Amnercan Jolunal of Pathology, Vol. 148, NVo. 1, Januiary 1996

Copyrght C) American Society for Investigative Pathology

Technical AdvanceImmunomagnetic Separation Can Enrich FixedSolid Tumors for Epithelial Cells

M. Lisa Yaremko,*Pond R. Kelemen,t Chris Kutza,*Darwin Barker,* and Carol A. WestbrookOFrom the Departments of Pathology,* Surgery,t andMedicine,* University of Chicago, Chicago, Illinois

Immunomagnetic separation is a highly specifctechniquefor the enrichment or isolation ofceUsfrom a variety offresh tissues and microorgan-isms or molecules from suspensions. Becausenew techniques for molecular analysis of solidtumors are now applicable to fixed tissue butsometimes require or benefit from enrichmentfor tumor ceUs, we tested the efficacy of immu-nomagnetic separationfor enrichingfixed solidtumorsfor malignant epithelial ceUs. We appliedit to two different tumors andfixation methodsto separate neoplasticfrom non-neoplastic ceUsin primary colorectal cancers and metastaticbreast cancers, and were able to enrich to a highdegree of purity. Immunomagnetic separationwas effective in unembeddedfixed tissue as weUasfixed paraffin-embedded tissue. The magneti-caly separated cells were amenable to fluores-cence in situ hybridization andpolymerase chainreaction ampliftcation oftheirDNA with minimaladditional manipulation. The high degree of en-richment achieved before amplification contrib-uted to interpretation ofloss ofheterozygosity inmetastatic breast cancers, and simplified fluo-rescence in situ hybridization analysis becauseonly neoplastic cells were hybridized andcounted. Immunomagnetic separation is effectivefor the enrichment offixed solid tumors, can beperformed with widely available commercial an-tibodies, and requires little specialized instru-mentation. It can contribute to interpretation ofresults in situations where enrichment by other

methods is difficult or not possible.Pathol 1996, 148:95-104)

(Am J

Enrichment is any procedure that isolates or in-creases the proportion of a specific cell type ormolecule from a heterogeneous population of cellsor molecules. In the molecular genetic analysis ofhuman solid tumors, enrichment for tumor cells canimprove sensitivity, particularly when looking for dif-ferences between tumor cells and normal tissue.Neoplastic and non-neoplastic cells are present invarying proportions in gross aliquots of solid tumor,and the presence of stromal or inflammatory cells ina sample may obscure these differences or renderthem inapparent. Microdissection from tissue blocksor slides1 is the most common method by which solidtumors are enriched, and it is widely used in studiesof loss of heterozygosity (LOH) by DNA polymor-phism analysis. Both frozen1 2 and paraffin34 sec-tions can be enriched by this method; however, it isinherently limited by the natural growth pattern of thetumor. Touch preparations and fine needle aspirates(FNAs) can enrich for neoplastic cells of a solidtumor to a limited degree, as the neoplastic cellstend to be less cohesive and thus release moreeasily than stromal cells.5 Because they result inslides with at least a proportion of monodispersedcells, touch preparations and FNAs are popularfor interphase fluorescence in situ hybridization

M. L. Y. performed a portion of the work with support from AmericanCancer Society training grant PRFT 145 and the American Society ofClinical Oncology Young Investigator Award. C. A. W. is supportedby Public Health Service grants CA53267 and CA56707, the JamesS. McDonnell Foundation, and the Bernard Kissel Fund. P. R. K. issupported by National Institutes of Health training grantT32HL07665.

Accepted for publication September 12, 1995.

Address reprint requests to Dr. M. Lisa Yaremko, University ofChicago, 5841 S. Maryland Ave. MC6101, Chicago, IL 60637.

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96 Yaremko et alAJPJanuary 1996, Vol. 148, No. 1

(FISH)6-8 of solid tumors. lnterphase FISH is a sta-tistical process, based on counts of hybridizationsignals in the nuclei of individual cells, and canbenefit from enrichment because it can be difficult todistinguish between neoplastic and stromal cells indispersions prepared for FISH; scoring only cellsknown to be tumor simplifies interpretation of num-bers.

In this study, we explored immunomagnetic sep-aration (IMS) as a method for enrichment of fixedsolid tumors for epithelial cells. IMS is a techniquethat applies the specificity of antibody-antigen inter-actions and the physical properties of super-para-magnetic beads to separate cells and is based ondifferences in the surface antigens between the dif-ferent cells in a heterogeneous population. It wasoriginally developed to deplete bone marrow of met-astatic neuroblastoma cells before autologous bonemarrow transplantation9 but has since been appliedto many other situations, including separation of bil-iary epithelial cells from hepatocytes of liver,10 cellsfrom specific segments of the kidney tubule,11 isola-tion of macrophages from synovial tissue,12 and iso-lation of pulmonary neuroendocrine cells from lungtissue,13 before culture in vitro. In the explant tissueculture of human tumors, IMS has been used toisolate malignant colon epithelial cells from colorec-tal carcinomas.14'15 Other applications of IMS havebeen to isolate DNA segments from solution for mini-sequencing,16 specific lymphocyte subsets for func-tional studies17 and, in clinical studies, to isolatemicroorganisms from tissue, food, or blood for cul-ture and identification,18 platelets from blood for bio-chemical analysis,19 and metastatic breast carci-noma cells from lymphocytes in FNAs of lymphnodes.20These applications have all used IMS for the en-

richment of fresh tissue and have generally usedspecialized antibodies. Because of its efficacy, how-ever, we were interested in whether IMS could enrichfixed solid tumors to the same degree as fresh andwhether we could use widely available commercialantibodies for its application. Specifically, we wantedto determine (1) whether IMS was effective for sep-arating epithelial from stromal cells in fixed humansolid tumors, (2) whether it could be performed withroutinely available antibodies, (3) whether resultingsuspensions would be amenable to interphase FISHand polymerase chain reaction (PCR)-formattedpolymorphism analyses, and (4) whether IMS couldsolve problems of enrichment when other methodswere not feasible. This report demonstrates the useof IMS to enrich two common tumors, colon andbreast carcinoma, fixed in two different ways and

with two different routinely available monoclonal an-tibodies. IMS was capable of a high degree of en-richment for the tumor cells and the end productsamenable to both FISH and PCR-formatted DNAanalysis. The method will be useful in molecular ge-netic studies for certain situations with fixed or archi-val samples and may have diagnostic applicationsfor genetic analysis when fresh tissue is not avail-able.

Materials and Methods

Specimens

Colorectal Carcinomas

Aliquots (0.5 to 1.5 cm3) of gross tumor wereharvested from eight colorectal carcinomas resectedat the University of Chicago. Fresh aliquots of tumorwere minced, fixed in fresh 4% paraformaldehyde for4 to 18 hours at 40C, washed three times in Trisbuffer, 0.1 mol/L, pH 7.5, and three times in calcium-and magnesium-free phosphate-buffered saline(PBS; Sigma Chemical Co., St. Louis, MO). Sampleswere stored at 40C in calcium- and magnesium-freePBS with 0.01% sodium azide until further process-ing.

Metastatic Breast Carcinomas

Lymph nodes with metastatic tumor from threecases of breast cancer were sectioned from theirparaffin blocks, six 50-,um sections each, and sec-tions minced finely and transferred directly into mi-crofuge tubes. The primary tumors and negativelymph nodes (for normal tissue comparison) from thesame patients were sectioned, microdissected,deparaffinized, and rehydrated as previously de-scribed.3 After rehydration, tissue was suspended inPBS and 0.01% sodium azide and stored at 4°C untilfurther processing.

Antibodies

Monoclonal antibodies to carcinoembryonic antigen(CEA) and epithelial membrane antigen (EMA) wereused (Dako, Carpinteria, CA). Anti-CEA was used inthe colon cancer separations and anti-EMA in theseparations of the breast metastases. The same an-tibody was used for immunohistochemical analyses.

Immunomagnetic Separation for Enrichment of Solid Tumors 97AJPJanuary 1996, Vol. 148, No. 1

Preparation of Cell SuspensionsColorectal Carcinomas

Dispersion with 0.25% trypsin, 0.25% collage-nase, and graded pepsin concentrations from 0.1 to1% (all enzymes from Sigma) were tested in thecolorectal carcinoma samples to determine optimaldispersion conditions for IMS. All enzyme disper-sions were performed in 2 ml of freshly preparedenzyme solutions at 370C for 30 minutes with pipetshearing at 5-minute intervals. Enzyme activity washeat inactivated at 650C for 10 minutes, and thecloudy dispersion supernatant was collected,washed with calcium- and magnesium-free PBS, andstored at 40C in PBS/0.01% sodium azide.

Metastatic Breast Carcinomas

Rehydrated breast cancer lymph node metasta-ses were dispersed with pepsin, in volumes of 500 [lIto 1 ml because of the smaller amount of tissue.Concentrations between 0.25 and 0.5% were tested,and light vortexing at 5-minute intervals was per-formed instead of pipet shearing. Dispersion proce-dures were otherwise the same as for colon tumors.

SeparationConditions for separations were first optimized in thecolorectal cancers. Dispersed cells were incubatedin 500 ,ul to 1 ml of 0.1% bovine serum albumin incalcium- and magnesium-free PBS for 20 minutes toblock nonspecific binding. The cells were pelletedby centrifugation and suspended in 250 ,tl of mono-clonal anti-CEA antibody (Dako) at 1:5 to 1:50 dilu-tions in Tris-buffered saline, pH 7.5. The cell-anti-body suspension was incubated for 1 hour at roomtemperature with gentle agitation on a rocking plat-form, then washed twice in Hanks balanced saltsolution (Gibco BRL, Gaithersburg, MD). Varying vol-umes of PBS were used to resuspend the cell-anti-body mix, from 200 ,ul to 2 ml, and 15 ,tl of magneticparticles coated with anti-mouse antibody (Dyna-beads 450, coated with sheep anti-mouse Fc; Dynal,Oslo, Norway) were added. This amount was calcu-lated to provide a 3:1 bead:target cell ratio as rec-ommended by the manufacturer for 2 x 106 targetcells. The resuspended mixture was incubated for 30minutes on a rocking platform at room temperature.Tubes were placed in a magnet (BioMag Separator,Advanced Magnetics, Cambridge, MA) for 10 min-utes. Once the magnetic particles were pelletedagainst the side of the tube, the supernatant was

carefully removed and the magnetic pellets werewashed twice with PBS.

Separations on the metastatic breast cancerswere the same except that monoclonal anti-EMA wasused instead of anti-CEA and volumes were propor-tionally smaller (200 ,ul to 1 ml) because of thesmaller amount of tissue. These separations wereplaced in a magnet sized for microfuge tubes (DynalMPC-E) for 10 to 20 minutes, and supernatants andpellets were treated as above.

Cytological and ImmunohistochemicalAssessment

Cytospins were made with 50 jil of each sample ontoslides coated with poly-L-lysine (Sigma). One slidewas stained with hematoxylin and eosin (H&E) andimmunoalkaline phosphatase (IAP) reactions wereperformed on the other, with anti-CEA antibody (di-luted to 1:20 in Tris-buffered saline) or anti-EMA(diluted 1:50 in Tris-buffered saline; both antibodiesfrom Dako). The IAP reactions were completed with aVectastain kit (Vector Laboratories, Burlingame, CA)according to the manufacturer's instructions.

Slides from pre- and post-separation sampleswere examined microscopically for cellularity of thesample, adequacy of dispersion, and types of cellspresent. Semiquantitative assessment for the per-centage of tumor cells versus nontumor cells presentin the dispersion was made as follows: 30 to 50%,approximately 50%, 50 to 70%, or >95% tumor cells.Both H&E and lAP-reacted slides were evaluated inall colorectal cancer samples and in the first meta-static breast tumor; in the other two breast carcino-mas, only H&E was evaluated because of limitedmaterial.

Fluorescence In Situ HybridizationAdditional cytospins of the immunomagnetically sep-arated, paraformaldehyde-fixed colorectal carcino-mas were made and used for interphase FISH. Cellswere denatured in 70% formamide at 700C and per-meabilized with proteinase K according to publishedmethods.21 A 2-,ul volume of a chromosome-1 7-spe-cific a-satellite centromere probe labeled with biotin(Oncor, Gaithersburg, MD) was added to 10 plI of ahybridization solution of 50% formamide, 6X stan-dard saline citrate, and 10% dextran sulfate andincubated briefly at 65°C. Hybridization, post-hybrid-ization washes, and detection with fluorescein-con-jugated avidin (Vector Laboratories) were performedaccording to our published methods.22 Slides were

98 Yaremko et alAJP.Januarv 1996, Vol. 148, No. I

visualized with a Zeiss Axioplan fluorescence micro-scope, and individual cells evaluated for the numberof hybridization signals per nucleus. Between 65 and100 cells were scored.

DNA ExtractionDNA was prepared from metastatic breast cancers.Sections from normal lymph nodes, microdissectedsections of primary tumor, unenriched sections ofmetastasis, and cell suspensions from IMS-enrichedsections of metastasis were lysed by proteinase Kdigestion,3 according to our published methods.DNA concentrations were measured by fluorometryand DNA used directly for PCR without further puri-fication or concentration.

Microsatellite Polymorphism AnalysisD8S133, a microsatellite polymorphism for humanchromosome 8,23 was used to evaluate LOH in thebreast cancer metastases. Primers and PCR reac-

tions were performed as previously published.3PCR reaction products were fractionated on form-

amide-polyacrylamide gels, transferred to nylonmembranes, hybridized to 32P-labeled (CA)15, andexposed to autoradiography as described by Litt etal.24 Autoradiographs were inspected for the pres-

ence of alleles; LOH was considered present whenan obvious visible decrease in the intensity of a

tumor allele was noted relative to both of the alleles inthe normal sample and the remaining tumor allele. Allapparent losses were verified by repeat reactions.

Results

Efficacy of IMS in Fixed TissuesWe tested dispersion and separation steps in the firstfour fixed colorectal cancers under a variety of en-

zyme concentrations and antibody dilutions, as de-scribed above. The desired end point of dispersion,as determined by results of subsequent IMS, was

epithelial cell clusters no greater than 100 ,tm indiameter, with release of the stromal and inflamma-tory cells and preservation of the target antigen. Alsodetermined by the results of dispersion and subse-quent IMS were the size of the gross aliquots andtissue handling before dispersion. Aliquots less than1.0 cm3 were limited in their yield of usable cells, ie,enough for H&E, anti-CEA IAP, and FISH analysis,and were exhausted by a single round of dispersion-IMS treatment. Aliquots in the 1.0- to 1.5-cm3 range

more consistently yielded adequate cell amounts for

Table 1. Enzrichment of Fixed Colorcectal CarcinomaSpecimens bv Ilmmunomnagnetic Separation

% tumorCase Stage* Gradet pre-IMSt

1 C2 Mod 50%2 B2 Poor 50-70%3 C2 Well 50%4 B2 Mod 50-70%5 C2 Mod 50-70%6 B2 Mod 50%7 B2 Mod 50%8 C2 Well <50%

% tumorpost-lMSt

>95%>95%>95%>95%>95%>95%>95%50-70%

.Modified Astler Collins pathological stage.tHistological grade as determined on routine histological

examination of paraffin sections of tumor. Well, well differentiated;Mod, moderately differentiated; Poor, poorly differentiated.

tPercentage of tumor cells before or after IMS, as determinedby cytospin and CEA staining.

all intended analyses. Additionally, dispersion yieldswere significantly improved by a second fine minc-ing step of the tumor after fixation, to <2-mm3 sam-ples (a gross aliquot able to pass through the lumenof a 5-ml serological pipet). Trypsin produced excel-lent dispersion but destroyed the CEA on the tumorcells, collagenase was also effective but expensive,and 0.3% pepsin concentration, at both acid (pH1.5) and neutral pH, was adequate for dispersionand preserved anti-CEA reactivity. We thereforechose our optimal digestion as 0.3% pepsin, incu-bated at neutral pH because it eliminated the need toretitrate the pH for subsequent steps. In the separa-tion, a primary antibody dilution of 1:5 was required;lower dilutions were less effective in yield of targetcells. Because of this, we kept the volume of incu-bation with the dispersion and primary antibody to250 Al. For the secondary antibody-bead step, how-ever, larger volumes (1 ml and greater, dependingon the size of the cell pellet) were more effective forcell recovery.Once conditions for optimal separations were de-

termined in the first few cases, separations wereperformed in eight sequentially collected colorectaltumors (Table 1) to determine the consistency of themethod in a series of clinical tumors. The dispersedcolon cancers contained varying percentages of tu-mor cells, the majority between 50 and 70% (Figure1, a and b). Other cells present were inflammatorycells, red blood cells, and stromal cells, which wouldinclude smooth muscle cells, fibroblasts, and endo-thelial cells. After IMS, the small clusters of the CEA-positive epithelial cells remain, and the inflammatoryand stromal cells have been excluded (Figure 1, cand d). In 1 1 of the 12 cases of colorectal carcinomatested in the initial steps, enrichment for epithelialcells was >95%, assessed as fewer than 1 nonre-

Immunomagnetic Separation for Enrichment of Solid Tumors 99AJPJanuary 1996, Vol. 148, No. 1

Figure 1. Tissue enrichnent by inlmmuntomagnetic weparatiotn. a: Cytospin ofpepsin-disaggregated colorectal carcinonma. The specimen conitainsstinallclusters of tumor cells and sinigle tumor cells mixed with stromal celLs anid inflainniatory cells. H&E; magnijication, x 560. b: Anti-CEA immuni-alkaline phosphatase reaction performed on unseparated dispersed tumor in a. Nuiclei counterstained with hematoxvlin; x 560. c: (Cytospin oftumorcell suspensionz after imnmunomagnetic separation. Tumnor cell cltusters coated with antibodies and nmagnetic particles remnain; inflammatory andstromal cells havc bc-cn eliminated. H&E; x 480. d: Anti-CEA immunoalkaline phosphatase reaction performed on imniunomnagnetically separatedtumor cells, confinning the CEA reactivity of the cells pclleted by the magnlet and the eliniination of nonreactive cells. Nuclei counterstained withhematoxylin; x 560.

active cell or identifiable inflammatory cell per 25anti-CEA-reactive cells or obvious tumor cell in 10random high power fields. In one case, assessedenrichment was from 50 to only 70% after IMS; thiscase was a mucinous carcinoma. The abundant mu-cin in this tumor seemed to interfere with cell sepa-rations because cells embedded in mucin appearedto adhere to one another nonspecifically.

Finally, it was also noted that the larger tumoraliquots (1.0 to 2.0 cm3) provided sufficient gross

material for two individual dispersion-separationsteps in the colorectal samples to yield enough finalcells for immunohistochemical and FISH analysis.

In the paraffin-embedded metastatic breast carci-nomas, the results were quite similar, except that ahigher pepsin concentration, 0.5%, was required toachieve the same degree of dispersion. EMA wassuccessful as the target antigen in this situation, atan optimal dilution of 1:10, remaining intact afterpepsin digestion as determined by IAP immunore-

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100 Yaremko et alAJPJanuary 1996, Vol. 148, No. I

Table 2. FISH and PCR Polymorphism Analysis of Fixed Solid Tuimors Enriched by Immunomagnetic Separation

FISH result; number ofsignals/nucleus(colon cancers)

2LOH result

3 (breast cancers)

Colon cancers1 50-70%2 50%3 50%4 50%

Metastatic breast cancers1 30-50%2 30-50%3 50%

>95%>95%>95%>95%

6369

20

82713651

1

0

311

70-90%>95%70-90%

Not informativeLossRetention

^Percentage of tumor cells before or after IMS, as determined by cytospin and CEA staining.

action. IMS with anti-EMA antibodies resulted in a

significant increase in the number of epithelial versus

lymphocytic cells, although not to as high a level ofpurity as in the colorectal cancer samples. Yields ofcells were proportionally lower in these samples be-cause of the small sample size, and cell morphologywas somewhat deteriorated compared with the fixedspecimens that had never been paraffin embedded.

FISH Analysis

Four cases of the eight colorectal carcinomas with>95% epithelial cells were chosen randomly to eval-uate the feasibility of using IMS cell preparations forinterphase FISH. A biotinylated chromosome-17-specific a-satellite centromere probe was hybridizedto the four sample cases, and the number of signalsper nucleus was assessed for 60 to 100 cells (Table2). Immunomagnetic beads were not removed be-fore hybridization. Two cases were predominantlydisomic for chromosome 17 and two had a signifi-cant number of aneusomic cells. The hybridizationand detection reactions were not affected by thepresence of the beads, nor did the beads interferewith interpretation in adequately dispersed cell clus-ters, although they are visible because of faintautofluorescence (Figure 2). Efficiency of hybridiza-tion, as compared with control lymphocytes hybrid-ized by the same FISH protocol, was slightly lower inthe IMS samples, 95 to 98% in the controls versus 85to 90% in the IMS cancer preparations, measured as

the percentage of cells with only one signal (con-trols) and percentage of cells with no signal (tumors).Because of limited material and the lower efficiencyof FISH in paraffin tissues6 the metastatic breastcarcinomas were not hybridized.

PCR Analysis for LOH

DNA yields were between 10 ng/p.l in a 100-,ul vol-ume for the lowest yield bead-separated metastaticbreast cancer (case 2, Table 2 and Figure 3a, lane4), and 75 and 100 ng/Al in the other two samples.Unlike the colon tumors, aliquots of the paraffin-embedded tumors were identical, ie, six 50-,m sec-tions, so DNA yields were not dependent on the sizeof the original aliquot. Yields were high enough for 4(lowest yield) to 10 (highest yield) PCR reactions at100 to 200 ng per reaction.PCR analysis for LOH with a chromosome 8 mi-

crosatellite repeat polymorphism was performed onthe metastatic breast carcinomas and results be-tween normal tissue, primary tumors enriched bymicrodissection, metastases for which enrichmentby microdissection was attempted, and the same

metastases after IMS were compared. These cases

are part of a larger series of breast carcinomas that

Figure 2. FISH of the immunomagnetically separated colon tumorfrom Figuzre 1. A small cltuster ol'fotur cells, hybridized with a biotin-Ylated chronmosotte-17 .specific centromere probe anid detected uwithfluorescein-conjugated avidin. Each cell has too signals, imndicatingthey are disomic for this chromosonmi. The paramaguetic beads havefaint autofluorescence. Nuclei counterstaimned with propidium iodide;magnification, X 975.

Case% tumorpre-IMS*

% tumorpost-lMS*

Immunomagnetic Separation for Enrichment of Solid Tumors 101A/P january 1996, Vol. 148, No. I

Discussion

1 2 3 4Figure 3. LOH analysis in two cases C

rated metastatic breast cancers infonnathe short arm ofchromosome 8. In each

tion product of normal DNA from the sasected primary tunmor; lane 3, prodtuctmicrodissection of metastatic tutmorfronlane 4, IMS-enniched metastatic tumor.priniary tutmor(lane 2) and le,ss obvion;stasis (lane 3). The IMS-enriched sample s

same allele as theprimary (lane 4). In b,theprimary and the metastasis is seen, ai

metastasis is not a result of contaminsample extracted for DNA.

we have analyzed for 8p LOH,of the markers,25 and were se

they had an infiltrative pattercluded enrichment by microcinitial results of LOH analysis vi

the fact that, by microscopicsection of the metastasis appTwo of the three cases were irone (case 2, Table 2) showedTable 2) showed retention ofprimary tumor. Figure 3, a andpattern for the two informativemal tissue from the same patiemicrodissected primary tumortasis before enrichment in laneafter enrichment by IMS in Ian

lane with normal DNA from theof equal intensity. In case 5, thLOH for the larger (top) allele,metastasis, the upper allele itense than the lower, becausethe lost allele from the admixEIMS-enriched sample, howevEtensities between the allelesous, and it is clear that the meloss as its primary. In case 3 (Tboth alleles are retained; in tlstated with confidence that tItains the allele, sharing the idethis marker with its primary an(

D8S133 LOH and metastaticnot appeared.

We demonstrate that IMS can enrich fixed solid tu-mors to a high level of purity and yields samples thatare suitable for FISH or PCR analysis. We havealso shown that IMS does not require specializedantibodies but can be performed with monoclonalantibodies used routinely in clinical immunohisto-chemistry. The availability of a bead coated withanti-mouse immunoglobulin G, an effective second-

1 2 34 ary antibody, gives the method great flexibility and1 2 3 specificity. Potential applications are limited only byf immutnomagneticallv sepa- the identification of a suitable antigen that is tumor!tive for marker D8S1 33, fromiase, lane 1 istheampDl_car specific and survives fixation and dispersion and amnepatietnt; lane 2, microdis- corresponding antibody that recognizes it. Theof DNA from an attemnpted wealth of monoclonal antibodies applicable to stan-

na regional lmiph niode; and

.In a, LOH is obvious in the dard paraffin sections for routine immunohistochem-s in the microdissected nmetas-shows unequivocal LOH ofthe stry suggests that this limitation IS not a serious one.

retention ofthe alleles in both For colorectal cancer, the logical choice for targetnd retention ofthe allele in theation bv lyvnphocytes in the antigen was CEA, because almost all colorectal car-

cinomas express it strongly and it is absent fromother cells in the supporting stroma. However, less

using D8S133 as one specific antibodies may be used if tailored to thelected either because situation. The choice of EMA as the target antigen forn of growth that pre- the metastatic breast cancers was successful be-lissection or because cause in this situation the separation required wasvere equivocal despite malignant epithelial from lymphoid cells rather thanevaluation, microdis- from other epithelial cells.

)eared to be possible. An obvious advantage of a method for the enrich-iformative for D8S133, ment of fixed tissues is that samples can be stored

LOH and one (case 3, and the separations performed in convenient

heterozygosity in the batches, but IMS enrichment of fixed solid tumors

Ib, illustrates the allele provides certain specific advantages in the applica-cases. Alleles of nor- tions tested. Interphase FISH is increasingly applied

,nt are in lane 1, for the to study the genetics of solid tumors, for which karyo-r in lane 2, the metas- types are both difficult to make and to interpret. FISH

3, and the metastasis performed on dispersed samples such as touch

e 4. In both cases, the preparations, FNAs, or disaggregations of 50-p.m

patient has two bands sections of tissue blocks allows evaluation of entire

e primary tumor shows nuclei but may lose distinctions between tumor and

and in the unenriched stromal cells. To preserve these distinctions and al-

s only slightly less in- low identification of the tumor versus the nontumor

of the amplification of cells, histological sections are often used but must

Ld normal cells. In the be thin (4 to 6 ,um) because of problems of probeDr, the difference in in- permeability. Interpretation of losses and gains of

again becomes obvi- chromosomes or sequences around a sectioning ar-

:tastasis has the same tifact is difficult and interferes with the sensitivity ofFable 2 and Figure 3b), the result. In one study, up to 38% of cells in sectionshis situation, it can be from normal tissue had single or absent signal for a

ne metastasis also re- probe.26 This may account for discrepancies thatntical allele pattern for exist in the literature. In prostate cancer, for example,d that a new clone with there are numerous reports of chromosomal aneu-

ability conferred has somy by interphase FISH, but the studies using sec-

tions report frequent chromosome losses26'27 as well

a b

102 Yaremko et alA/P.januarv 1996, Vol. 148, No. 1

as gains, whereas those performing FISH on prepa-rations of whole nuclei report chromosome losses asvery rare28 or as common only in tetraploid tu-mors.829 IMS provides whole nuclei for evaluationand almost guarantees that all cells present in thedispersion analyzed are neoplastic. In our four ex-amples of colorectal carcinoma and chromosome17, two of the tumors were primarily disomic and twohad significant aneusomic populations. The beadsdid not interfere with the reactions and exhibited onlymild autofluorescence. Our slightly lower rate of hy-bridization efficiency for the IMS samples, 85 to 90%versus 95 to 98% for control lymphocytes, is similar toothers' experience with fixed epithelial cells6 and islikely a result of the residual cytoplasm and poorerprobe penetration. We took no extraordinary mea-sures to enhance probe penetration so that we mightknow the baseline efficiency of hybridization with aminimum of sample manipulation.

Enrichment is also important when using PCR-formatted assays to study solid tumors. The sen-sitivity of PCR means that a mutation or abnormal-ity in the tumor epithelial cells may be obscuredbecause of product amplified from DNA contrib-uted by stromal cells. This causes false negativeresults. In our examples of metastatic breast car-cinomas not amenable to microdissection, a highdegree of enrichment was important not only in thecase with allele loss but also in the case withretention of alleles as well. Confident of the degreeof enrichment we achieved, we could be certainthat the retention in the metastatic disease wasreal and not an artifact of contaminating lympho-cytes, which might obscure a newly acquired lossin the metastatic clone and cause us to overlookan event in tumor progression. Although microdis-section will probably remain the most popularmethod for the enrichment of solid tumors for LOHbecause of its technical simplicity and the fact thatit is adequate for enrichment in most situations,some tumors do not lend themselves well to micro-dissection. This was true in our cases of metastaticbreast carcinomas, but other tumors may presentthis problem. An example is pancreatic carci-noma, which frequently grows in a pattern ofwidely separated malignant ducts within a reactiveand often highly cellular stroma. Other examplesinclude breast carcinomas that have prominentlymphocytic responses, Brenner tumors of theovary, lymphoepithelial lesions of the salivaryglands, thymomas, or any tumor that may elicitactive desmoplastic stromal reactions, all of whichpresent with growth patterns or cell compositionsthat render microdissection unreliable.

This is, to our knowledge the first report of usingIMS in fixed and paraffin-embedded tissue, withwidely available antibodies, although others haveused IMS to enrich clinical samples similar to ourown. Durrant et al 14 and Kemmner et al15 usedmonoclonal antibodies against CEA and other epi-thelial antigens, directly bound to the magneticbeads, to isolate colon carcinoma epithelial cellsfrom fresh tumor dispersions before plating in cellculture. Their aim was to prevent the overgrowth offibroblasts in explant cell cultures. Maas et al20 de-scribes the separation of metastatic breast carci-noma cells from lymphocytes from fresh, unfixedFNA samples of lymph nodes in breast cancer pa-tients using IMS. Because they used FNAs, no dis-persion step was necessary, and they used a spe-cialized antibody against a breast epithelialmembrane differentiation antigen. Their requirementfor a high degree of separation in their tumors orig-inated with their objective to determine by reversetranscriptase PCR the level of expression of theMDR1 gene in the breast carcinoma cells indepen-dently of the lymphocytes, which also express thegene. Our success in separating the same cell typesfrom fixed tumors was similar to that of these otherinvestigators, except that the enzyme inactivationstep in our samples was simpler (heat inactivationversus enzyme inhibitors or repeated cell washing)because of the protection that fixation gives againstthermal injury to cells.

Cell and DNA yields impose a limitation on themethod, although from our most limited of samples, ametastatic breast cancer only 300 ,um x 1 cm insize, we were able to extract enough DNA for fourseparate PCR reactions. Keeping volumes of ali-quots of the starting material as large as possible willhelp to overcome this limitation. Tumor cell hetero-geneity imposes another limitation to the method, asspecifically selecting and analyzing for neoplasticcells that express a particular antigen may restrictappreciation for subpopulations of tumor cells thatdo not. This, however, is a limitation that, with refine-ment of the method, may be turned to advantage asa tool to isolate tumor subpopulations on the basis ofantigen expression. One may then look for differ-ences in DNA mutations, gene amplification or dele-tion, or RNA expression in the subpopulations withvarying expression of antigens. These different pa-rameters can then be correlated to expression ofspecific antigens and new information regarding ge-notypic-phenotypic correlations may be uncovered.

IMS is a simple and effective method to enrichsolid tumors for epithelial cells. It is flexible andapplicable to tissues fixed in different ways, includ-

Immunomagnetic Separation for Enrichment of Solid Tumors 103AJP January 1996, Vol. 148, No. 1

ing routinely processed, paraffin-embedded tissue.The method is less technology-intensive than flow-sorting, can be performed with commercially avail-able antibodies, and can be valuable when samplesare not amenable to microdissection. It is also rapid;dispersion and separation can be completed in asingle day. It has potential for improving the molec-ular analysis of solid tumors by isolating pure popu-lations of tumor cells before study and can serve asa useful adjunct method for enrichment of fixed solidtumors.

Acknowledgments

We thank Dr. Anthony Montag for assistance withimmunohistochemistry and the Tissue Procurementand Oligonucleotide Synthesis core facilities of theUniversity of Chicago Cancer Research Center, sup-ported by grant CA14599 from the National CancerInstitute.

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