Southern Blot Analysis of DNA Exfracted from Formalin-Fixed Pathology Specimens'

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(CANCERRESEARCH46, 2964-2969, June l986J Southern Blot Analysis of DNA Exfracted from Formalin-fixed Pathology Specimens'

Louis Dubeau,2 Lois A. Chandler, Julie R. Gralow, Peter W. Nichols, and Peter A. Jones Urological Cancer Research Laboratory, USC Comprehensive Cancer Center IL D., L A. C., I. R. G., P. A. I.], and Department ofPathology, Kenneth Norris, Jr., Hospital(P. W. N.], University ofSouthern California, LosAngeles, California 90033

ABSTRACF probes can be obtained from embedded specimens.In the pres ent paper, we have characterized the effect of formalin fixation We havedevelopeda methodfor the extraction ofDNA fromformalin and paraffin embedding on the availability ofDNA for Southern fixed, paraffin-embedded pathology specimens. High-molecular-weight blot analysis and have developed an extraction method which DNA was recovered from well-fixed nonautolyzed samples of viable tissue. DNA recoveredfrom samples exposed to picric acid or mercuric allows for the removal of degraded and irreversibly modified chloride containing fixatives was not intact. Increasing the formalin DNA from the preparation. The purified DNA is suitable for fixation time decreased the amount of intact DNA available. When these quantitative as well as qualitative Southern blot analyses. limitations were taken into consideration, the procedure allowed for the removal ofdegraded and chemically modified DNA from the preparation, and the final productwassuitable for quantitativeand qualitativeanalysis MATERIALS AND METHODS by Southern or dot blotting techniques. Digestion with methylation sensitive restriction endonucleases showed that DNA methylation pat Source and Handling of Tissue. Fresh tissue was obtained from the term were not altered after formalin fixation. Department of Pathology of the Kenneth Norris Hospital in Los Angeles. Unfixed tissue samples were frozen in isopentane and kept at â€”70T until DNA was extracted using the method previously described INTRODUCFION by Jones and Taylor (16). Fixed samples were sliced, washed several times with 10% buffered formalin, fixed in an excess volume of for The field of molecular biology has expanded rapidly over the main, and processedforparaffin embeddingin parallelwith routine last few years, and a large amount of new information has surgical pathology specimens.Old blocks of paraffin-embedded, for accumulated on the organization of the human genome. The main-fixed tissue were obtained from the Kenneth Norris Hospital or genome is not as static as previously thought, and gene rear from the archives of the Los Angeles County Hospital. rangement (1), amplification (2), deletion (3), mutation (4), and Digestion with Restriction Endonucleases.DNA was incubated over altered DNA methylation patterns (5, 6) are examples of deter night at 37C using 4 units ofenzyme per @gofDNA as recommended minants now thought to be important for the regulation of gene bythe manufacturer (New England Biolabs, Beverly,MA). The follow expression. Similar modifications are also thought to take place ing morning one additional unit of enzyme was added per @gofDNA, and incubation was continued for 8 h or until completeness of digestion in neoplastic processes and may have prognostic significance. was demonstrated by running aliquots of the digest on a minigel. For example, gene deletions (3), amplifications (7), and the Gel ElectrophoresisandSouthernBlotting. Aliquots of restriction presence of some restriction fragment length polymorphisms endonucleasedigests were electrophoresed on 1% agarosegels in 40 (8) have been associated with a poor prognosis for specific mM Tris-acetate-l mM EDTA, pH 7.4. After electrophoresis the DNA tumors. was stained with ethidium bromide and visualized by UV illumination. The useof this new knowledge for the study of tumor biology The DNA was then transferred to nitrocellulose filters (Millipore, and also as a clinical tool has been delayed becauseof the need Bedford,MA) usingthe procedureof Southern(17). The filters were to use fresh or frozen tissue as a source of DNA, making it air dried and baked between 2 sheets of Whatman No. 3MM paper difficult to conduct retrospective studies on a large number of undervacuumfor 3 h at 80C. patients. These restrictions would be overcome if DNA was Preparation of 32P-labeled DNA Probes. The c-Ha-ms probe (clone pT24-C3) was obtained from Dr. Mariano Barbacid from the National obtainable from the vast archives of material available in pa Cancer Institute. The probe was labeled with [32PIdCTPto specific thology departments throughout the world. Since clonable and activities >i0@ cpm/@@gusing the procedure of Feinberg and Vogelstein restrictable DNA has been obtained from sourcesas ancient as (18). Egyptian mummies (9), we were encouraged to develop an DNA Hybridization Conditions. Nitrocellulose filters were prehy extraction method for DNA from formalin-fixed, paraffin-em bridized overnight at 68T in a solution containing 6x SSC,3 0.5% bedded tissue. We are particularly interested in analyzing gene SDS, 5x Denhardt's solution (19), and denatured salmon sperm DNA rearrangements, methylation patterns, and amplification using (200 @@g/ml)(Sigma).The prehybridization buffer was then discarded, Southern blotting and dot blotting techniques. and the 32P-labeledprobeswere hybridized to the filters for 60â€”65hat Formylation of nucleic acids produces Schiff baseson free 68T in 6x SSC-0.01 M EDTA-5x Denhardt's solution-0.5% SDS amino groups of nucleotides (10.-i 2), and exposure of nucleo denatured salmon sperm DNA (100 @ig/ml).Hybridization mixes were filtered through Millex-HA 0.45-sm filter units (Millipore) as recom proteins to formaldehyde results in the formation of cross-links mendedbyMeinkoth andWahl (20) prior to addition to the nitrocel between proteins and DNA (13). The fact that both of these lulose filter to aid in the reduction of background. Following hybridi processesare reversible in aqueoussolutions (12â€”14)suggested zation, the filters were washed at room temperature once in 2x SSC to us that DNA could be recovered from formalin-fixed tissue. 0.5% SDS for 5 mm, once in 2x SSC-0.1 % SDS for 15 mm, and then Indeed, Goelz et a!. (15) have recently shown that DNA which for 1â€”3hat 55C in 0.lx SSC-0.5% SDS with multiple bufferchanges. can be cut by restriction endonucleasesand hybridized to DNA The filters were exposed to Kodak XAR-5 X-ray film with 2 DuPont Lightning-Plus intensifying screens for 10â€”14days at â€”80'C. Received 10/17/85; revised 1/21/86; accepted 3/5/86. Dot Blotting. Ten-pg samples of formalin-fixed DNA were precipi The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked advertisement in tated in ethanol and resuspendedin 9 @zlof0.lx SSC. One @tlof2 N accordancewith 18 U.S.C. Section 1734solelyto indicatethis fact. NaOH was added, and the samples were boiled for 5 min. Twenty j@l I This work was supported by Grant CA40422 from the National Cancer Institute and by the Dorothy and Hugh EdmondsonResearchFund. 3The abbreviations usedare: SSC, 0.15 M NaCl-0.0l5 M sodium citrate; SDS, 2To whom requestsfor reprints should be addressed. sodium dodecyl sulfate. 2964

DNA FROM FORMALIN-FIXED TISSUE of 3 M NaC1 were added, and the tubes were put on ice. Each sample and SSC. The final pellet was resuspended in 5 ml of SSC containing was spotted onto Millipore (Bedford, MA) type HA, 0.45-@tmnitrocel 1% SDS and 100 @Lgofproteinase K per ml (E. Merck, Darmstadt, lulose paper. The paper was dried on a piece of Whatman No. 3MM Germany) (Solution A). If the block contained only small amounts of filter paper for 30 mm, washed twice for 15 mm in 6x SSC, and dried tissue (less than 25% of the volume of the block), it was resuspended for an additional 30 mm. The nitrocellulose paper was baked at 80C in 2.5 ml of Solution A insteadof 5 ml. The preparationwasthen under vacuum for 3 h between 2 sheets of Whatman No. 3MM paper. incubated at 37C in a plastic centrifuge tube that was taped inside an Prehybridization and hybridization with a 32P-labeledc-Ha-ras probe 850-cm2 tissue culture roller bottle (Falcon, Oxnard, CA) and rotated were done as for Southern blots. at a speed of one revolution every 2.5 min. All of the DNA present in Extraction of DNA from Formalin-fixed, Paraffin-embedded Speci the tissue was slowly released in a soluble form during this process mens.A flowdiagram of the various steps used for the DNA extraction (Fig. 2). The rate of release was not the same for different types of procedureisshownin Fig. 1. Commentsabouteachof thesestepsare tissue and depended on the density and abundance of extracellular given in the rest of this section as well as in â€œResults.â€• stroma. For highly cellular tissue containing little stroma, such as Selection of Paraffin Blocks. The single most important cause of spleen (Fig. 3a), over 80% of the DNA was released within 24 h (Fig. failure to recover intact high-molecular-weight DNA from formalin 2). However, it took 6 days before a similar percentage of the total fixed pathology specimens is the use of inadequately fixed tissue. DNA could be recovered from a uterine leiomyoma containing large Histological sections of paraffin blocks should therefore be examined quantities of dense stroma (Figs. 2 and 3). DNA from a metastatic prior to DNA extraction, and the blocks should not be usedif autolysis teratoma of moderate cellularity containing areas of loose stroma was is evident. Examination of histological sections may also reveal the solubilized at an intermediate rate (Fig. 2). The rate of solubilization presence of tissue necrosis. Such areas should be removed with a scalpel and the amount of total DNA recovered were not altered when the blade before starting the extraction. Intact DNA could not be recovered length oftime the tissue had been exposed to formalin was varied from from tissue that had been exposed to picric acid (Bouin) or mercuric 12 h to 5 days (results not shown). chloride (Zenker, B5) containing fixatives. The amount of intact DNA Incubation in Solution A was done with 2 consecutive extractions in available from the tissue decreased as the length of fixation in formalin order to remove low-molecular weight DNA from the preparation. was increased. After 6 to 12 h of incubation (24 h for very fibrous tissue),the Tissue Homogenization. One effect of formalin fixation is to produce preparation was centrifuged, and the supernatant was discarded. The a hard rubbery tissue difficult to homogenize. We have taken advantage pelletwasthenresuspendedinfreshSolutionA andput backat 37T. of the fact that the fixed tissue was embedded in paraffin blocks to cut This step also allowed removal of pigments such as hemoglobin. The it into thin slicesusingthe microtome.Five-tim-thicksliceswerecut second incubation was stopped after 2 to 6 days, depending on the until the whole paraffin block was exhausted or enough tissue was density of the tissue stroma. obtained,andthesliceswerecollectedintoacentrifugetube.Practically Extraction with Chloroform-Isoamyl Alcohol. The preparation was every cell present in the tissue was cut at this setting, thus providing a centrifuged to remove insoluble debris after the second incubation in first homogenization step. SolutionA, andthe supernatantwastransferredto a round-bottomed Solubilization of DNA. The thin tissue slices were deparaffinized and glass flask. An equal volume of a mixture of 24 parts of chloroform rehydrated by resuspension in 50 ml ofxylene(J. T. Baker, Phillipsburg, and one part of isoamyl alcohol was added, and the flask was shaken NJ) and washing 3 times by centrifugation in xylene, absolute ethanol, manually for 15 mm. The suspension was then transferred to a Corex tube (Corning, Palo Alto, CA), and the phases were separated by centrifugation at 7000 rpm for 5 mm in the 55-34 rotor of a Sorvall CUT EMBEDDED TISSUE WITH MICROTOME centrifuge. The top phase was removed using a bent-tip Pasteur pipet, transferredto anotherCorex tube,and precipitatedovernightwith 2 volumes of absolute ethanol at â€”20Â°C.Theprecipitate was recovered DEPARAFFINIZE AND REHYDRATE by centrifugation for 30 mm at 7000 rpm in a Sorvall centrifuge and resuspended in 0.5 to 1.0 ml of SSC for each paraffin block originally used. Larger volumes did not allow for spooling of the DNA after the phenol extraction (see later). FIRST INCUBATION IN SOLUTION A (discard supernatant)

0 Â¶1? â€˜U N SECOND INCUBATION @NSOLUTION A -J (save supernatant)

U) EXTRACT WITH CHLOROFORM - ISOAMYL ALCOHOL z 0 .-J DIGEST WITH RNase AND PRONASE 0

EXTRACT WITH PHENOL

DAYS Fig. 2. Effect of stromal density on the rate of extraction of DNA from SPOOL formalin-fixed tissues. Paraffin blocks from the spleen (â€¢),teratoma (A), and (discard unspoolable DNA) uterine leiomyoma (U) shown in Fig. 3 were cut, and the tissue slices were incubated at 37C in Solution A after rehydration. The samples were centrifuged at the indicated time intervals, the supernatants were saved, and the pellets were resuspendedand reincubatedin fresh Solution A until the next time point. The DIALYZE amount of total DNA present in each supernatant as well as in the insoluble pellet remaining after 10 days was determined using the diphenylamine method (29). Fig. 1. Flow diagram for extraction of DNA from formalin-fixed, paraffin Total DNA originally present in each sample wascalculated by adding the amount embedded tissues. of DNA in each supernatant to the DNA left in the residual pellet after 10 days. 2965 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. @@ .@

DNA FROM FORMALIN-FIXED TISSUE

- .â€˜ ,. ,, .@ ,â€˜.1.@.;,. â€˜,@â€˜ heat-treated RNase and self-digested Pronase was done as for DNA @@ @@;@::â€˜â€œ%!@extraction from unfixed tissue. Extraction with Phenol. After digestion with Pronase, the preparation was extracted with phenol as for DNA from fresh tissue. One volume of ethanol was added to the aqueous phase of the phenol extract, and @@@@@ @; â€˜..: -â€˜@: the DNA was spooled with a glass rod, washed once with 70% ethanol, @ ..â€˜@.â€˜... ,@, ., @@@@ @...... â€˜ y dissolved in 0.lx SSC, and dialyzed extensively against this buffer under sterile conditions. Variable quantities of unspoolable DNA were always seen in the phenol extract. This unspoolable material was not suitablefor further analysisandwasdiscarded. RESULTS It should be stressed that intact high-molecular-weight DNA could not be recovered from autolyzed and/or inadequately fixed tissues, and any area of tissue necrosis in the original sample generated increased amounts of degraded low-molecu lar-weight DNA in the preparation. When care was taken to select well-fixed viable tissue specimens, DNA preparations could be obtained that had UV absorbancespectra very similar to those obtained from unfixed DNA samples (results not shown). Thus formalin fixation and processing for histology did not damage the DNA so that its UV absorbance properties were appreciably altered. Effect of Extraction Time. Because variable amounts of de graded DNA were always present in our preparations, we wanted to see if this degraded material was solubilized at the same rate as high-molecular-weight DNA during the incubation step in Solution A and to determine if this step could therefore be used to remove the low-molecular-weight material. Paraffin blocks from 3 different inadequately fixed breast tumors were selected in order to obtain DNA preparations with increased amounts of degraded low-molecular-weight material. The blocks were sectioned with the microtome, rehydrated, and incubated in Solution A for 24 h. The preparations were then centrifuged, and the pellets were resuspended in fresh Solution

@ ;- - -:l.@'@ A. The supernatantsandthe resuspendedpelletswereput back at 37C for an additional 60 h. DNAs from each tube were purified using the method described in â€œMaterialsandMeth @ â€˜:@â€¢@: @ . â€˜:â€˜â€¢ @. .:@ odsâ€•exceptthat the purified samples were collected by centrif

@ ; fl@ : ...â€œ,â€˜ â€¢r@@' - ugation instead of spooling after the phenol extraction step. @@@ ;.s. - :@:@. . ,4_ @ @â€˜.: . - @. â€” DNA from each purified sample was electrophoresed on a 1% @@@ ..â€˜. , , @.: -: â€¢ @â€¢ agarose gel (Fig. 4). The results clearly show that, for all 3 @@@@@@@@ ., - . @â€œ4@1.' @, â€¢.. ;: cases, low-molecular-weight DNA with high electrophoretic iI@ @,, j! @ \, S , % Ii mobility was recovered during the first 24 h, but not during the @ â€˜ â€œ@ - @. .-.@. â€œâ€¢â€¢4@@â€¢ (I second incubation period. Thus, low-molecular-weight DNA .. unsuitable for Southern blot analysis can be removed by per forming 2 consecutive incubations in Solution A and discarding @@@ : â€¢@ @, I :,. the supernatant obtained after the first incubation. It should be : emphasized that poorly fixed autolyzed material was used for . @: this experiment and that most of the DNA recovered from

.. : adequately fixed tissues would not have penetrated I % agarose gels unless it had first been digested by restriction endonucleases (results not shown). Fig. 3. Histological sections of tissues with different amounts and quality of stroma. Histologicalsectionsof the 3 differentorgans used for Fig. 2 are shown. Effect of Length of Fixation in Formalin. When tissue speci a, Normal spleen removed for staging of Hodgkin's disease; the section shows mens are processed in surgical pathology laboratories, formalin portions of the splenic white and red pulps, note the degree of cellularity and fixation times often vary from a few hours up to 5 days. Certain relative paucity of stroma b, metastatic teratoma proliferating glandular struc tures and mesenchymalcell types are seen;extracellularstroma is abundant, but types of tissue such as lymph nodes are routinely fixed for at its density is less than in c; c, uterine leiomyoma showing proliferating smooth least 48 h in some centers. We determined the effect of fixation muscle cells in a dense stroma. x570. time on DNA extractability, because it seemed likely that the length of fixation would be an important determinant of the Digestion with RNase and Pronase. The resuspended pellet was quality of DNA present in a specimen. Tissue samples from a vortexed gently and allowed to dissolve for 30 to 60 min at room metastatic ovarian carcinoma were fixed for 12, 36, 60, and temperature. After this period of time most of the precipitate was still 110 h. The corresponding DNAs were spooled on a glass rod in an insoluble form. The suspension was transferred to a 37C water after phenol extraction. After spooling, variable quantities of bath, and RNase digestion was started immediately. Digestion with precipitate were seenremaining in each sample. These precip 2966

a b C b C d. 12 12 12 â€¢a@Ã¶ kb

e f @. h.

23.1 -

6.6 - Fig. 5. Dot blots ofspoolable and unspoolable DNA samples. DNA from each sample of spoolable and unspoolable material in Table 1 was dot blotted onto nitrocellulose, hybridized to 3@P-labeledc-Ha-re., probe, and autoradiographed. 2.3- Dots a, b, C,and d represent spooled samples fixed for 12, 36, 60, and 110 h, respectively. Dots e,f, g, and h are the corresponding unspooled samples. 0.9- 0.6- Ha-ras probe (Fig. 5). All spoolable DNAs showed good hy bridization, although dot intensity of the sample fixed for 110 h wasslightly decreased.Hybridization intensities from samples of unspoolable DNA were reduced but showed a gradual in 0.2- creasewith increasing fixation time. DNA Samples a, b, c, d, and h were digested with MspI, electrophoresed on an agarose gel, stained with ethidium bromide, and visualized under UV light (Fig. 6). Although the staining intensity of the DNA entering the gel was similar for each sample of spooled DNA, Fig. 4. Electrophoresis of DNA extracted for different lengths of time in Solution A. Paraffin blocks of poorly fixed specimens from 3 different breast most ofthe DNA in Sample h, which is the unspoolable sample tumors (a to c) were cut with the microtome, rehydrated, and incubated for 24 h that showed the highest hybridization intensity in Fig. 5, could in Solution A. The 3 samples were then centrifuged, and the pellets were resuspended in fresh Solution A. The supernatants and resuspended pellets were not penetrate the gel. When similar experiments were done put back at 3TC for an additional 60 h. DNA was extracted from each of the using the other unspoolable samples, the data were either resulting 6 samples but was not spooled after the phenol extraction step. Each sample was electrophoresed through 1% agarose, and the gel was stained with similar or showed mainly low-molecular-weight DNA (results ethidium bromide. For each tumor, Lane 1 is the supernatant recovered after the not shown). Thus, it was important that samples be kept con first incubation, and Lane 2 is the resuspended pellet. kb, kilobase(s). centrated enough during the extraction procedure to allow for spooling of the DNA and that any unspoolable material be DNADNATable 1 Effectoftime offixation on the ability to spool discarded. was extracted from paraffin blocks of a metastatic ovarian adenocarci Digestion with Restriction Endonucleases. Fig. 7 is a stained extractionstep,noma that had been fixed for 12, 36, 60, and 110 h. After the phenol samplewasDNAs were spooled, and the unspoolable material left behind in each gel of DNA extracted from unfixed and fixed samples of a DNAwasprecipitated overnight at â€”20C in 2 volumes ofabsolute ethanol. Total metastatic ovarian adenocarcinoma. A certain amount of in quantitatednm.TotalLength in each sample by measuring uv absorbance at 260 completely digested material was present in the fixed sample totalfixationof spoolable Total un- % of even after taking the precautions mentioned above. Although DNA(h) DNA spoolable the nature of this material is not clear, it was observed in spoolable12 (,ig) DNA (jig) samples from paraffin-embedded tissue that had been fixed in 5736 829 619 ethanol without exposure to formalin (results not shown). Some 3560 474 889 23110 211 696 of this undigestible material may therefore represent single 49 568 8 stranded DNA generated by exposure to elevated temperatures during the embedding process. Increasing either the amount of enzyme or the length of digestion did not improve the com itates were grossly indistinguishable from those of intact DNA, pletenessof the reaction. The amount of DNA loaded on a gel but they did not stick to glass rods. This unspoolable material should be 2 to 3 times the amount normally used for DNA was precipitated overnight in 2 volumes of ethanol at â€”20Â°C. from unfixed samples to account for the presence of this unre DNAs were quantitated by UV absorbanceafter removing the strictable material. Most of this material did not transfer to phenol by dialysis. The proportion of total DNA from each nitrocellulose filters during Southern blotting procedures (re sample that could be spooled decreasedasthe length of fixation sults not shown). was increased (Table 1). Although 57% of the total DNA extractable after 12 h of fixation could be spooled, only 8% of Southern blots ofDNA obtained from fresh and fixed tissues, the material recovered after 110 h was spoolable. digested with either MspI or HpaII endonucleases and hybrid The rate of solubilization of the DNA during incubation in ized to 32P-labeledc-Ha-ras gene probe, were also compared Solution A and the percentage of total DNA recovered after (Fig. 8). The mobility of the fixed material was slightly slower incubation in this solution were not altered when the length of than that of DNA from fresh tissue, as also reported by Goelz fixation was varied from 12 h to 5 days (results not shown). et a!. (15). The reasons for this decrease in mobility are not Importance of DNA Spooling. Since one of the effects of clear, but this was found with all of the DNA samplesextracted increasing the length of fixation was to increase the amount of from paraffin blocks. The banding patterns of fresh and fixed unspoolable DNA in the preparation, we wanted to determine tissues were otherwise very similar, and comparison of the if this unspoolable material was still suitable for analysis. Equal results with the 2 enzymes showed that DNA methylation amounts of spoolable and unspoolable DNA from each of the patterns were not altered by formalin fixation and could be samples listed in Table 1 were analyzed by dot blots with the c analyzed by methylation-sensitive endonucleases. 2967

a b c dh ab

k kb

23. 23.3- 9.

Fig. 7. Electrophoresis of restriction DNA fragments from fixed and unfixed tissues. DNAs from a fresh (Lane a) or fixed (Lane b) bladder tumor specimen were digested with MspI, electrophoresed, and stained with ethidium bromide. kb, kilobase(s).

nization that allowed the recovery of over 95% of total DNA present in embedded tissues. We have also characterized the effect of stromal density on the rate of recovery of the DNA from fixed tissues. Despite these improvements we were not successful in ob Fig. 6. Effect of DNA spooling on electrophoretic mobility of restriction taming DNA suitable for analysis in every case. This was most fragments.DNA samples used for Dots a, b, c, d, and h in Fig. 5 were digested with Mspl, electrophoresed, and stained with ethidium bromide. kb, kilobase(s). often due to tissue autolysis or necrosis. Although our initial success rate in obtaining usable high-molecular-weight DNA DISCUSSION from old paraffin blocks was less than 50%, this was improved to almost 100% when care was taken to select blocks of well The results of our studies clearly show that DNA suitable for fixed viable tissues. The amount of usable material will un dot blotting and Southern blotting analysis can be obtained doubtedly depend on the quality of fixation of surgical pathol from formalin-fixed, paraffin-embedded material. The results ogy specimens available at any given center. Unfortunately, also show that DNA methylation patterns are preserved intact intact DNA was not obtained from tissues that had been ex during histological processing. The procedure we have devel posed to picric acid containing (Bouin) or mercuric chloride oped contains the following modifications over a simpler one containing (Zenker, B5) fixatives. recently reported by Goelz et a!. (15). (a) Degraded low-molec The number of surgical pathology specimens kept as for ular-weight DNA was removed by sequential extractions with maIm-fixed, paraffin-embedded tissue is considrable, and this Solution A; this was also an effective way to remove tissue material is available throughout the world. The ability to extract pigments (e.g., hemoglobin). (b) Chemically modified DNA intact high-molecular-weight DNA from such specimens brings unsuitable for restriction endonucleasedigestion or hybridiza a new dimension to the use of molecular biology for the study tion studies was removed by spooling out the intact DNA. (c) of tumor biology and as a diagnostic tool. Probes for immuno We have brought attention to the fact that the amount of DNA globulin genes(21â€”23)andmore recently T-cell receptor genes usable for hybridization studies decreasedmarkedly when the (24â€”27)have been used for the typing of B- and T-cell lympho time of fixation in formalin was increased to as little as 5 days. mas, and this technique can now be expanded for use on (d) We used a simple and effective method for tissue homoge formalin-fixed samples. Because DNA methylation patterns of 2968

DNA FROM FORMALIN-FIXED TISSUE

2. Brodeur, G. M., Seeger, R. C., Schwab, M., Varmus, H. E., and Bishop, J. M. Amplification of N-myc in untreated human neuroblastomas correlates abcd with advanced disease stage. Science (Wash. DC), 224: 1121â€”1124,1984. 3. Cavanee, W. K., Hansen, M. F., Nordenskjold, M., Kock, E., Maumenee, I., Squire,J. A., Phillips, R. A., and Gallie, B. L. Genetic origin of mutations . predisposing to retinoblastoma. Science (Wash. DC), 228: 501â€”503,1985. 4. Santos, E., Martin-Zanca, D., Reddy, E. P., Pierotti, M. A., Della Ports, G., and Barbacid, M. Malignant activation ofa k-ras oncogene in lung carcinoma but not in normal tissue of the same patient. Science (Wash. DC), 223: 661â€” kb 664, 1984. 5. Feinberg, A. P., and Vogelstein, B. A technique for radiolabelling DNA restriction endonucleasefragmentsto high specificactivity.Anal. Biochem. 132:6â€”13,1983. 6. Riggs, A. D., and Jones, P. A. 5-Methylcytosine, gene regulation, and cancer. Adv. Cancer Res., 40: 1â€”30,1983. 7. Little, C. 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Separation ofnewly synthesized nucleohistone by equilibrium centrifugation in cesium chloride. Biochemistry, 13: 3952â€” 3956, 1974. 14. Jackson, V. Studies on histone organization in the nucleosome using form aldehydeas a reversiblecross-linkingagent. Cell, 15:945â€”954,1978. 15. Goelz, S. E., Hamilton, S. R., and Vogelstein, B. Purification of DNA from Fig. 8. Southern blotting of fresh and fixed DNA after digestion with restric formaldehyde-fixedandparaffin-embeddedhumantissue.Biochem.Biophys. tion enzymes. DNA extracted from fresh (Lanes a and c) and fixed (Lanes b and Res. Commun., 130: 118â€”126,1985. â€˜0samplesofauterineleiomyomawasdigestedwitheitherMspI(Lanesaandb) 16. Jones, P. A., and Taylor, S. M. Cellular differentiation, cytidine analogs, and or HpaII (Lanes c and d). The restricted DNAs were electrophoresed, transferred DNA methylation. Cell, 20: 85â€”93,1980. to nitrocellulose, hybridized to 32P-labeled c-Ha-rat, and autoradiographed. kb, 17. Southern, E. M. Detection of specific sequences among DNA fragments kilobase(s). separated by gel electrophoresis. J. Mol. Biol., 98: 503â€”517,1975. 18. Feinberg, A. P., and Vogelstein, B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature (Lond.), 301: 89â€”92,1983. certain genesare tissue specific (seeRef. 6), methylation studies 19. Denhardt, D. T. A membrane-filter technique for the detection of comple of metastatic tumors may allow us to determine their tissue of mentary DNA. Biochem.Biophys.Res. Commun.,23: 641-646, 1966. origin. Viral infections, which often produce nonspecific mor 20. Meinkoth, J., and WahI, G. Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem., 138: 267â€”284,1984. phological changes only, can be readily detected by using the 21. Korsmeyer, S. J., Hieter, P. A., Ravetch, J. V., Poplack, D. G., Waldman, appropriate DNA probes. In certain cases, the method can be T. A., and Leder, P. 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A., Davis, M. M., Bengiovanni, F., and Korsmeyer, S. J. and Dr. Emil Bogenmann from the Children's Hospital of Los Angeles Rearrangementsof genes for the antigen receptor on T cells as markers of for supplying us with the fresh leiomyoma specimen. We also thank lineage and clonality in human lymphoid neoplasm& N. Engl. J. Med., 313: Ligaya Pen for her technical assistance. 776â€”783,1985. 28. Vogelstein, B., Fearon, E. R., Hamilton, S. R., and Feinberg, A. P. Use of restrictionfragmentlength polymorphismsto determinethe clonal origin of REFERENCES human tumors. Science (Wash. DC), 227: 642â€”645,1985. 29. Burton, K. A study of the conditions and mechanism of the diphenylamine 1. Klein, G. Specific chromosomal translocations and the genesis of B-cell reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. derived tumors in mice and men. Cell, 32: 311â€”315,1983. J., 62: 315â€”323,1956.

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Louis Dubeau, Lois A. Chandler, Julie R. Gralow, et al.

Cancer Res 1986;46:2964-2969.

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