00019606-200803000-00002ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 by Lippincott Williams & Wilkins.17March 2008
p 3-13Optimization of the Affymetrix GeneChip Mapping 10K 2.0 Assay for Routine Clinical Use on Formalin-fixed Paraffin-embedded TissuesOriginal ArticlesLyons-Weiler, Maureen MSc* †; Hagenkord, Jill MD‡; Sciulli, Christin BS* §; Dhir, Rajiv MD†; Monzon, Federico A. MD* †*Clinical Genomics Facility†Department of Pathology§University of Pittsburgh Cancer Institute‡Department of Biomedical Informatics, University of Pittsburgh, Pittsburgh, PASources of support: University of Pittsburgh Cancer Institute, Cancer Center Support Grant 5P30CA047904-18 NCI/NIH (Ronald Herberman PI).Maureen Lyons-Weiler and Jill Hagenkord made equal contributions to this paper.Part of this work was presented at the Annual Meeting of the Association for Molecular Pathology, November 10 to 13, 2005, Scottsdale, AZ, and it obtained an AMP Technologist Award for Maureen Lyons-Weiler.Reprints: Federico A. Monzon, MD, The Methodist Hospital, Molecular Diagnostics Laboratory, 6565 Fannin St, MS205, Houston, TX 77030 (e-mail:
[email protected]).A supplementary file is available online on http://www.molecularpathology.com.AbstractThe use of chromosomal copy number changes as markers for tumor behavior or as prognostic markers for patient outcome has been suggested. However, current clinically used technologies cannot perform genome-wide assessment of chromosome copy number and analysis of loss of heterozygosity in the same assay for paraffin-embedded tissue. We have optimized the Affymetrix GeneChip Mapping Assay for the 10K 2.0 array for use with formalin-fixed, paraffin-embedded (FFPE) tissues. This technology uses single nucleotide polymorphism (SNP) arrays to assess the changes in chromosomal copy number and loss of heterozygosity. DNA from 3 paired tumor/normal samples of adrenal tumors and 4 samples of renal tumors were processed with modifications to the manufacturer's protocol. Modifications at different steps were evaluated for their effects on SNP signal-detection and call rates. Frozen samples showed 99.6%±0.3% signal-detection rates and 94.7%±3.0% SNP call rates. FFPE samples labeled with the original protocol failed to produce enough polymerase chain reaction products for hybridization, whereas the same samples processed with the optimized protocol had signal-detection rates of 97.4%±0.018% and SNP call rates of 90.9%±0.034%. The average SNP call concordance between fresh and matching FFPE samples was 96%. Chromosomal aberrations detected in the frozen tumors were also detected in the FFPE tissues. Our optimized protocol significantly improves the performance of the FFPE samples in the Affymetrix GeneMapping Assay with the 10K 2.0 SNP array. This optimized protocol opens up the potential for the GeneChip Mapping assay to be used in the development of clinical test assays.ArticlePlusClick on the links below to access all the ArticlePlus for this article.Please note that ArticlePlus files may launch a viewer application outside of your web browser.http://links.lww.com/PDM/A5Detection of acquired chromosomal gains/losses in human tumors is clinically helpful for the confirmation of suspected diagnoses such as 1p/19q deletions in oligodendrogliomas and chromosomal gains associated with bladder cancer in urine specimens. Most of the methodologies in clinical use detect a specific target for deletion or amplification; hence, they cannot detect multiple chromosomal copy number (CCN) changes with high resolution in 1 assay. In research, array-based technologies are being used to identify recurrent CCN changes in tumors, with prognostic or diagnostic implications.1,2 Array comparative genomic hybridization (aCGH) is already in clinical use for the detection of constitutional CCN alterations.3 Single nucleotide polymorphism (SNP) arrays allow researchers to determine both the copy number status and the genotype of almost a million SNPs, detecting regional gains/losses and the loss of heterozygosity (LOH) in a single assay. Although SNP arrays have been primarily used for genetic association studies, they have also proved to be useful for studying the gains and losses of genetic material in human tumors.2,4–7 In addition, SNP arrays have several advantages over other methods for detecting copy number changes such as CGH, aCGH, spectral karyotyping, and fluorescent in situ hybridization. The advantages include high-resolution coverage of the entire genome, scalability and automation, ease of scoring, need for minimal total genomic DNA, stringent quality control in manufacturing, and relatively low cost. It is likely that array-based technologies for CCN analysis will become an additional tool for cancer diagnosis in molecular diagnostics laboratories.The Affymetrix SNP mapping array platform has been rapidly increasing in density, with the 10K array being released in 2003 (10,000 SNP probes), to the 6.0 array in June 2007 (more than 906,600 SNP probes and more than 946,000 probes for the detection of copy number variations). These latest, ultrahigh-density arrays are remarkably powerful and have several research applications. However, several issues make them less attractive for developing routine clinical assays at this time, such as cost, longer turnaround time, more complex protocols, limited availability and maturity of analysis software, and interlaboratory variability in the CCN analysis applications. In our laboratory, we have found that the Affymetrix GeneChip 10K Xba 2.0 arrays provide sufficient resolution for detecting large chromosomal aberrations of clinical significance in specific tumor types.To develop a protocol for the clinical application of CCN analysis with SNP arrays, we have optimized the Affymetrix GeneChip Mapping assay for use with formalin-fixed and paraffin-embedded (FFPE) archived tissues, by performing a detailed evaluation of the impact of the modifications at each step of the protocol. To evaluate the performance of the modified protocol, we performed CCN analyses on both frozen and FFPE tissues of adrenal tumors, to confirm that chromosomal aberrations seen in the frozen tissues were also detected in the FFPE samples. Additionally, we analyzed 4 FFPE renal tumors and validated the CCN changes and LOH, using aCGH and microsatellite analyses, respectively. In this manuscript, we present a reliable, robust, reproducible, and relatively inexpensive method with rapid turnaround time, for the assessment of chromosomal aberrations in archived tumor samples, using the Human Mapping assay with the GeneChip 10K 2.0 array.MATERIALS AND METHODSTissue SamplesThree FFPE tumor/normal pairs of adrenal pheochromocytomas were obtained from the Health Sciences Tissue Bank of the University of Pittsburgh. These FFPE samples were matched, when available, with their frozen tumor/normal counterparts. However, in one case, a frozen sample for normal tissue was not available. In addition, FFPE tumor/normal pairs for 4 renal tumors were obtained and processed in a similar manner: 1 conventional (clear cell)
renal cell carcinoma (CRCC), 1 oncocytoma (OC), 1 chromophobe
renal cell carcinoma (CHRCC), and 1 papillary
renal cell carcinoma (PRCC). The archival time for the FFPE samples ranged from 1 to 5 years in routine storage conditions for diagnostic samples. Samples were deidentified and obtained with Institutional Review Board approval through an honest broker system. In this system, investigators using materials and requesting additional information on a sample have to route the request through a pathology tissue bank “honest broker” (someone not involved as a collaborator in the study, who has administrative access to the tissue bank database). Codification of all the stored materials is performed, and the “codes” are made available only to the tissue bank team. This ensures compliance with the regulations under the Health Insurance Portability and Accountability Act (HIPAA).Sample Preparation and ExtractionFrozen tissue samples were embedded in optimal cutting temperature compound, and a hematoxylin and eosin (H&E)-stained frozen section was obtained. For FFPE samples, ten 10-μm slides were obtained for all samples with corresponding H&E-stained slides. Slides from all the frozen and FFPE tissues were evaluated by a pathologist (F.A.M.), and areas that had more than 90% tumor or that were 100% normal were selected and marked on the coverslip.For frozen samples, the marked H&E frozen section slide was placed over the frozen tissue block, and the area of interest was identified. Once selected, approximately 10 μL of prechilled RNA Later-ICE (Ambion, Austin, TX) was added to the area of interest directly on the optimal cutting temperature block, to promote easy removal of the frozen tissue by scraping with a scalpel blade. RNA Later-ICE was selected to soften the tissue, to ensure that the tissue would be preserved for future RNA extraction. Approximately, 100 mg of tissue were scraped for each sample (average 3 mm3). The scraped frozen tissue was immediately placed into a nuclease-free 1.5-μL tube and placed on ice. DNA from all the frozen tissue samples was extracted using the Qiagen DNeasy kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. Briefly, the frozen tissue was placed into 250 μL of the buffer, ATL. To this, 50 μL of proteinase K was added, and the sample was then incubated overnight at 37°C at 60 rpm in a shaking air incubator for 16 hours. After the overnight incubation, the DNA was extracted by following the instructions in the DNeasy DNA kit manual.FFPE samples were prepared for extraction according to a deparaffinization protocol developed in our laboratory and outlined below. Five 10-μm slides for each sample were initially heated to 56°C for 20 minutes, to melt the paraffin, and were then placed into a reservoir containing 100% xylene (Fisher Scientific, Pittsburgh, PA) for 5 minutes. This incubation was repeated 2 more times (for a total of 3 washes). The slides were allowed to air-dry briefly in a fume hood. Next, the area of interest (tumor or normal) was identified by superimposing the marked H&E-stained slide, and the area was manually dissected with a scalpel. A minimum of 1 cm2 of tumor surface area was scraped from each slide (fewer slides were needed if the tumor area in each slide was larger). The scraped tissue was placed into a 1.5-μL nuclease-free tube, and was then washed twice with 1 mL of 100% xylene, to ensure that all the paraffin was removed. Samples were then spun in a microcentrifuge for 3 minutes at maximum speed, and the xylene was removed. Once deparaffinization was complete, the samples were rehydrated by sequentially adding 1 mL of 100%, 75%, and 50% ethanol, after a centrifugation step and the removal of the previous ethanol. The first 2 ethanol washes were centrifuged for 3 minutes at 14,000×g, and the final wash was centrifuged for 5 minutes at 14,000×g. After the removal of the 50% ethanol, the tubes containing the tissue samples were allowed to briefly air-dry before extraction was carried out.All FFPE samples were extracted using the Qiagen DNeasy kit (Qiagen, Valencia, CA), with a modified protocol developed in our laboratory. The deparaffinized, rehydrated FFPE tissue was placed into 300 μL of buffer ATL and 100 μL of Qiagen proteinase K (600 mAU/mL). The samples were incubated overnight at 56°C in an air incubator, with shaking at 60 rpm. Once lysis was complete, 400 μL of buffer AL was added to the sample, which was then vortexed for 15 seconds, and then incubated at 70°C for 10 minutes. About 400 μL of 100% ethanol was then added to the sample and mixed by vortexing. Approximately half the volume of the sample was placed into the spin column provided by the manufacturer, was then centrifuged for 1 minute at 8000×g, and the flow-through was discarded. The second half of the sample was then added to the spin column and the step was repeated. The AW1 and AW2 wash steps were performed according to the standard DNeasy protocol, using 500 μL of each wash buffer. AW1 was followed by a 1-minute centrifugation at 8000×g, and the AW2 wash was followed by a 3-minute 14,000-g centrifugation. The DNA was then eluted with 2 sequential 50-μL Buffer AE incubations, for a final volume of 100 μL.DNA obtained from both the frozen and the FFPE tissues were quantitated on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). All samples processed for downstream analysis in this study had an OD-260/280 ratio higher than 1.8.Affymetrix 10K 2.0 Genotyping ProtocolFrozen tissue samples were processed according to the standard protocol provided by the GeneChip Mapping 10K Xba Assay Kit (Affymetrix, Santa Clara, CA), using 250 ng of total genomic DNA.8 The only modification done to the manufacturer's protocol was the use of the Agilent Bioanalyzer DNA 7500 kit, to determine polymerase chain reaction (PCR) performance (as opposed to gel electrophoresis).FFPE samples were run using the standard 10K assay listed above; however, these samples failed to produce sufficient PCR product for array hybridization when we used 4 PCR reactions. We used a subset of samples to optimize the following parameters of the 10K 2.0 assay: initial DNA amount (250 ng, 500 ng, and 1 μg), duration of restriction enzyme digest (2 and 16 h), adapter concentration (2× and 4× of the suggested concentration), and number of PCR cycles (35, 40, and 45 cycles). The additional steps in the assay, fragmentation, labeling, hybridization, washing, staining, and scanning were not modified. Only one modification was introduced at a time, and PCR yields were used as a quality-control (QC) measure for the success of the assay modification. Only modifications that yielded a sufficient template (between 11 and 20 μg) were hybridized to 10K 2.0 arrays and underwent downstream analysis for SNP detection and call rates.SNP Array Data AnalysisData acquired from the Affymetrix GeneChip Operating System v4.0 were analyzed using the Affymetrix GeneChip Genotyping Analysis Software (GTYPE) 4.0. Mapping array reports, with SNP call rates and signal detection rates, were generated for both frozen and FFPE samples. The parameters evaluated for each sample were signal detection rate (the percentage of SNPs that meet quality requirements), the SNP call rate (rate of successful genotype identification), and concordance (rate of agreement between successful genotype calls). The SNP call rate is a good indicator of the overall performance of the assay. According to the manufacturer, a call rate above 92% indicates that all the steps (DNA isolation to scanning) have worked well. Call rates less than 85% indicate that problems might have occurred and that the sample should be evaluated before being included in further analyses. Poor call rates can be caused by poor genomic DNA quality, deviation from protocol, or contaminated DNA.LOH and copy number estimates on the array data were performed using the publicly available analysis package, Copy Number Analyzer for Affymetrix GeneChip arrays 2.0 (CNAG).9 Results from the CCN changes on all the FFPE samples were obtained, with the data generated from the optimized method being described in “results”. The LOH likelihood threshold in CNAG was set to 14. Best-fit references were selected by the algorithm from all available data files for normal (nontumor) tissues. For SNP copy number analysis, moving averages were set to 20 adjacent SNPs. Fragment lengths were restricted to 300 to 700 bp for all samples. For the pheochromocytoma samples, diploid regions were set for each sample on the basis of visual inspections of the copy number output, as described in Nannya et al.9 For the CHRCC sample, the diploid region was set based on the results of the allele specific analysis using self-reference. This is important to avoid artifactual trisomies in hypodiploid samples.aCGHWe validated copy number changes on 1 sample from each renal tumor subtype with aCGH. The same DNA, extracted from FFPE tissues, was used for both the SNP arrays and aCGH. aCGH was performed on a subset of samples by the Microarray Core at the Comprehensive Cancer Center, University of California, San Francisco, using the method described in Snijders et al.10 2464 bacterial artificial chromosome clones, spotted in triplicate with an average spacing between clones of 1.4 Mb, were used. Data analysis was performed by the UCSF Microarray Core, using SPOT and SPROC software.11Microsatellite AnalysisLOH was validated on a subset of renal tumors by the Molecular Anatomic Pathology Laboratory at the University of Pittsburgh Medical Center. Briefly, PCR was performed on tumor and normal genomic DNA, using primers targeting short-tandem repeat units for each locus of interest with commercial markers (Integrated DNA Technologies, Coralville, IA). Amplification products were detected using capillary electrophoresis (ABI 3100, Applied Biosystems, Foster City, CA). The allelic peak height ratios were calculated between normal and neoplastic samples. The sample was considered to have LOH if the allelic ratio for a specific microsatellite marker was below 0.5 or above 2.0. All selected samples were tested for LOH on chromosome 1, using 3 markers (D1S1183, D1S407, and D1S1172). The CRCC sample was tested using 2 markers for the presence of characteristic -3p (D3S1539, D3S1234). The CHRCC sample was tested for LOH on chromosome 13 (D13S319), chromosome 17 (D17S1844, D17S1161), and chromosome 21 (D21S1256, D21S1244).RESULTSProtocol OptimizationThree tumor/normal pairs of adrenal pheochromocytoma tissue (frozen and FFPE) were used for the optimization of the GeneChip 10K 2.0 Mapping assay for paraffin-embedded samples. This analysis led to the optimization of 3 major aspects of the assay: the amount of starting material (to 1 μg), the restriction enzyme digest, and the PCR conditions, as described below.The first step to be optimized was the DNA-extraction process and restriction enzyme digestion. We found that a 3-day incubation with proteinase K did not yield significantly more DNA than an overnight incubation, given the additional days added to the overall turnaround time (data not shown). However, we did find that increasing the amount of DNA added to the restriction enzyme digestion resulted in a better representation of DNA fragment sizes, as shown in the Bioanalyzer profiles in Figure 1. The DNA fragment profile from the 1-μg digestions was similar to the ones obtained with 250 μg of DNA from frozen tissues (Fig. 1). In addition, increasing the amount of Xba I enzyme to 40 U and extending the restriction enzyme digest from 2.5 to 16 hours resulted in increased PCR yields (data not shown). In the optimized method, 1 μg of whole genomic DNA from FFPE tissue is thus digested using 40 U of Xba I (New England BioLabs, MA) in 30 μL of NEB Buffer II (New England BioLabs, MA) for 16 hours at 37°C, followed by 20 minutes at 70°C.JOURNAL/dimp/04.03/00019606-200803000-00002/figure1-2/v/2021-02-17T195944Z/r/image-jpeg
Capillary electrophoresis profiles of the Xba restriction enzyme digests. A, Frozen tissue (250 ng of DNA) and paraffin-embedded tissue (B—250 ng and C—1 μg of DNA). Increasing the amount of input DNA increases representation of DNA fragments from more sizes in the digest.We then proceeded to optimize the PCR reaction, including the ligation of adapter oligos. Increases in the adapter concentration for ligation up to 4-fold did not improve the performance of the PCR reactions (data not shown), and in the optimized protocol, ligation was performed as per the standard Affymetrix 10K 2.0 Genotyping Protocol. Tumor and normal FFPE samples were processed, using the standard 35 PCR cycle protocol, modified 40-cycle protocol, and the modified 45-cycle protocol. FFPE samples, processed with the manufacturer's protocol, failed to yield enough PCR products for hybridization on an array. For all 45 cycle reactions, yields were consistently higher, and only 4 PCR reactions were needed to achieve PCR yields comparable with those obtained from frozen tissue, using the manufacturer's recommended protocol (Table 1). Distribution of PCR product sizes was not affected by the number of PCR cycles (not shown). However, PCR products from FFPE tissues were consistently shorter than those generated by DNA from frozen tissues (Fig. 2). In terms of assay performance, a significant increase in call rates was seen with 40 and 45 cycles, compared with 35 (analysis of variance, P=0.0057, Table 1). Increasing the number of cycles from 40 to 45 did not increase the call rate; however, it decreased the number of PCR reactions needed to obtain sufficient PCR products for hybridization (more than 12 μg of PCR product). Consequently, in the optimized protocol, all the PCR reactions used 45 cycles.JOURNAL/dimp/04.03/00019606-200803000-00002/figure2-2/v/2021-02-17T195944Z/r/image-jpeg
Electropherogram of PCR products from formalin-fixed, paraffin-embedded, and frozen tissues. Range of amplicon size for paraffin-embedded tissue is different from that for frozen tissue. Larger DNA fragments are not represented in the amplified DNA.JOURNAL/dimp/04.03/00019606-200803000-00002/table1-2/v/2021-02-17T195944Z/r/image-tiff Performance of GeneChip Mapping Assay in Frozen and Paraffin-embedded TissuesAlthough the manufacturer recommends at least 18 μg of labeled PCR product for hybridization, we successfully hybridized samples with as little as 10.2 μg, when using 35 PCR cycles. On the basis of the SNP call rate performance, we established a threshold of 12 μg as the minimum amount of PCR product for hybridization (data not shown). Fragmentation, labeling, hybridization, washing and staining, and scanning procedures were not modified. The entire optimized protocol is presented in a Supplementary File.Because the unmodified protocol did not yield sufficient PCR products for hybridization, array quality metrics are only presented for frozen tissue and the optimized protocol for FFPE tissues. The optimized 45-cycle protocol yielded SNP signal-detection rates of 97.35%±1.00% and SNP call rates of 90.85%±3.57% (Table 1). Frozen samples had, on average, 99.55%±0.29% signal-detection rates and 94.62%±3.29% SNP call rates, using the recommended protocol by the manufacturer (n=34, including repeats). Normal tissue samples, both frozen and FFPE, consistently showed better SNP call rates when compared with tumor tissue (Table 1). Agreement of SNP calls between tumor/normal pairs of frozen tissues and same sample repeats was 99.45%±0.22% (Table 2). Agreement of SNP calls between frozen/FFPE pairs was 95.98%±1.746%. As expected, unrelated frozen and/or FFPE samples had agreement rates of 52.764%±0.841%.JOURNAL/dimp/04.03/00019606-200803000-00002/table2-2/v/2021-02-17T195944Z/r/image-tiff Agreement in SNP CallsLOH and CCN AnalysisLOH and copy number changes were evaluated using the software tool CNAG 2.0 in 2 phases. In our first analysis, we determined the ability of the assay to detect similar LOH and copy number changes in matched frozen/FFPE samples. For this, we compared the results of the frozen tissue with those of the corresponding FFPE tissues for both normal and tumor samples (3 pheochromocytomas). In the second phase, we performed the optimized assay and subsequent copy number analysis on 4 FFPE renal epithelial tumors and validated the results with aCGH and microsatellite analyses.Assay Performance Using Matched Frozen and FFPE PheochromocytomasWe assessed all normal samples from both the pheochromocytoma and renal tumor cohorts, to ensure that no chromosomal aberrations were detected in either the frozen or the FFPE normal tissue samples. The genomes from all normal samples were diploid throughout, and neither copy number changes nor LOH were detected (data not shown). For the pheochromocytomas, frozen tumor samples were compared with their corresponding FFPE tumor samples, to ensure that we could reliably detect, in the FFPE tissues, chromosomal aberrations that were present in the frozen tissues. Chromosomal aberrations seen in the frozen tissues were also seen in their FFPE counterparts. Figure 3 shows the comparison between the fresh tumor and the FFPE tumor for each of the 3 pheochromocytoma samples. The data are presented in a whole genome view arranged in chromosomal order, with chromosome 1 on the far left and chromosome X on the far right. Each sample has the data represented in 4 ways: the topmost plot shows the estimated copy number at each SNP (log2 ratio) averaged over 20 SNPs; the dark green bars represent heterozygous SNPs present in the tumor; the first yellow bar represents the Hidden Markov Model (HMM) for copy number estimate (yellow=copy number 2, aqua=copy number 1, and pink=copy number 3); and the bottom yellow bar represents the HMM for LOH likelihood (yellow=no LOH, the more blue the bar=the more likely the LOH). Although the FFPE tissues tend to have a higher standard deviation (SD) of the log2 ratio (0.17 vs. 0.42, after fragment length restriction), the signals are still clear and correlated with the changes seen in frozen tissue. Tumors have varying degrees of normal stroma and vasculature present in them (“normal contamination”). This higher normal stroma content results in a dilution of the log2 ratio, and thus the estimated copy number for each SNP is closer to the 0 line (copy number 2). Pheochromocytoma GB253 seems to have more normal tissue present than tumors C109 and D399. Consequently, although the gains and losses can be detected in the copy number plot, the log2 values do not consistently trigger the HMM for copy number estimates in either the fresh or the frozen samples of this tumor.JOURNAL/dimp/04.03/00019606-200803000-00002/figure3-2/v/2021-02-17T195944Z/r/image-jpeg
Copy number and LOH analysis on pheochromocytoma showing correlation between frozen and FFPE samples. Pheochromocytoma samples C109, D399, and GB253 are shown with the frozen copy number results in the top plot (A) and the corresponding FFPE results in the lower plot (B) for each sample. The SNPs are arranged in chromosomal order along the x-axis with chromosome 1 on the far left and chromosome X on the far right. The samples are not sex-matched to the reference samples, so the copy number of X chromosome should not be considered in this image. The y-axis of the uppermost plot for each sample represents the estimated copy number as a log2 ratio averaged over 20 SNPs (a), thus the 0 line indicates a copy number of 2, and deviations up or down represent gains or losses. The dark green bars (b) are heterozygous calls in the tumor. The upper yellow bar (c) represents the copy number data in a color-coded HMM (yellow=copy number 2, pink=copy number 3, and aqua=copy number 1). The lower yellow bar for each sample (d) represents the HMM for LOH likelihood (yellow=no LOH, the more blue the bar=the more likely the LOH in that region). Chromosomal aberrations detected in the fresh tumor sample were also detected in the corresponding FFPE sample for each case.Analysis of FFPE Renal Epithelial Tumors and Validation with aCGH and LOH Analysis by PCRWe analyzed 4 FFPE renal epithelial tumors: 1 conventional clear cell carcinoma (CRCC), 1 PRCC, 1 CHRCC, and 1 OC. Each tumor showed characteristic CCN changes in agreement with known chromosomal imbalances in renal cell tumors (Fig. 4).12 The clear cell tumor shows the expected −3p, as well as +5q, trisomy 7, and monosomy 14. PRCCs are known for trisomies of chromosomes 7 and 17, both of which are readily detected by the SNP arrays. The SNP arrays also detected +3q, −4p, −5p, −8p, +8q, and +12q in this sample. CHRCCs are characterized by multiple losses of entire chromosomes, which is demonstrated by the SNP array results. OCs are either cytogenetically normal or show a monosomy 1 by conventional karyotyping. SNP array analysis of 1 OC shows the expected monosomy 1 and a trisomy 7.JOURNAL/dimp/04.03/00019606-200803000-00002/figure4-2/v/2021-02-17T195944Z/r/image-jpeg
Copy number and LOH analysis of FFPE renal epithelial tumors. DNA from paraffin-embedded samples from each of the 4 subtypes of renal cell neoplasms was analyzed: OC, chromophobe carcinoma (CHRCC), PRCC, and conventional
renal cell carcinoma (CRCC). Please refer to Figure 3 for an explanation of the copy number/LOH plots. Results show characteristic findings for these tumors and illustrate the application of this protocol to a clinically relevant problem.The copy number changes and LOH detected in the renal tumors by the SNP arrays were validated by aCGH and microsatellite analysis, respectively. All chromosomal gains and losses detected by the SNP arrays were confirmed by aCGH (Fig. 5—only CRCC and PRCC shown). Array CGH detected a gain of chromosome 19 in CRCC4, which the SNP array did not. Chromosome 19 is represented by only 149 SNPs on the 10K mapping array and represented by 39 probes on the bacterial artificial chromosome array, which leaves both methods with low resolution on this chromosome. Ten microsatellite markers were used to assess LOH in the 4 renal tumors (see Methods). LOH detected by SNP arrays were confirmed by microsatellite analysis at all the loci examined (Table 3).JOURNAL/dimp/04.03/00019606-200803000-00002/figure5-2/v/2021-02-17T195944Z/r/image-jpeg
Validation of SNP array copy number results by aCGH for renal epithelial tumors. Representative data from samples validated by aCGH. The top plot in each box is the aCGH copy numbers for each bacterial artificial chromosome plotted in chromosomal order from left to right. The smaller bottom plot in each box is the copy number for each SNP on the SNP 10K mapping array plotted in chromosomal order from left to right with each chromosome being color-coded. A, CRCC; B, PRCC.JOURNAL/dimp/04.03/00019606-200803000-00002/table3-2/v/2021-02-17T195944Z/r/image-tiff Correlation of LOH Analysis by SNP Arrays and PCRDISCUSSIONParaffin-embedded tissues are readily accessible and are associated with complete surgical pathology reports, ancillary study results, treatments, and outcome data, which make them attractive and desirable specimens for cancer research. Furthermore, in routine surgical pathology practice, the extent of the need for ancillary studies is not known until after the specimens have been formalin fixed and paraffin embedded. Consequently, for both research and clinical applications, it is desirable to have the option of analyzing FFPE tissues. However, DNA extracted from FFPE is fragmented and has been troublesome to use in downstream reactions requiring long DNA fragments. SNP genotyping arrays are now being used to study CCN changes in several cancer tissues.2,4–7,13 However, the SNP array assays were designed for high-quality DNA, preferably from frozen tissues. In this manuscript, we describe an optimized protocol for SNP array analysis with FFPE tissues.We followed a systematic approach to the modification of the manufacturer-recommended protocol by individually modifying different steps in the assay and evaluating their effects on signal-detection and call rates. In summary, increases in adapter concentration had no effect on these parameters, whereas increased proteolytic digestion time, greater amount of starting DNA, and increased number of PCR cycles all had positive effects. Our results show that signal-detection rates, SNP call rates, copy number changes, and LOH calls, comparable with those of frozen tissues, can be reliably obtained from archived FFPE samples. Our optimization improves the performance of the FFPE samples in the Affymetrix GeneChip Mapping Assay with the 10K 2.0 SNP array.Although no clinical assays are as yet available with this platform, several investigators have described CCN changes of clinical significance in human tumors.1,14–16 It is thus foreseeable that CCN analysis will be performed as a clinical test in the near future. Recently, other protocols have been published for using DNA from FFPE tumors for whole genome SNP arrays.13,17 However, our protocol and our results differ from others in several ways that are important for the possible clinical application of this technology. First, our DNA extraction is performed overnight rather than in a 3-day period. This is a significant issue for specimen turnaround time in clinical testing. Second, we start with a larger amount of genomic DNA (1 μg vs. 250 ng), which should be readily obtainable from clinical specimens. Third, we do not require a PCR-based QC test on the genomic DNA. The PCR performed as part of the assays serves as a good control for performance. This also economizes on reagents and decreases the time it takes to complete the entire protocol. Fourth, we increase the number of the PCR cycles alone, rather than the number of PCR cycles and the number of PCR reactions, thus reducing PCR costs, again. Although other alternative protocols use a variable number of PCR reactions (4 to 9) and a variable cycle number (35 to 45) to achieve 20 μg DNA,13,17,18 we found that we could get reliable performance using a consistent protocol of 4 PCR reactions and 45 cycles, without necessarily adhering to a 20 μg limit.With these modifications, the average SNP call rate of our optimized protocol is 94% compared with 83% in other published protocols.13 Moreover, 93% of the samples that we have processed in our laboratory with the optimized protocol achieved call rates >75%, making this a reliable assay. Comparatively, other authors have described that only about one-half to two-thirds of samples pass the genomic DNA QC test, and that then only three-quarters of these work when hybridized on the arrays.19 Importantly, for the clinical application of this technology, in this study we used FFPE samples obtained from the routine surgical pathology archives in our institution, with archival periods of 1 to 5 years. No significant correlation was observed between storage time and performance in the assay (data not shown), which indicates that our method can be successfully applied to samples in routine fixation and storage conditions for diagnostic material. In contrast, Thompson et al13 specifically prepared samples for their study with known formalin fixation times (<24 h), and all but one had a storage time of 3 months, which is not representative of routinely archived tissues. It is unclear whether the modifications introduced by these authors will yield successful results in routine diagnostic material from surgical pathology laboratories.The SNP call rate is considered to be an indication of the overall performance of the assay; according to the manufacturer's protocol, this parameter should be >85%. Other authors have asserted that the overall call rate is not an indication of successful performance on the mapping array when using FFPE DNA, and that one should expect and accept call rates in the range of 70% to 95%.18 This is understandable, because FFPE DNA is fragmented, and because large fragments are not present for amplification, which results in these decreased call rates.17 Although samples with call rates <85%, in our experience, do yield adequate CCN results, results from samples with call rates >85% show better detection of LOH owing to fewer SNP no-calls in the regions of genomic loss (data not shown). Higher call rates thus do improve the detection of chromosomal losses by improving the correlation between copy number estimates and LOH.To confirm the accuracy of the copy number variations obtained from the SNP genotyping array data, we validated the copy number status of 4 renal epithelial tumors using aCGH. As shown in Figure 5, all copy number changes seen using SNP arrays were also seen with aCGH, except for a gain of chromosome 19 in CRCC4 seen by aCGH, which the SNP array did not detect. Both methods have a relatively low number of probes in this chromosome; hence, it is not possible to determine which of the 2 results is more reliable. We opted to validate copy number changes using aCGH rather than quantitative PCR because the copy number changes seen in these tumors were relatively small changes from the normal diploid state (ie, copy number 3 or copy number 1). Therefore, the ΔΔCt between normal and copy number changes would be too small to reliably detect with quantitative PCR. The LOH detected by SNP arrays were confirmed by microsatellite analysis at all the loci examined.Although our optimized protocol significantly improves the performance of the GeneChip Mapping assay on FFPE tissues, there are considerations that are not addressed by our study. Excessively long formalin fixation times might affect the results, and the need for increased starting DNA can be difficult to achieve, if there is scant tissue available in the block. The resolution of the 10K 2.0 arrays might not be adequate for all tumor types, depending on the expected chromosomal changes, which can be deduced from the pathophysiology and known genetic lesions. Renal epithelial tumors have small copy number gains and losses over large chromosomal regions (eg, monosomies/trisomies of entire chromosomes or chromosomal arms), which are readily detected with lower resolution arrays. Other tumors studied by our group have focal, high amplifications, which can be difficult to discriminate from noise, and they might be better suited for higher density arrays, which would provide more local SNP coverage (data not published). Tumors with significant chromosomal alterations less than 300 kb most likely require arrays with higher resolution (250 K and above). In future clinical applications, it is possible that custom arrays with increased SNP coverage in diagnostically relevant areas of the genome will be used. This would be desirable, as SNP arrays designed for research keep changing and might be phased out.It is important to note that DNA obtained from frozen tissue is always preferable to DNA from FFPE tissues. Results from frozen tissue show higher call rates and less SD. The fact that only 250 ng of DNA are required for the frozen tissue protocol allows this technique to be performed with limited amounts of tissue. However, assessment of tumor/normal content in a frozen-section slide is not always straightforward when dealing with small frozen samples (preparation of the frozen sample might consume all available material). The effect of different levels of normal tissue contamination is an important parameter that requires further study. It necessitates careful consideration of tumor architecture and growth pattern, and pathologists need to be consulted in the early stages of the experimental design. Renal cell tumors and pheochromocytomas both tend to grow into large tumor balls with pushing edges and relatively little intervening normal stroma. Some tumors, however, grow in crablike extensions through normal tissue and/or with a marked host response. These latter tumors will be difficult to macrodissect, and they are thus likely to have much more normal contamination, to confound the downstream analysis. If such samples are used, or if frozen tissue not evaluated for tumor content is used, recent algorithms optimized to deal with normal contamination should be used.20Equally important as the method used to generate the SNP genotyping data is the method used for the computational analysis. To obtain SNP probe intensity data, the array image files are analyzed with the GTYPE software (Affymetrix), which generates the .cel and/or .chp files that can be used for further analysis of copy number and LOH. Currently, there are few software packages available for this task. In their paper, Thompson et al used the Affymetrix Chromosome Copy Number Analysis Tool v1.1 (CNAT) and cited analysis limitations that were due to the amplification of background noise in their FFPE samples, with a higher amplitude of the signal being seen in the copy number changes. In our study, we used the CNAG 2.0 software package, which includes robust compensations for systematic deviations of raw signal ratios across different experimental conditions, optimizes selection of best-fit references, and allows for smoothing of the data by means of a moving average.9 This allowed for high-quality copy number analysis with improved signal-to-noise ratio, as evidenced in the frozen/FFPE comparisons in Figure 3, and robust performance with 2 different types of tissues. Tumor samples from both the pheochromocytoma cohort and the renal tumor cohort were analyzed using both the paired normal sample and the CNAG-generated best-fit references. For all samples, the lowest SD of the log2 ratio was obtained using the best-fit references.In summary, we have optimized the GeneChip Mapping assay for use on formalin-fixed, paraffin-embedded tissues. The data shows comparable SNP call rates, signal-detection rates, copy number changes, and LOH estimation for frozen and FFPE tumors. Additionally, the copy number results obtained using SNP arrays on FFPE tissues are corroborated by the expected chromosomal changes in specific tumor morphologies, and are validated by aCGH and LOH analyses. This optimized protocol will not only allow researchers to tap into the pathology archives for adequate research samples but, given that most clinical material exists as FFPE tissue, will also open the possibility of diagnostic assay development with this technology.ACKNOWLEDGMENTSThe authors thank the Health Sciences Tissue Bank at the University of Pittsburgh for their help in retrieving tissue samples and deidentified patient information. 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Capillary electrophoresis profiles of the Xba restriction enzyme digests. A, Frozen tissue (250 ng of DNA) and paraffin-embedded tissue (B—250 ng and C—1 μg of DNA). Increasing the amount of input DNA increases representation of DNA fragments from more sizes in the digest.
Electropherogram of PCR products from formalin-fixed, paraffin-embedded, and frozen tissues. Range of amplicon size for paraffin-embedded tissue is different from that for frozen tissue. Larger DNA fragments are not represented in the amplified DNA. Performance of GeneChip Mapping Assay in Frozen and Paraffin-embedded Tissues Agreement in SNP Calls
Copy number and LOH analysis on pheochromocytoma showing correlation between frozen and FFPE samples. Pheochromocytoma samples C109, D399, and GB253 are shown with the frozen copy number results in the top plot (A) and the corresponding FFPE results in the lower plot (B) for each sample. The SNPs are arranged in chromosomal order along the x-axis with chromosome 1 on the far left and chromosome X on the far right. The samples are not sex-matched to the reference samples, so the copy number of X chromosome should not be considered in this image. The y-axis of the uppermost plot for each sample represents the estimated copy number as a log2 ratio averaged over 20 SNPs (a), thus the 0 line indicates a copy number of 2, and deviations up or down represent gains or losses. The dark green bars (b) are heterozygous calls in the tumor. The upper yellow bar (c) represents the copy number data in a color-coded HMM (yellow=copy number 2, pink=copy number 3, and aqua=copy number 1). The lower yellow bar for each sample (d) represents the HMM for LOH likelihood (yellow=no LOH, the more blue the bar=the more likely the LOH in that region). Chromosomal aberrations detected in the fresh tumor sample were also detected in the corresponding FFPE sample for each case.
Copy number and LOH analysis of FFPE renal epithelial tumors. DNA from paraffin-embedded samples from each of the 4 subtypes of renal cell neoplasms was analyzed: OC, chromophobe carcinoma (CHRCC), PRCC, and conventional
renal cell carcinoma (CRCC). Please refer to Figure 3 for an explanation of the copy number/LOH plots. Results show characteristic findings for these tumors and illustrate the application of this protocol to a clinically relevant problem.
Validation of SNP array copy number results by aCGH for renal epithelial tumors. Representative data from samples validated by aCGH. The top plot in each box is the aCGH copy numbers for each bacterial artificial chromosome plotted in chromosomal order from left to right. The smaller bottom plot in each box is the copy number for each SNP on the SNP 10K mapping array plotted in chromosomal order from left to right with each chromosome being color-coded. A, CRCC; B, PRCC. Correlation of LOH Analysis by SNP Arrays and PCROptimization of the Affymetrix GeneChip Mapping 10K 2.0 Assay for Routine Clinical Use on Formalin-fixed Paraffin-embedded TissuesLyons-Weiler Maureen MSc; Hagenkord, Jill MD; Sciulli, Christin BS; Dhir, Rajiv MD; Monzon, Federico A. MDOriginal ArticlesOriginal Articles117p 3-13
