Formalinfixed Paraffinembedded Clinical Tissues Show Spurious Copy

Clin Genet 2007: 72: 441–447 # 2007 The Authors Printed in Singapore. All rights reserved Journal compilation # 2007 Blackwell Munksgaard CLINICAL GENETICS doi: 10.1111/j.1399-0004.2007.00882.x Short Report Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles

Mc Sherry EA, Mc Goldrick A, Kay EW, Hopkins AM, Gallagher WM, EA Mc Sherrya,b, A Mc Goldrickb, Dervan PA. Formalin-fixed paraffin-embedded clinical tissues show EW Kayc, AM Hopkinsb,d, spurious copy number changes in array-CGH profiles. WM Gallaghera and PA Dervanb,d Clin Genet 2007: 72: 441–447. # Blackwell Munksgaard, 2007 aUCD School of Biomolecular and b Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens Biomedical Science, and UCD School of Medicine and Medical Science, are invaluable in discovery of prognostic and therapeutic targets for UCD Conway Institute, University College diseases such as cancer. However, the suitability of FFPE-derived genetic Dublin, Belfield, Dublin, Ireland, material for array-based comparative genomic hybridization (array- cDepartment of Histopathology, CGH) studies is underexplored. In this study, genetic profiles of matched Beaumont Hospital and The Royal FFPE and fresh-frozen specimens were examined to investigate DNA College of Surgeons in Ireland, integrity differences between these sample types and determine the Beaumont Hospital, Dublin, Ireland, and impact this may have on genetic profiles. Genomic DNA was extracted dMater Misericordiae Hospital, Dublin, from three patient-matched FFPE and fresh-frozen clinical tissue Ireland samples. T47D breast cancer control cells were also grown in culture and processed to yield a fresh T47D sample, a fresh-frozen T47D sample and a FFPE T47D sample. DNA was extracted from all the samples; array- Key words: comparative genomic hybridization – formalin fixation – CGH conducted and genetic profiles of matched samples were then genetic profiling compared. A loss of high molecular weight DNA was observed in the FFPE clinical tissues and FFPE T47D samples. A dramatic increase in Corresponding author: Professor William absolute number of genetic alterations was observed in all FFPE tissues Gallagher, UCD School of Biomolecular relative to matched fresh-frozen counterparts. In future, alternative and Biomedical Science, UCD Conway Institute, University College Dublin, fixation and tissue-processing procedures, and/or new DNA extraction Belfield, Dublin 4, and CGH profiling protocols, may be implemented, enabling Ireland. identification of changes involved in disease progression using stored Tel.: 1353 1 7166743; clinical specimens. fax: 1353 1 2837211; e-mail: [email protected] Received 14 May 2007, revised and accepted for publication 6 July 2007

Genomic instability is a hallmark of human cancer ization (CGH). Array-CGH is conducted by (1). Amplification or deletion of distinct sub- differentially labelling total genomic DNA from chromosomal areas can lead to over- or under- test and reference samples followed by cohybrid- expression of key genes, thus conferring a growth ization onto an array consisting of DNA probes advantage to malignant cells (2). Conversely, spanning the genome. The ratio of fluorescent deletions of tumour suppressor genes, such as intensities allows identification of copy number those involved in cell death, may also push the change between test and reference samples. genetic balance towards malignant growth. Ampli- Classically, this technique has been successfully fied genes are, therefore, important targets for used on DNA extracted from fresh-frozen clinical therapeutic invention, and identification of such tissue specimens, as these yield higher quality copy number alterations (CNAs) can help elucidate nucleic acids (3–5). However, as the availability potential mechanisms involved in tumour develop- of fresh-frozen tissue is often limited, studies of ment and progression and identify cancer subtypes. archival formalin-fixed paraffin-embedded (FFPE) The most widely used and successful technique clinical tissue specimens with accompanying ret- to study CNAs is comparative genomic hybrid- rospective data would be of enormous benefit in

441 Mc Sherry et al. elucidating key genes involved in disease pro- were immediately frozen using liquid nitrogen gression. Array-CGH studies using FFPE DNA upon reaching the histopathology laboratory have been reported (6–8), although little is known from surgery. Samples were stored at 280°C for of the true impact of tissue fixation and process- a maximum of 2 months prior to frozen sectioning ing on resulting genetic profiles. on a cryostat. DNA was immediately extracted The present study was primarily performed to after sectioning. Matched colon cancer FFPE assess the correlation of array-CGH profiles of tissues were fixed in 10% formalin for an average DNA extracted from matched FFPE and fresh- of 16 h before embedding in paraffin. Blocks were frozen tissues, investigate the number of CNAs stored at room temperature for a maximum of present in DNA extracted from FFPE tissues with 2 months prior to sectioning. DNA was then respect to their matched fresh-frozen counter- extracted within 1 week of sectioning. parts, and to assess any difference in DNA quality between these sample types. DNA extractions Materials and methods DNA from all above sources was extracted using Cell line and tissue preparation the NucleoSpin Tissue Kit (Clontech, Saint- T47D breast cells (European Collection of Cell German-en-laye, France). The following additional Cultures, UK) were cultured in Dulbecco’s Mod- protocol was applied prior to DNA extraction ified Eagle’s Medium 1 10% foetal bovine serum, from FFPE tissue. FFPE tissue sections were 200 mM L-glutamine. Three tissue culture flasks placed in 1.5 ml tubes. Xylene (1 ml) was added were grown to confluency. Three identical cell and mixed at room temperature for 30 min. Tubes T47D cell pellets were prepared, and genomic were microcentrifuged for 3 min at 11,000 3 g to DNA was immediately extracted from the first. A pellet tissue, and subsequent supernatant was FFPE cell sample was prepared from the second removed. Ethanol (96% v/v; 1 ml) was added to pellet, by first fixing T47D cells by suspension in each tissue, mixed, and microcentrifuged for 10% neutral buffered formalin (NBF) for 1 h at 3 min at 11,000 3 g then ethanol was removed. room temperature. The pellet was centrifuged at This step was repeated, and after removal of 18,000 3 g r.p.m. for 5 min, formalin removed, ethanol, the tissue was incubated at 37°C until all 70% ethanol added, mixed and left overnight. The ethanol had evaporated. The standard Nucleo- pellet was then dehydrated through increasing Spin Tissue Kit manual procedure was then concentrations of ethanol (70%, 95%, 100%, and applied to all tissues for extraction of DNA. 100%) at room temperature for 1 h 45 min each Extracted DNA was quantified using a NanoDrop and then finally in xylene at room temperature for ND-1000 Spectrophotometer (Nanodrop Tech- two steps of 1 h 45 min each. Liquid paraffin was nologies, Wilmington, DE). DNA integrity was added to the pellet, and cells were maintained at assessed by analysis of 100 ng DNA via 2%agarose 60°C (melting point of paraffin) and replaced gel electrophoresis. every 20 min for a period of 2 h to ensure removal of residual xylene. The pellet was then allowed to solidify at room temperature in fresh paraffin and Array-CGH was then implanted in a new paraffin block from which sections were then cut for DNA extraction The GenoSensor array-CGH platform (Vysis; from FFPE cells. Fresh-frozen T47D cells were Abbott Laboratories, Des Plaines, IL) was utilized prepared by snap-freezing the third pellet of T47D in this study. In brief, test and reference DNA cells in optimum cutting temperature (OCT) (100 ng) were labelled with Cy3 and Cy5 fluoro- matrix. The frozen block was stored at 280°C phores (1 mM). Labelled targets were DNase for 1 week, then trimmed to remove excess OCT treated for 1 h at 15°C then purified by sodium matrix from the frozen cell pellet, and fresh-frozen acetate and ethanol precipitation steps. The quality DNA was extracted. of purified, labelled DNA was assessed by agarose Three cases of patient-matched fresh-frozen gel electrophoresis. Equal concentrations of and FFPE colon cancer tissue samples were labelled test and reference DNA were then com- also acquired for array-CGH investigation from bined and loaded onto the GenoSensor Array 300 the Department of Histopathology, Beaumont array. Hybridization was conducted in the pre- Hospital, Dublin. DNA was extracted from a sence of humidified 23 standard saline citrate (SSC)/ 20 mm section of each fresh-frozen tissue and each 50% (v/v) formamide for 60 h at 37°C. Arrays matched FFPE tissue, which had been processed were washed in 23 SSC/50% (v/v) formamide, as follows. Colon cancer tissues for frozen storage followed by 13 SSC. Spots were counterstained

442 Array-CGH of FFPE tissues with DAPI, and scanning was performed using the differences in quality by agarose gel electropho- GenoSensor Reader System. resis (Fig. 1). Lanes 1–3 show a complete lack of high molecular weight DNA in FFPE clinical samples. Lanes 4–6 show the retention of high molecular weight DNA in fresh-frozen clinical Image acquisition and data analysis samples, with minor degradation as indicated by The GenoSensor reader was used to acquire the appearance of lower molecular weight DNA. a three-colour image of each microarray using DNA samples extracted from three alternatively a charge coupled device camera and a 175W xenon processed T47D breast cancer cell samples were light source. Automated analysis was then con- also compared. Lane 8 shows the retention of ducted by the GENOSENSOR READER software high molecular weight DNA in the DNA as follows. Spot segmentation and identification extracted from fresh T47D cells. Lane 9 shows was conducted to isolate and relate every DNA that snap-frozen OCT-embedded T47D cells spot on the microarray to a particular DNA target produced DNA of high molecular weight with clone. Spot intensity and brightness was measured little degradation. Lane 7 shows a large DNA for each spot and ratios of Cy3 to Cy5 fluores- smear with the loss of the majority of high cence were calculated. Ratio values were then molecular weight DNA. This DNA, which was normalized, subjected to statistical analysis using extracted from FFPE T47D cells, displayed the an analogue of the t-test and placed in a report highest degradation of the three matched T47D format. This t-test analogue takes account of samples. These results indicate that formalin- modal target variability, replicate spot ratio fixation and paraffin-embedding may have a neg- variability of the target being assessed and ative effect on the quality of extracted DNA. assumes a normal distribution of modal target DNA yields from differentially processed pellets mean ratios, thus providing an unbiased signifi- of 3.5 3 107 T47D cells also differed significance estimate for non-modality. cantly. Yields of 43.2, 7, and 1.6 ug were The resulting target report contained the mean quantified for fresh, OCT fresh-frozen, 1 h and coefficient-of-variation of the normalized formalin-fixed cells, respectively. CGH ratios of the three replicate spots, together We next sought to identify any differences in with a significance or p value that the mean ratio the array-CGH profiles of DNA from alterna- corresponds to a gain or loss of copy number for tively processed samples (Fig. 2). A dramatic each target clone on the array. Only those targets whose computed p values were less than 0.01 for all samples were selected in this study. This was conducted to improve the stringency of results, and thus, the comparability of resulting genetic profiles of alternative sample types. Subsequent target report tables from matched fresh-frozen and FFPE specimens were compared, and numbers of genetic alterations and areas of genomic change were recorded. Additionally quality parameters, as set by the array manufacturers, were recorded for each array. Only those arrays passing all quality parameters were included in this study.

Results Fig. 1. Assessment of genomic DNA integrity via agarose This study was conducted to primarily investi- gel electrophoresis. Genomic DNA (100 ng) extracted from cell lines and clinical tissue, with varying degrees of gate the possible impact of formalin-fixation and degradation is shown. High molecular weight and low paraffin-embedding on DNA degradation and molecular weight DNA are indicated. DNA of 100 bp or subsequent DNA profiling. Prior to profiling 1 kb ladders from Promega were used as DNA size markers using array-CGH, we assessed possible differ- (M). Lanes 1–3: DNA extracted from formalin-fixed ences in integrity of DNA extracted from FFPE paraffin-embedded (FFPE) clinical tissues. Lanes 4–6: DNA and fresh-frozen matched samples by agarose gel extracted from fresh-frozen clinical tissues. Lane 7: DNA extracted from FFPE T47D cell line sample. Lane 8: electrophoresis using equal amounts of DNA. DNA extracted from fresh T47D cell line sample. Genomic DNA extracted from matched FFPE, Lane 9: DNA extracted from snap-frozen optimum cutting and fresh-frozen clinical samples show dramatic temperature-embedded T47D cell line sample.

443 Mc Sherry et al.

Fig. 2. Numbers of genetic changes detected in DNA from matched formalin- fixed paraffin-embedded (FFPE) and fresh or fresh-frozen samples using array-CGH. (a) Total number of statistically relevant genetic changes detected in matched T47D cell line samples and matched clinical samples. (b) Number of DNA amplifications vs deletions detected in matched T47D cell line samples and matched clinical samples.

difference in the total number of statistically ples. It can be seen that the array-CGH profiles of relevant genetic changes between matched FFPE all three FFPE clinical samples display an obvious and fresh-frozen samples was observed [Fig. 2(a)]. increase in genetic changes relative to their Of the additional genetic changes observed in all patient-matched fresh counterparts [Fig. 3(b)]. FFPE specimens relative to matched counterparts, there was an almost equal increase ampli- Discussion fications and deletions [Fig. 2(b)]. No pattern of alterations or chromosomal region bias was We have shown that suitable quantities of DNA evident in these FFPE tissues, as alterations for investigation using the GenoSensor array- occurred on almost all chromosomes. Notably, CGH platform can be obtained from both snap- an increase in the number of genetic alterations, frozen and FFPE samples. Prior to conducting from 28 to 122, was observed for fresh- and FFPE- array-CGH, test genomic DNA was visualized by derived DNA from identical T47D breast tumour agarose gel electrophoresis to assess potential cell line samples. The absolute numbers of genetic degradation. As expected from previously pub- changes, as well as regions where these occurred, lished studies (11–14), all FFPE samples showed in the fresh T47D DNA sample correlated well an increase in degradation and a loss of high with those previously reported in the literature molecular weight DNA in comparison with using standard chromosome CGH spreads and matched fresh-frozen specimens. A complete loss of spectral karyotyping techniques (9, 10). high molecular weight DNA was observed in all Differences can also be observed when compar- FFPE clinical tissues examined, indicating a sub- ing array-CGH profiles of DNA extracted from stantial deleterious effect of formalin fixation on the three alternatively fixed and processed T47D DNA quality. In our hands, different DNA cell line-derived samples [Fig. 3(a)]. Profiles of extraction kits had negligible effects on DNA fresh- and snap-frozen samples are almost iden- quality after formalin fixation, producing similar tical; however, a large increase in genetic changes highly degraded DNA (data not shown). For- can be seen in the FFPE DNA sample. We also malin fixation alone was shown to be sufficient sought to examine the difference in array-CGH to produce degraded DNA, which showed mul- profiles of alternatively processed clinical sam- tiple spurious genetic alterations in array-CGH

444 Array-CGH of FFPE tissues

Fig. 3. Array-CGH profiles of fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue DNA. (a) Array-CGH spectral profiles of differentially processed T47D cancer cell pellets. A. Fresh T47D cell line DNA. B. Snap-frozen optimum cutting temperature-embedded T47D cell line DNA. C. FFPE T47D cell line DNA. (b) Array-CGH spectral profiles of matched fresh-frozen and FFPE DNA extracted from colon cancer clinical samples. A. Tissue 1 fresh-frozen DNA. B. Tissue 1 FFPE DNA. C. Tissue 2 fresh-frozen DNA. D. Tissue 2 FFPE DNA. E. Tissue 3 fresh-frozen DNA. F. Tissue 3 FFPE DNA. Green, statistically significant genetic amplifications; Red, statistically significant genetic deletions. profiles (Figs S1 and S2, supplementary material It was concluded that samples fixed for 20 h online). Additionally array-CGH genetic profiles showed similar, but notably not identical, profiles of clinical archival FFPE and fresh-frozen colon to freshly harvested DNA. Although the above tissues showed a notable increase in total reports cited discordance between array-CGH numbers of statistically relevant genetic alter- profiles of FFPE and fresh-frozen tissue, the issue ations in FFPE tissues compared with patient- of false-positive target discovery was not men- matched fresh-frozen specimens. tioned nor were alternative tissue fixation or Previous reports have noted differences in DNA embedding processes suggested. extracted from matched FFPE and fresh-tissue Tissue processing by formalin fixation followed specimens. Inoue et al. and Serth et al. (12, 14) by paraffin embedding provides an opportunity to both reported a reduction in the yield of total store large banks of tissue and allows retrospective genomic DNA from FFPE specimens compared genomic studies of tumours to be performed, in with matched frozen counterparts. Discordances which chromosomal imbalances associated with between array-CGH profiles of alternatively tumour development and progression may be iden- processed samples have also been reported, noting tified. In contrast, the creation and hosting of bio- the appearance of spurious copy number gains banks of fresh-frozen tissue comes with limitations and losses in FFPE material only. Lee et al. (15) such as storage space and power requirements. noted that matched FFPE and frozen samples Currently in the clinical setting, emphasis is exhibited similar array-CGH profiles, but also based on preservation of tissue morphology for observed several discordant aberrations in FFPE pathological diagnosis, rather than the preserva- samples. Little et al. (16) reported the same tion of nucleic acids within tissue specimens. Ten phenomenon; citing tumour heterogeneity, alter- percent buffered formalin, an aqueous dilution of nate tissue sampling, or a drop in reliability of formaldehyde, is the most widely used fixative as DNA extracted from FFPE tissue as possible it preserves a wide range of tissues and tissue reasons for the aberrations. Ghazani et al. (11) components and is inexpensive (17, 18). However, showed aberrant changes in array-CGH profiles formaldehyde is known to interact with DNA of cultured cells fixed for 30 min, 20 h and 1 week. and initiate DNA degradation (19). Numerous

445 Mc Sherry et al. additional factors can also influence the recovery Supplementary material of high-quality DNA from tissues. Longer Fig. S1. Assessment of genomic DNA integrity via agarose gel pre-fixation times, or time from surgery to electrophoresis. Lane 1: DNA extracted from fresh T47D pellet. fixation, can have detrimental effects on DNA Lane 2: DNA extracted from snap-frozen optimum cutting integrity (20). Although it has been reported that temperature-embedded T47D cell line sample. Lane 3: DNA limited tissue fixation times have minimal impact extracted from T47D pellet fixed for 1 h in 10% NBF. Lane 4: on array-CGH profiles (11), in the hospital DNA extracted from T47D pellet fixed for 24 h in 10% NBF. Lane 5: DNA extracted from HOPE-fixed T47D pellet. laboratory, standard operating procedures can Fig. S2. Array-CGH profiles of DNA non-embedded differen- vary between hospitals and routine fixation times tially fixed T47D pellets. 1. Fresh T47D cell line DNA for can vary from 6 to 48 h. comparison. 2. DNA extracted from Hepes-Glutamic acid buffer Numerous alternative fixatives to NBF have, mediated Organic solvent Protection Effect-fixed T47D cell however, been reported to have minimal impact of pellet. 3. DNA extracted from 1 h 10% NBF-fixed T47D cell pellet. 4. DNA extracted from 24 h 10% neutral buffered formalin- nucleic acids while maintaining tissue morphology fixed T47D cell pellet. Green, statistically significant genetic for diagnosis (21–27). Alcohols such as ethanol amplifications; Red, statistically significant genetic deletions. and methanol have been shown to preserve high- quality RNA and DNA. However, alcohols are often not sufficient to maintain morphology for Acknowledgements diagnosis (21, 25, 26). The AMeX method, which The authors wish to thank Dr Robert Cummins for provision of is based on cold acetone fixation followed by clinical tissue samples. This work was funded by Cancer clearing with methyl benzoate and xylene, then Research Ireland, the Irish Research Council for Science, embedding in paraffin, has been shown to be Engineering and Technology under the National Development a versatile multipurpose tissue-processing pro- Plan of Ireland and the Health Research Board of Ireland, the latter under the auspices of the ÔBreast Cancer Metastasis: cedure, maintaining tissue morphology and DNA Biomarkers and Functional Mediators’ research programme. integrity (23). This method does, however, require The UCD Conway Institute, University College Dublin. longer fixation at colder temperatures than formalin fixation, but could be easily implemented in the clinical setting. References The Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect (HOPE) tech- 1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000: 100: 57–70. nique utilizes a protection solution comprised of 2. Schwab M, Amler LC. 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