flow and image cytometric dna analysis in...

(CANCER RESEARCH 50, 6023-6027. September 15, 1990]

Flow and Image Cytometric DNA Analysis in RhabdomyosarcomaAreta Kowal-Vern,1 Frank Gonzalez-Crussi, Joyce Turner, Yolanda P. Trujillo, Pauline Chou, Chester Herman,

Melanie Castelli, and .lami WallochDepartment of Pathology, Loyola L'nirersity Medical Center, Maywood, Illinois 60153 [A. K.-V., J, T., C. H., M. C., J. H'./,- Department of Pathology, Children'sMemorial Hospital, Chicago, Illinois 60614 [F. G.-C., P. C.J; and Department of Pathology. Lutheran General Hospital, Park Ridge. Illinois 60068 />'. P. T./

ABSTRACT

Rhabdomyosarcoma is the most common malignant soft-tissue tumorin childhood, with an overall 3-year disease-free survival of 73%. DNAcontent is known to correlate with prognosis and therapy response inmany cancers. To determine the role of DNA content in rhabdo-ntyosarcoma, 23 tumor samples were studied retrospectively: 18 primarytumors and 5 post-chemotherapy recurrences or specimens obtained atsecond-look surgeries. The DNA analysis was performed on disaggregated paraffin-embedded tissue nuclei by flow and image cytometry andcorrelated with the histology and clinical history. Of the primary tumors4 were diploid, 4 polyploid, and 10 aneuploid (9 with a single aneuploidG0G| peak and 1 multiploid) by flow cytometry. The concordance ratebetween flow and image cytometry was 19 of 23 (83%); one case did nothave flow cytometry available. Most embryonal rhabdomyosarcomaswere aneuploid (10 of 12; 83%), and they had a high incidence ofrecurrence in Stages III and IV (4 of 12; 33%). Although aneuploid) inpediatrie cancers may predict a therapeutic response and good prognosis,this was not supported by our findings in rhabdomyosarcoma. The tumorDNA content correlated with the clinical stage but not with the patient's

clinical course or tumor histopathological type. DNA content did notappear to be as important a prognostic tool as tumor stage.

INTRODUCTION

Rhabdomyosarcoma is the most common malignant soft-tissue tumor in children (1-5). Multiple factors have been usedto predict patient outcome and response to treatment: cellmorphology; histological patterns; and clinical staging. At present, prognosis for these children is greatly improved by multi-modal treatment using surgery, intensive chemotherapy, andradiation therapy. An event-free 3-year survival of 73% hasbeen reported by the Intergroup Rhabdomyosarcoma Study HI(6).

It has been suggested that aneuploidy in rhabdomyosarcomamay be a predictor of good prognosis in patients who have thiscancer, based on the significantly favorable survival of childrenwith this tumor (7). Our initial experience with 2 patients withStage III embryonal rhabdomyosarcoma of favorable histology,in whom treatment failed, prompted us to further investigatewhether DNA ploidy could be used in the prognostic assessmentof rhabdomyosarcoma. We measured DNA ploidy by both flowand image cytometry.

MATERIALS AND METHODS

Eighteen primary and 5 recurrent or post-chemotherapy persistentchildhood rhabdomyosarcomas were obtained from Children's Memo

rial Hospital, Chicago, IL; Loyola University Medical Center, May-wood, IL; and Lutheran General Hospital, Park Ridge, IL. The meanage of the children at the time of diagnosis was 6.9 years with a rangeof 4 months to 17 years. The male-to-female ratio was 3:1. Theanatomical sites of the primary tumors included head and neck (5

Received 3/26/90; accepted 6/19/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 17.14 solely to indicate this fact.

' To whom requests for reprints should be addressed, at Department ofPathology, Loyola University Medical Center, 2160 South First Avenue, May-wood, IL 60153.

cases), trunk (3 cases), urogenital (8 cases), and extremities (4 cases).The cell types of the rhabdomyosarcoma were embryonal ( 12 cases),alveolar (4 cases), spindle cell:leiomyomatous (2 cases), undifferentiated(1 case), and mixed alveolar/embryonal (1 case) according to theIntergroup Rhabdomyosarcoma Study (6, 8, 9). In addition to the usualhistological studies, the following Â¡mmunostaining procedures wereperformed to confirm the nature of the tumor: (a) desmin; (h) vimentin;and (c) muscle-specific actin stains. Electron microscopy was done inselected cases. The tumors at initial diagnosis were staged as I (8 cases),II (2 cases). III (8 cases), and IV (2 cases). The mean follow-up was 5.1years with a range of 2 months to 14.5 years. All the tissue utilized inthis study were fixed in 10% buffered formalin and embedded in paraffinusing standard histological techniques.

Nuclear Isolation for Quantitative DNA Analysis. Isolated nuclearsuspensions were prepared using a modified Hedley technique (10. 11).Three to 6 SO-^m sections were cut from each paraffin-embedded tissueblock. An adjacent section was obtained for standard hematoxylin-eosin staining for microscopic tissue confirmation. The tissue was thendeparaffinized using xylene or Americlear and sequentially rehydratedin a graded series of ethanol and washed in distilled water. The tissuewas centrifuged at 400 x g for 2 min, the supernatant was aspirated,and the tissue pellet was resuspended after each rehydration change.Following deparaffinization, a tissue suspension in 0.5% pepsin solution [pepsin, 0.050 g (Sigma Chemical Co., St. Louis, MO), NaCl 0.090g to 10 ml in IX PBS2 without Ca2VMg2*, and adjusted to pH 1.5 with2 N HCI] was incubated for 60 min at 37Â°Cwith vigorous vortexing

every 10 min. The suspension was filtered through a 30-Â«imnylon mesh(Small Parts Inc., Miami. FL) and washed twice in Ix PBS to removedebris. A nuclear count of 1 x 106/ml was considered optimal. Two mlof the nuclear suspension in PBS with Ca2* was incubated in 0.4 ml of

0.1% RNase solution [0.001 g RNase (Type II-A from bovine pancreas;Sigma) in 1 ml of Ix PBS without Ca2VMg2*] at 37Â°Cfor 30 min. A

100-^1 aliquot of the nuclear suspension and 100 ^1 of bovine serumalbumin (Sigma) were used to prepare a cytospin for Feulgen staining(1500 rpm for 5 min; Shandon. Sewickley, PA).

Flow Cytometry. The remaining suspended nuclei were stained in a2-ml propidium iodide solution [0.005 g PI (Sigma) in 100 ml of IxPBS without Ca2+/Mg2+ stored in the dark at 4Â°C]for 30 min and

filtered through a 30-^m nylon mesh immediately followed by FCManalysis. The propidium-iodide-stained cells were analyzed on a FAC-Scan flow cytometer (Becton-Dickinson Immunocytometry Systems.Mountain View, CA) with a 488-nm argon laser for excitation and totalfluorescence above 610 nm collected for DNA. For each histogram, 1x IO4events were analyzed. The DNA histogram display and analysis

were performed on a Consort 30 Hewlett-Packard minicomputer (Becton-Dickinson).

Image Cytometry. The prepared cytospin slide was Feulgen-stainedwith a CAS Quantitative DNA staining kit (Cell Analysis Systems,Inc., Lombard, IL). The air-dried cytospin was fixed in 10% bufferedformalin for 30 min, then acid-hydrolyzed in 5 N HCI for 60 minfollowed by Feulgen staining for 60 min. It was rinsed in 3 changes of0.05 N HC1 for 30 s, 5 min, and 10 min. respectively, followed bydistilled H2O for 5 min. It was then placed in 0.05 N HCI/70% ethanolsolution for 5 min and dehydrated in 100% ethanol, changes of 3 mineach. This was followed by clearing with xylene, Permount, and cov-erslipping. The samples were analyzed on the CAS-200 image analyzerusing the single-cell quantitative DNA analysis program (Cell AnalysisSystems, Lombard, IL). Analysis was performed by measuring theintegrated absorbance of morphologically identified rhabdo-

2The abbreviations used are: PBS. phosphate-buffered saline: FCM. flow

cytometry; 1C. image cytomelry.

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DNA ANALYSIS IN RHABDOMYOSARCOMA

myosarcoma cell nuclei. For each histogram, 130-225 nonlymphocytic prognostic indications, ploidy, and survival by rectangular contingencynuclei were measured. Image cytometry allowed for morphologicalsubclassification of nuclei by size, configuration, and chromatin qualitysince it utilized a standard light microscope that was observer controlled.

DNA histograms were generated for nuclei classified into morphologies type I (NCI) and type II (NC2) (Fig. 1). Nuclei were designatedas type I when they appeared larger than 8 ^m, irregular, pleomorphic,and of vesicular, lacy chromatin pattern; type II when they were smaller,ovoid, generally regular in contour, and of dark, dense chromatin. Thisclassification of nuclei was adopted because of the suggestion thatnuclear morphology may be relevant to prognostic assessment (12).

DNA Ploidy Determination. Calibration and instrument linearitywere performed with a human lymphocyte/chick RBC mixture for flowcytometry and rat hepatocyte nuclei for image cytometry. DNA ploidywas determined by the number and relative position of the peaks(nuclear subpopulations with similar DNA content) on the histogram.If only one peak was present, it was considered the GoG, peak and thehistogram was DNA diploid. If 2 or more peaks were present and a 2"

(n = 0, 1,2) integer multiple of the G0G, DNA content, then the

histogram was considered DNA polyploid. If 2 peaks were present andnot a 2" (n = 1,2...) integer multiple of the Go-G, peak DNA content,

then the histogram was considered DNA aneuploid. If more than 2peaks were present and not a 2n (n = 1,2...) integer multiple of theGo-Gi peak DNA content, then the histogram was considered multiple

aneuploid. The coefficient of variation was calculated for the G0G,peak of each histogram. The mean coefficient of variation for flowcytometry was 7.6 (range, 3.9 to 14.4) and for image cytometry 9.9(range. 3.6 to 22.0). The ploidy of each histogram was determinedindependent of the patient's histology, stage or outcome.

Statistical Analyses. Statistical significance comparing conventional

NC1

*NC2

Fig. 1. Representative cell nuclei depicted bv (a) H&E (x 1000) and (A)Feulgen(x 1000) staining for nuclear classification (iVC) studies. NCI, large nuclei; A'C2,

small nuclei (sec text).

tables were calculated by the x2 method.

RESULTS

Table 1 lists the results of DNA analysis by flow and imagecytometry for each case according to age, anatomical site, stage,cell type, ploidy pattern, follow-up, and survival status.

Comparison of Flow and Image Cytometry. Twenty-one of the23 tumors from the 20 patients were analyzed by both FCMand 1C. Case 11 was not analyzed by FCM due to hypocellular-ity of the specimen. Of the 23 tumors analyzed by FCM, 5 werediploid, 4 polypoid, 10 aneuploid, and 4 multiple aneuploid.Of the 23 tumors analyzed by 1C, 4 were diploid, 3 polyploid,11 aneuploid, and 5 multiple aneuploid (Fig. 2). The correlationbetween the 2 DNA analysis methods was 83% (19 of 23). Ofthe cases that did not correlate, 2 were diploid, 1 polyploid, 1aneuploid, and 1 multiple aneuploid by FCM. By 1C, 1 wasdiploid, 3 were aneuploid, and 1 was multiple aneuploid.

In the correlations between ploidy and prognostic indicators,and survival, the worst ploidy pattern in the 5 discordant FCMand 1C histograms was used.

Correlation between DNA Content, Conventional PrognosticVariables, and Survival. DNA content was compared with conventional prognostic indicators including anatomical location,patient's age, cell type, and stage (Table 2). Only the stage was

significantly related to aneuploidy (P < 0.05). Although 5 ofthe 9 Stage I and II tumors (55%) were aneuploid, 9 of the 10Stage III and IV tumors (90%) contained an aneuploid cellpopulation. No significant relationship between ploidy andanatomical site (P < 0.25), patient's age (P < 0.25), and cell

type (P < 0.10) was found.Fourteen of the 20 patients (70%) were still alive at the time

of this study with a mean follow-up of 5.1 years (2 months to14.5 years). Of the surviving patients, the mean follow-up was4.7 years (5 months to 14.5 years) (Table 1). Clinical stage wassignificantly associated with survival (P < 0.025). All 10 of theStage I and II tumor patients were alive (100%) and only 4 ofthe 10 Stage III and IV tumor patients were alive (Table 3). Nosignificant relationship between survival and anatomical site.

Table 1 Sludy patient characteristics

No.1234567891011121314151617181920Age(yr)/sex1.3/F"0.7/M8/M3.5/F1.3/M1/M2/M7.5/M14/F18/M6.5/M4/M11/M0.6/F5/F2/M12.5/M5.5/M17/M0.3/MPloidySiteHNIRGÃ¬EXGUCUGUHNTRHNGUHNGUEXEXHNTRGUGUEXTypeEEEEEEEEEEEEE/ALALALALALSSUStage1IIIIIIIIIIIIIIIHIIIIIIIVIIIIIIIIVIIIIIPCMrâ€¢AAADPA

AADADMAA

MAPMADPPMAAAD1Cr

â€¢AAAAPA

AAAADMAA

MAMAMADPPMAAnD-ollow-up

(yr)Status53.50.4111220.74840.20.66720.63214.5NEDNEDNEDNEDNED++NEDNED+NED++NEDNEDNED+NEDNEDNED

Â°F. female: M, male; A. aneuploid; AL. alveolar: D. diploid: E. embryonal:

MA, multiple aneuploid: NED. no evidence of disease: P. polyploid: S. spindlecell, leiomyomatous: II. undifferentiated: +. dead of disease: GU, genitourinary(prostate, testes): TR. trunk (axilla, pericardium); EX, extremity (foot, thigh):HN, head and neck (nasopharynx, neck, scalp): I", primary tumor.

* Recurrence.

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DNA ANALYSIS IN RIIABOOMYOSARCOMA

Table 3 Significance of variable!, compared Kith survival

DI: 1.00CV: 14.4

DI: 1.42CV: 9.2

DI: 1.97CV: 7.0

DI: 2.83/CV: 10.3

DI: 1.00CV: 10.1

A

D

DNA MASS

Fig. 2. Representative histograms by flow and image cytometry of: a. diploidy;b. polyploidy; c. aneuploidy; and d. multiple aneuploidy. CT. coefficient ofvariation. DI, DNA index.

Table 2 Significance of variables compared with ploidy

VariableAnatomical

siteHNÂ°TRGUEXAge<2>2TypeEALUSE/ALStageIII111IVTotal

no.of

patients53848121241218282No.

ofdiploids(%)0(0)1

(33)0(0)2(50)2(25)1(8)1

(8)1(25)1

(100)0(0)0(0)2(25)0(6)1(12)0(0)No.

ofpolvploids

(%)1(20)0(0)1(12)1

(25)2(25)1

(8)1

(8)2(50)0(0)0(0)0(0)1(12)2(100)0(0)0(0)No.

ofaneuploids or

multipleaneuploids

(%)P4

(80)<0.252(67)7(88)1

(25)4

(50)<0.2510(84)10(84)

<O.IO1(25)0(0)2(100)1

(100)5

(63)<0.05*0(0)7(88)2(100)

VariableAnatomical

siteHNTRCilEXAge<2>2TypeEALUSE/ALStageIIIIIIIVPloidyDiploidPolyploidA

or MATotal

no. ofpatients538481212412182823314NED"(%)3

(60)2(67)5

(62)4(100)6(75)8(67)8(67)4(100)1

(100)2(100)0(0)8(100)2(100)4(50)0(0)3

( 100)3(100)8(57)DOD

(%)2(40)1

(33)3(38)0(0)2(25)4

(33)4

(33)0(0)0

(0)0(0)1

(100)0(0)0(0)4(50)2(100)0

(0)0(0)6(43)P<0.25<0.25<0.25<0.025*<0.25

"UN. head and neck: E. embryonal; AL. alveolar: I'. undifTcrcntiatcd: S.

spindle cell:lei(imyomatous; TR. trunk: EX. extremity: Cil!, genitourinary.* Significant.

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Â°NED. no evidence of disease: DOD, dead of disease: E. embryonal: AL.

alveolar: S. spindle cell:lciomyomatous; 11, undifferentiated: A. aneuploid: HN.head and neck: TR. trunk; EX. extremity: Gil. genitourinary.

* Significant.

cell type, age or ploidy (P < 0.25) was found. Although ploidywas not significantly related to survival, a trend could be discerned. All the diploid and polyploid patients were alive,whereas 6 of the 14 aneuploid patients had died. Three of the4 patients with multiple aneuploid tumors were dead; the lonesurvivor had a recurrence, but was presently free of disease atthe 3.5-year follow-up.

Nuclear Subclassification. Subclassification of the nuclei byimage processing did not correlate with the tumor histolÃ³gica!classification or with ploidy. It is of interest that upon recurrence a greater percentage of type I bizarre, large nuclei werepresent. These 2 types of nuclei may actually have been cells inactive proliferation and degeneration, as a possible explanationfor their noncorrelation with ploidy. Our nuclear classificationschema could only identify 2 types of tumor cells present; butthese were not present in significant numbers to correlatespecifically with either the embryonal or alveolar tumors orwith the diploid or aneuploid peaks. Upon further analysis,nuclear classifications I and II were present in both the diploidand aneuploid peaks in different proportions.

DISCUSSION

DNA ploidy in a variety of pediatrie tumors has been investigated as to its prognostic significance and correspondingresponse to chemotherapy (Table 4) (7,13-23). Previous studiesof rhabdomyosarcoma have noted that the major factor forpredicting outcome was extent of disease (4). Children withtumor confined to the tissue of origin had a predicted 5-yearsurvival of 86%, whereas children with metastatic disease orextension of tumor beyond the primary site had a poorerprognosis, with a 5-year survival rate of 21% (4). Treatmentfailure is usually a result of local recurrence or extension oftumor. Alveolar rhabdomyosarcoma is known to have a higherincidence of mÃ©tastasesto bone marrow (24). HistolÃ³gica! typeand site of primary tumor affect prognosis but are not independent of extent of disease. Molenaarefa/. (7) have postulated




Table 4 DNA-ploidy related to prognosis in selected pediatrie tumors

No. ofTumor cases Good prognosis Poor prognosis

RhabdomyosarcomaMolenaar 11 AneuploidPresent study 20 Diploid and aneu- Aneuploid

ploid Stage I Stage IIIand II and IV

MedulloblastomaTornila and Masaharu 53 Aneuploid and Diploid and

total resection subtotalresection

Soft-tissuesarcomasPerssonKreicbergsNeuroblastomaLookGanslerBone

tumorsKreicbergsMankinWilm's

tumorKumarRenalSchmidtKumar382173815217Â°795914DiploidDiploidAneuploidAneuploidDiploidDiploidDiploidAneuploid

(low S)AneuploidAneuploidDiploidDiploidAneuploidAneuploid

andtetraploidAneuploidAneuploid

and(highS)Diploid

" Not separated by age.

CELL

COUNT

DI: 1.00CV: 6.49

DI: 1.64CV: 7.50

Â¡X

eoabDMI1

1 l 1DI:

1.00CV: 5.4Dl:

2.201 f\ CV:5.8V~-^

V,~-"-TT'' l ' | ' ' ' ' 1

DI: 1.00CV: 10.69

found that 61% of the embryonal pattern were aneuploid; 59%of the alveolar cell type were diploid.

It is possible that at recurrence there may be a shift in theaneuploid peak, or a selection of an aneuploid population bychemotherapy that prior to chemotherapy was an insignificantpopulation. Molenaar et al. (27) have demonstrated that chemotherapy selectively eliminates the undifferentiated tumor cells.This phenomenon has been explored in tissue culture. Humantumors grown in soft agar cultures have demonstrated a greatvariability of tumor cell subpopulations, often exhibiting diploidand aneuploid peaks not seen in the original tumors (28). Onecan postulate that the tumor cells were modified by chemotherapy and radiation thus becoming more resistant to treatment,or that occult resistant cell lines were present from the beginning, hidden by the more numerous treatment-sensitive cells.With the obliteration of the original sensitive aneuploid tumorcell line, these resistant numerically insignificant aneuploidcells proliferate and predominate. Patients may fail treatmentbecause their aneuploid tumors either do not respond to therapy, or do respond but unmask a more aggressive aneuploidpopulation following treatment (Fig. 3). Our data showed thatthe resistant aneuploid population predominated during recurrence.

Unlike recent reports demonstrating that certain pediatrieDNA aneuploid neoplasms may have a better prognosis (Table4), our data do not support aneuploidy as a predictor of a goodprognosis in rhabdomyosarcoma. The basis for better prognosishas been postulated to be the instability of the aneuploid DNAand its high susceptibility to chemo- and radiotherapeutic modalities (28, 29). At this time, it has been confirmed that themajority of embryonal rhabdomyosarcomas are aneuploid;however, due to the small number of cases thus far studied,DNA ploidy cannot yet be firmly correlated with prognosis orhistological type.

One important finding in this study was that 1C was moresensitive than FCM in identifying the near diploid populationand aneuploid populations not seen by FCM. It also permittedthe identification of individual cell ploidy and could be utilizedin cases where there were too few cells for analysis by FCM.

In summary, it appears that extent of disease and successfulsurgical excision is probably the single best prognostic indicatorin rhabdomyosarcoma (8). Further work with a larger series isneeded to definitively ascertain whether DNA analysis mayhave the value of an independent variable in evaluating prognosis.

DNA MASSFig. 3. a, Pre-treatment and b, post-treatment histograms by flow and image

cytometry. indicating a tumor line with a different DI upon recurrence CV.coefficient of variation; DI. DNA index.

ACKNOWLEDGMENTS

We would like to thank Ann Devitt for typing the manuscript.

that with the improved survival of rhabdomyosarcoma patients,aneuploidy of the tumor cells may be used to predict a goodprognosis; however, 3 of their patients with high stage aneuploid tumors died of disease: a Stage III alveolar, Stage IVbotryoid, and Stage III embryonal rhabdomyosarcoma. Moreover, 3 of the tumors were identified as both alveolar/embryonalor alveolar/botryoid. Molenaar et al. (25) have demonstratedthe variability of differentiation present in rhabdomyoblasts,reflected in different patterns of desmin and vimentin positivity.The majority of our alveolar pattern tumors, however, werediploid and polyploid in contrast to the findings of Molenaaret al. (25), but in concordance with recently published work.Schmidt et al. (26) studied 73 rhabdomyosarcoma tumors and

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1990;50:6023-6027. Cancer Res Areta Kowal-Vern, Frank Gonzalez-Crussi, Joyce Turner, et al. RhabdomyosarcomaFlow and Image Cytometric DNA Analysis in

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