDKN1B (p27kip1) and MDM2 are observed in an equal frequency (10%). Losses of DNA copies of RB1, CDNK2A, ATM, or TP53 are observed in 38, 31, 26, and 10 % of cases respectively. Some genes are almost exclusively gained (MYC,CDK2), deleted (RB1, ATM, TP53, CDNK2A) or both (CDK2, CDKN1B). Deletions of ATM and RB1 appear strongly associated (21% of cases, p=.001). CDKN2A (p14arf and p16ink4a) homozygous deletions and CDKN1B gains are more frequently observed in blastoid variants (p=.04 and .005 respectively). According to the MIPI score, the number of gene copy abnormalities tends to increase in the high/intermediate risk group (median = 1, range 0–6), as compared to the low risk group (median=0, range 0–3, p=.07). More specifically, MYC gain is exclusively observed in the high risk group (p=.04) and CDKN2A deletions are observed in patients with the highest MIPI. The prognostic relevance of the assay was tested in 42 patients. With a median follow-up of 22 months, CDK2 (3y OS=0%) or MDM2 (3y OS=0%) gains and CDKN2A (3y OS=20%) or TP53 (3y OS=25%) losses correlate to a shorter OS (p <.0001, p=.0007, p=.003 and p=.03 respectively). CDKN2A deletions remain predictive of the outcome in patient with a high MIPI (p =.0005). Furthermore, PCR performed in 5 cases at the time of relapse showed an increase of gene copy number abnormalities compared with initial diagnosis (median = 4 vs.1; p =.02), suggesting that gene losses/gains are involved in a dynamic and selected process. To conclude, we developed a reliable and routinely applicable PCR assay which delineates distinct MCL oncogenic pathways with strong prognostic impact that could be used in combination with the recently defined MIPI.

Fluorescent resonance energy transfer (FRET) based detection of a multiplex ligation-dependent probe amplification assay (MLPA) product

A fluorescent resonance energy transfer (FRET)-based hybridization assay for detecting multiplex ligation-dependent probe amplification (MLPA) products has been developed, extending the diagnostic power of the technique and demonstrating the possibility of combining MLPA with microarrays for the detection of multiple mutations. FRET is one of the most commonly used detection techniques for hybridization assays. To investigate the applicability of FRET based detection of MLPA products, a sandwich assay was designed to detect gene copy number by exploiting an immobilized probe labeled with an acceptor dye, Alexa Fluor 555, which hybridises to specific PCR amplicons, followed by hybridization of a second probe labeled with the donor dye, Alexa Fluor 488. Following excitation of the Alexa Fluor 488, a FRET signal was produced only if a DNA sequence specific to the BRCA1 exon 13 was present in the test sample. We have verified this assay on a DNA sample of a patient carrying a heterozygous BRCA1 exon 13 deletion using male genomic DNA as control. Here we demonstrate that the DNA sample containing the heterozygous deletion generated a considerably reduced FRET signal as compared to the control male human DNA. Our results show that the FRET design presented in this study can differentiate between reduced copy numbers any genomic DNA sequence after MLPA analysis, and the reported format is applicable to multiplex detection of MLPA products, using microarrays, or optical biosensor arrays, and future work will focus on the demonstration of this.

Deletions of N33, STK11 and TP53 Are Involved in the Development of Lymph Node Metastasis in Larynx and Pharynx Carcinomas

Background: Lymph node metastasis is the mayor cause of mortality in patients with head and neck squamous cell carcinomas (45%). The genetic changes underlying metastasis are still largely unknown and genetic markers to predict lymph node positivity still need to be found. The aim of this study was to search such markers by using Multiplex Ligation-dependent Probe Amplification (MLPA), a semi-quantitative PCR technique to detect gene copy number alterations. Methods: Thirty-seven genes were analysed by MLPA in 34 larynx and 22 pharynx carcinomas. Results: Losses of CDKN2A (9p21) and MLH1 (3p22) and gains of CCND1, EMS1 (both at 11q13), RECQL4 and PTP4A3 (both at 8q24) were the most frequent aberrations in both larynx and pharynx carcinomas. Amplifications were detected at EMS1, CCND1 and ERBB2 (17q21). A correlation between loss of N33 (8p22) and poor survival was found (p=0.02). Gain of EMS1 had the same relation with survival but not significant (p=0.08). Lymph node positive tumors presented a specific pattern of genetic alterations, with losses of N33, STK11 (19p13) and TP53 (17p13), the latter especially in larynx tumors. Conclusion: We propose that these 3 genes might play a role in the development of metastasis in larynx and pharynx squamous cell carcinomas.

Molecular Characterization of Genomic Structural Alterations through CGH Arrays in Acute Myeloid Leukemia with Normal Cytogenetic Findings.

Acute myeloid leukemia (AML) is an heterogeneous disease. Cytogenetic findings are one of the main prognosis criteria beside age, leucocytosis, and antecedent of previous hemopathy. More than 50% of AML patients, mainly with normal cytogenetic, belong to the intermediate cytogenetic risk group. Even in this group, cryptic molecular abnormalities as mutation are frequent with prognosis value. In this work we have studied 31 AML patients (Pts) with normal caryotype by high resolution comparative genomic hybridization array CGH using agilent 185k chips to detect gene copy number alterations (CNA). Tumor DNA was obtained from bone marrow diagnosis samples (>60% of blasts) and control DNA from pooled blood samples obtained from 30 healthy donors or from AML patients in CR during the follow up. The design of the study was first to hybridize tumor DNA from the 31 patients against pooled control DNA to detect both copy number polymorphisms (CNP) and acquired CNA in AML and in a second time to hybridize tumor DNA from the 10 pts with the greater number of abnormalities against autologous DNA obtained at CR time to distinguish acquired CNA from CNP. Briefly DNA labelling was performed using Cy3dUTP and Cy5 dUTP respectively for control and tumor samples. Fluorescence ratios were normalized and spots with value that deviated significantly from background were retained as significant evidence of DNA copy number alterations after statistical analysis using Feature Extraction and CGH Analytics AGILENT software. BAC from the Human Genome Center (Wellcome Trust Sanger) were used to obtain FISH probes to confirm abnormalities observed by CGH arrays on metaphase spreads obtained from bone marrow diagnosis sample when available. In each case, two colour FISH was performed by the simultaneous hybridization on a green BAC probe located in the CNA regions and a red control BAC probe located in the vicinity of the CNA regions obtained from the UCSC genome browser data.Results98 DNA copy number alterations were observed with 56 losses and 42 gains cases. Those CNA were variable in size from 8,6 Mb to <100 Kb. 8 CNA regions were recurrent (5 losses in 7q31 (12pts), 4q13 (3pts), 1q31 (4pts), 2q37 (2pts) et 22q13 (3pts) and 3 gains in 1q23 (3pts), 2p11(2pts) et 17q21(2pts). Non recurrent CNA were located in 1p36.2, 1p34.3, 1p21.1, 1p22, 1q31.2, 3q12.2, 4p13, 5p15.3, 5q14.1, 6p22.1, 7p22.1, 7q11.23, 10q26.3, 11q13.2, 15q11.2, 15q15.3, 15q25.3, 15q26.1, 16q24.2, 17p13.2, 17p11.2, 22q13,33 for gains and in 1p33, 1q32.1, 1q32.2, 3p14.1, 3p13-p12.3, 4q26, 7p11.2, 7p14.3, 7q35, 8p23.2, 8q23.1, 9p24.1, 9p13.1-p12, 9q34.11-q34.12, 12p13.31, 13q14.2, 14q21.2, 14q22.1, 14q23.2, 15q22.31, 16q22.1, 21q21.2, 21q22.12 for losses. No one of the patients had a genome without CNA and 6 patients (20%) had more than 5 CNAs. The CGH data showed clearly that 80% of the CNA observed with pool control DNA were not present when tumor DNA was hybridized against the autologous CR DNA confirming the high frequency of CNP as reported on http://projects.tcag.ca/variation website. All of the acquired CNA regions observed were concordant with the FISH results involving genes known to be crucial in leukemia as RUNX1, Abl, CEBPa but also many other genes. In conclusion cryptic abnormalities are frequent in normal caryotype AML and 20% of the patients studied had more than 5 CNA traducing a great instability of the genome of leukemic cells.

A Comparative Analysis of Chromosome 13 Gene Expression and Copy Number Changes in Multiple Myeloma Using Array-Based Methods.

Chromosome 13 (Δ13) abnormalities are found in greater than 50% of patients with Multiple Myeloma (MM). MM is most commonly defined by chromosome 13 monosomy or 13q loss (85%). Interstitial deletions comprise the remaining 15%. Many studies have revealed that Δ13 in MM are associated with poor survival and reduced response to therapy. Genes mapping to chromosome 13 may be involved in pathogenesis and/or progression of the disease due to loss of function from gene mutation or from epigenetic effects such as haploinsufficiency. In this study, array-based comparative genomic hybridization coupled with microarray technology (aCGH) is used to detect gene copy number loss on chromosome 13 from nine MM patient samples. Whole genome long-oligo microarrays constructed by Agilent Technologies were used which contain 40,000 genes that span the human genome with an average spatial resolution of ~75 kb. Using genomic DNA isolated from MM patients with interstitial deletions on chromosome 13, DNA was amplified, labeled and hybridized with a differentially labeled normal DNA reference to determine gene/genomic copy number changes. Arrays were analyzed to search for the minimum region of loss based upon single copy loss for a series of nearby mapping transcripts. A common region of loss of 2.2 Mb, at 13q14.2 was detected. Additionally, we investigated the correlation between genomic copy number change and the expression level for MM patients in the13q region. From an independent gene expression data set whose expression measurements were conducted with Affymetrix HG-U133A v2 microarrays, data was selected that corresponded to the samples used for current aCGH. Expression values from MM samples were divided by the mean expression values from 12 normal bone marrow samples for each gene and the resulting values were treated as surrogate ratios between MM and normal samples. Probes from both microarrays were then aligned according to their chromosomal positions and merged if their chromosomal positions overlapped. Composite chromosomal maps were generated that displayed the expression levels and copy number changes. The maps were used to differentiate chromosomal regions in 13q where copy number changes and expression levels show high correlation and regions where such correlation was not observed. Although the number of probes sampled in the expression microarray was much smaller than those in aCGH microarray, a chromosomal region of great interest, that encompasses 13q14.2, arose naturally from this analysis. Although the mechanism by which loss of 13q effects tumorigenesis in MM could be a haploinsufficiency model, we are not ruling out the presence of a tumor suppressor gene in this region. We are evaluating candidate tumor suppressor genes in the region for loss of function by mutational analysis and hypermethylation studies.

Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes

In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values < or = 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.

Detection of gene copy number in head and neck squamous cell carcinoma using multiplex amplifiable probe hybridisation

Detection of gene copy number in head and neck squamous cell carcinoma using multiplex amplifiable probe hybridisation