(CANCER RESEARCH 48. 6614-6619. Nmember 15. 1988]

Nucleic Acid Flow Cytometry in Large Cell Lymphoma1

Peter McLaughlin,2 Barbara M. Osborne, Dennis Johnston, Paul Jennings, James J. Butler, Fernando Cabanillas,

and Bart BarlogieFrom the Departments of Hematology [P. .V/., F. C., B. B.]; Pathology- ¡B.M. O., J. J. B.]; Biomathematks (D. J.]; The University of Texas, M. D. Anderson Hospitaland Tumor Instílate.Houston, Texas 77030; and the L'niversity of Texas Medical School/\1. D. Anderson Hospital ¡P.J.¡,Houston, Texas 77030

ABSTRACT

Between 1978 and 1985, 140 patients with large cell lymphoma (21follicular, 92 diffuse, 5 immunoblastic. and 16 transformed) had DNA-RNA cytometry performed on involved tissue. DNA-RNA features werecorrelated with treatment outcome and compared to other establishedprognostic factors in 63 newly diagnosed patients who received uniformlyintensive therapy. Significantly better outcome was noted for previouslyuntreated patients with intermediate RNA content (RNA index, 1.0-1.8),diploid DNA content, and (during the initial 12-month follow-up) lowproliferative activity. Of patients followed beyond 12-24 months, thosewith high proliferative activity appeared to have the most durable remissions, although this was not statistically significant. These findingssuggested a preferential impact of intensive chemotherapy on patientswith intermediate RNA content and possibly those with high proliferativeactivity, since previous studies and our own experience with relapsingpatients have indicated a progressively worse outlook with higher proliferative activity and RNA index values. In newly diagnosed patients,multivariate analysis identified RNA content as the most importantprognostic factor, followed by proliferative activity and serum láclatedehydrogenase. Thus, for patients with large cell lymphoma. DNA-RNAcytometry appears to be a valuable prognostic parameter for identifyinga subset of patients who have a high likelihood of cure with intensivechemotherapy.

INTRODUCTION

Despite the clinical usefulness of histológica! classificationsof the malignant lymphomas (1), patient outcome varies considerably in many of the subtypes, including LCL.' The LCLs

are particularly important because of their frequency and theirpotential curability. Numerous attempts have been made tostratify prognostic groups within the LCLs, including analysisof morphological subcategories (2-5), phenotypic subcategories(6, 7), clinical features such as stage and extent of disease (8-11), and cellular DNA content from paraffin-embedded tissuesections (12).

FCM of DNA content permits an objective and quantitativeanalysis of a large number of tumor cells with establishedcorrelations between DNA ploidy and chromosome numberand between proliferative compartment size and labeling index(13-15). The derived information on DNA ploidy and proliferative activity has been shown to be a useful gauge of theaggressiveness of the malignancy for a number of solid tumorsand hematological malignancies (16-21). AO provides a quantitative analysis not only of DNA but also of RNA content,both of which have been shown to relate to prognosis in the

Received 12/23/87: revised 4/29/88: accepted 8/9/88.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 1734 solely to indicate this fact.

1Supported in part by Grant CA-16672 from the National Cancer Institute.

Presented in part at the 78th Annual Meeting of the American Association forCancer Research. Atlanta. G A. May. 1987.

•¿�To whom reprint requests should be addressed, at Department of Hematol-

ogy. University of Texas M. D. Anderson Hospital. 1515 Holcombe Boulevard.Houston. TX 77030.

1The abbreviations used are: LCL, large cell lymphoma: FCM, flow cytometry;

AO, acridine orange; LDH. láclatedehydrogenase; DLCL. diffuse LCL; DI, DNAindex; RI, RNA index; CR. complete remission; FFS. failure-free survival; FLCL.follicular LCL: IBL. immunoblastic lymphoma: PI, propidium iodide.

malignant lymphomas (22, 23). Thus, these studies add valuablebiological data that can augment routine histopathologicalanalysis.

In the present study, we extend the use of FCM of DNAcontent and provide the first extensive assessment of tumor cellRNA content in patients with LCL. The AO FCM studiesreported here were done prospectively, using freshly biopsiedspecimens; the results were subsequently correlated with thepatients' clinical features and treatment outcome.

MATERIALS AND METHODS

Between 1978 and 1985. AO FCM analyses were performed on 140biopsy specimens of LCL. Excluded from the remission analyses werethree patients whose treatment response was uncertain. Excluded fromremission and survival analyses were 10 patients who were known tohave received either no therapy or suboptimal (e.g.. single alkylatingagent) therapy. Tissue was obtained by excisional biopsy in 98% of thepatients and fine needle aspiration in 2%. The source of tissue waslymph node in 72% and involved extranodal solid tissue in 28%. Bonemarrow, blood, and body fluid samples were not included in thisanalysis.

The biopsy material was reviewed at the time of this analysis andclassified according to the Working Formulation (1).

Clinical features at the time of the AO FCM analysis were recordedand correlated with treatment outcome. In particular, distribution andbulk of nodal disease, extranodal involvement, and serum LDH levelwere assessed, in keeping with their known prognostic value for patientswith newly diagnosed Stage III-IV DLCL (8).

The treatment approach was uniformly intensive for these patients,although specific therapy varied according to stage, current protocols,and. for relapsing patients, prior treatment history. All newly diagnosedpatients received combination chemotherapy: 92% received doxorubi-cin-based programs: 43% also received VP-16/methotrexate noncross-resistant alternating combinations (24); 38% received chemotherapy inconjunction with radiotherapy (Stage I-III patients). Of relapsing patients. 43% received VP-16/methotrexate-based combinations (25),19% received further doxorubicin-based combinations, 14% receivedcytarabine-based combinations (26), 10% received Phase II singleagents, and the remainder received a variety of other therapies.

The technique for AO FCM has been previously described (22, 27.28). Specimens were minced and syringed to produce a single cellsuspension. Representative involvement by lymphoma of the specimenswas confirmed by examination of immediately adjacent tissue sections.An admixture of a fraction (not quantitated) of nonneoplastic lymphocytes in the specimens is expected; these background cells play a rolein the morphological diagnosis of the lymphoma and likewise probablycontribute to the ability of FCM studies to stratify the morphologicsubtypes of lymphoma (22). Acridine orange staining was done according to a previously reported two-step procedure (27). Cytometric analysis was carried out with an ICP-22 mercury-arc cytometer (Phywe,Goettingen. W. Germany) with appropriate excitation and emissionfilters. At least 10.000 cells were measured in an optimum study. Themedian coefficient of variation of the Go-i DNA peak was 4.4% (range.2.9-6.6). The analysis of the DI of the sample G0-i cells, the quantita-tion of cells in the S+G?M compartment, and the calculation of RI ofthe Go-i compartment were all done as previously described (22). Threetechnical points deserve comment: (a) the admixture of nonneoplasticcells can influence (usually reduce) the derived estimates of (S+G2M)%,and, to a lesser extent, RNA index: (b) the (S+G2M)% estimate for

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NUCLEIC ACID FLOW CYTOMETRY IN LARGE CELL LYMPHOMA

Table 1 \ucleic acid features by histolog)'

HistologyPreviously

UntreatedFollicularLCLDifTuse

LCLImmunoblasticRelapsingFollicular

LCLDiffuseLCLImmunoblasticTransformedNo.

ofpatients18502942316%Aneuploid61485056423350(S

+ GjM)%±SD8.1

±4.916.3±8.626.5±3.55.9

±4.913.2±8.819.3

±16.211.5±9.1RNA

Index±SD1.4

±0.81.8±0.91.

8±0.41.4

+0.51.8±1.23.1±1.01.4±0.7

aneuploid specimens derives from a more homogeneous neoplastic cellpopulation and thus is less prone to this dilution effect than diploidpopulations: (c) the boxogram method of estimating (S+GiMJ't is a

satisfactory approximation for studies of freshly biopsied lymphomasamples in which the (S+G:M)cc is generally low, but the same method

would carry a high potential for error in situations of high proliferativeactivity, such as in some cell culture experiments. These issues havebeen addressed elsewhere (29-31). While these facts indicate potentiallimitations of the FCM-derived estimates of cell cycle phase, it hasbeen demonstrated that the FCM estimates of (S+G2M)% correlatewell with tritiated thymidine labeling index (13-15).

Lymphocyte surface marker studies were available for 80 of thebiopsy specimens. During the early part of this time period, markeranalyses were done on single-cell suspensions of the biopsy specimens,utilizing a panel of monoclonal and polyvalent antibodies. More recently, frozen section immunoperoxidase studies were done (32, 33).

CR was defined as the resolution of all symptoms and signs ofdisease. Survival was measured from the time of the biopsy to the timeof death or last follow-up. FFS was measured from the time of thebiopsy to the time of treatment failure (progression of disease, deathdue to toxicity, or abandonment of therapy due to toxicity, patientrefusal, or physician choice) (34). Survival and FFS curves were calculated according to the method of Kaplan and Meier (35). Comparisonswere made using \~ tests for complete remission rates and Gehan'smodification of the generalized Wilcoxon test for survival (36). Cox's

model was used for the multivariate analysis (37).

RESULTS

Half of the FCM studies were done on specimens from newly-

diagnosed patients. Table 1 illustrates the FCM parametersaccording to histological category, for both newly diagnosedpatients and those studied at time of relapse. The FLCL grouphad a significantly lower S phase than the DLCL and IBLgroups.

The CR rates for newly diagnosed patients with FLCL andDLCL were 75 and 67%, respectively; the corresponding 2-yearsurvival figures were 72 and 41%, and the 2-year FFSs were 42

and 44%. The FLCL group had a trend for longer survival (P= 0.18) than the DLCL group. The two previously untreatedpatients with IBL both achieved CR and remain in remissionwith 9- and 12-month follow-up.

The overall CR rate for relapsing patients was 24%, and therewere no significant differences among the histological categories. The median survival from relapse was 22 months forFLCL, 13 months for DLCL, 6 months for IBL, and 9 monthsfor LCL transformed from low-grade lymphoma. The respective median FFSs were 7, 5, 3, and 4 months. For all histologicalcategories, survival and FFS were shorter for patients withmultiple relapses than for those in first relapse.

Newly Diagnosed Patients

DNA Index. Table 2 and Fig. 1 illustrate the analysis of theimpact of DNA Index on attainment of CR, survival, and FFSfor the 63 previously untreated patients who received intensivetherapy. Patients with diploid LCLs had significantly bettersurvival and FFS rates than patients with aneuploid malignancies.

RNA Index. Analysis of the study population in two cohorts,those above and those below the median RI value, failed todemonstrate prognostic differences. Since the lymphomas arediseases with a continuum of clinical behavior, and treatmentfailure is frequent both in the very indolent (low grade) andvery' aggressive (high grade) categories, similar heterogeneity

was sought within LCL by dividing the population into threeapproximately equal groups. It then became apparent thatpatients with intermediate RNA content had the best prognosis(Table 2 and Fig. 2). Patients with very low RI had a slightlymore indolent course than those with very' high RI, but neither

of these groups had a high proportion of durable remissions,whereas 80% of patients with RI 1.0-1.8 were projected to befailure free with a median follow-up period of 18 months. Bothsurvival and FFS were superior for the group with intermediateRI. whether the analysis included all LCLs or was restricted toDLCL only.

Proliferative Activity. As with the analysis by RI, the analysisof survival and FFS by (S+G2M)% failed to demonstrate asingle cutoff (S+G2M)% value that identified prognosticallydifferent groups (Table 3). Again, dividing the population intothree groups revealed prognostically important differences.

Table 2 and Figs. 3 and 4 illustrate the analyses of the impactof (S+G2M)% on CR, survival, and FFS. For the overall group,low (S+G2M)% (<10) was prognostically favorable, in partbecause of the large proportion of FLCL patients in the low(S+GjM)% group and their generally longer survival.

Table 2 Outcome for previously untreated patients by nucleic acid features

DLCL Patients%

2-YearNucleicacidfeaturePloidydiploid

aneuploidRNA

index<11-1.8>1.8(S

+G2M)%<1010-18>18No.212461920131418%CR79

5860795877SO75Survival

P«

|0°)72

[0.0128j38]13

>0.1660JFFS

P«|

0,10«]81

]•0.00226|39]21

>0.6159JNo.29

34132426251820r;CR8262758356805078All

LCLs%

2-YearSurvival

Pïï!

o-47178

}-0.00229[61]20

f0.0163JFFS651

27|18185

1"j39]31

r62JP0.01<0.0010.29

" See Fig. 2: No surviving patients with 24-month follow-up in this subgroup.

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NUCLEIC ACID FLOW CYTOMETRY IN LARGE CELL LYMPHOMA

100-h 100

80

Fig. 1. Outcome by DNA index for patientswith diffuse large cell lymphoma. Left, survival; right, failure-free survival.

oat

20

DIPLOID (N = 21)

40

ANEUPLOID (N = 24)

0

DIPLOID (N = 21)

ANEUPLOID (N = 24)

24 36

MONTHS

24 36

MONTHS

100

80

60

Fig. 2. Outcome by RNA index for patients ^with diffuse large cell lymphoma. Left, sur- yvivai: right, failure-free survival. ac.

40

20

100

Rl 1.0-1.8 (N-.19) go

Rl > 1.8 (N=20)

RI S 1.0 (N=6)

24 36 48 60

MONTHS

40

20

Rl 1.0-1.8 (N=19)

Rl >1.8 (N= 20)

RI S 1.0 (N= 6)

12 24 36 48 60

MONTHS

Table 3 Diffuse large cell lymphoma: survival by (S •¿�untreated patients

GiM)%for previously Table 4 Prognostic factors affecting survival in LCL using the Cox RegressionModel

Cutoff(S + G2M)%10

1214

16182022

24No.

patientsbelow/above

cutoff13/32

17/2821/2423/2227/1829/1634/1135/10ct

2 year survivalbelow/above

cutoff38/43

35/4429/5033/4927/6024/6832/6433/60P

Value0.19

0.530.880.680.590.250.690.91

When the analysis was restricted to DLCL patients only,patients with (S+G:M)% <10 were seen to have somewhatbetter short-term survival (particularly for the first 12 months)than other patients. However, the highest projected fraction oflong-term survival and FFS was from the group with high(S+G2M)% (>18) (Fig. 4), although this was not statisticallysignificant. The median follow-up of this high (S+G2M)%group with the highest apparent potential for cure was 15months.

Multivariate Analysis. The nucleic acid features of the newly-

diagnosed patients (FLCL, DLCL, and IBL) of all stages wereassessed along with the clinical features listed in the left half ofTable 4. Prior analyses of Stage III-IV patients had shown theparticular importance of serum LDH level and of tumor burden.

UnivariateanalysisRNA

Index(1.0-1.8favorable)(S

+ G2M)%(10- 18adverse)DNA

Index(Diploid favorable)P0.0010.0040.007Multivariate

(in order ofentry)RNA

Index(1.0- 1.8favorable)(S

-1-GjM)%(10- 18adverse)LDH(£225

mU/ml favorable)P0.0010.0050.001

Tumor burden (Low favorable) 0.02

Extranodal sites 0.03(Zero favorable)

Stage (111 favorable) 0.03

LDH (<225 mU/ml favorable) 0.06Constitutional symptoms NS°

Age NS

which is a composite measure of number of extranodal sitesand extent of nodal involvement (8). Because only three patientshad T-cell LCL, the impact of phenotype could not be assessedreliably.

Table 4 summarizes the analysis of factors influencing survival. The univariate analysis identified six factors [Rl,(S+G2M)%, DNA index, tumor burden, extranodal involve-

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NUCLEIC ACID FLOW CYTOMETRY IN LARGE CELL LYMPHOMA

100

80

Fig. 3. Survival by <S+G2M)%. Left, all ¡5large cell lymphomas; right, diffuse large cell j¿lymphoma. ^

20

1001

80

60

401

X S >18 (N= 1B)

?.S 10-18 (N= 1B)

10(N=13)

* S 10-18 (N=14)

12 24 36

MONTHS

60 12 24 36

MONTHS

1001

80

O 60

U

LU 40O.

20 ^

>18(N=18)

H

5CS S 10(N=13)

—¿�I H-

10-18 (N=14)

12 48 6024 36

MONTHSFig. 4. Failure-free survival by (S+G2M)% for patients with diffuse large cell

lymphoma.

ment, and stage] that significantly correlated with survival; inaddition, LDH was of borderline significance. The multivariateanalysis identified RI as the most significant factor, followedby (S+G:>M)% and serum LDH. These three were the onlyfeatures that were significant in the multivariate analysis.

Relapsing patients

Table 5 summarizes the results for the 67 relapsing patientswho received salvage therapy, according to their FCM features.The results for the subset with DLCL were similar. Low RIcorrelated significantly with longer survival and FFS. Therewas a trend for longer survival and FFS for those with low(S+G2¡VI)%.DNA ploidy had no bearing on outcome for relapsing patients.

DISCUSSION

The current study demonstrates the prognostic utility of AOFCM in patients with large cell lymphoma. Each FCM param-

Table 5 Outcome for relapsing patients by nucleic acid features

Nucleic acidfeaturePloidyDiploid

AneuploidRNA

indexT-1.8>l.8(S

+G:M)%<IO10-18No.37

3018

3118341914%CR27

21332318271629All

LCLsMedian

survivalP¡2J

NS°221

11 \0.01611419

}•0.095ÕMedian

FFS51

5110

i3)613Õ,NS0.020.25°

NS. not significant.

éter[RNA index, (S+G2M)%, and DNA index] provided significant prognostic information. This ability to stratify patientswithin one histológica! subtype of lymphoma indicates that AOFCM can be a valuable adjunct to routine histopathologicalexamination of biopsy specimens. The dominant prognosticvalue of the RNA content in LCL has not previously beenreported.

The RNA index was the strongest predictor of outcome inthe current study. Cellular RNA content correlates with cellularprotein content and cell cycle compartment; quiescent cellshave the lowest amount of RNA (38,39). In broad-based studiesof malignant lymphoma, increasing RNA content has beennoted to correlate with increasing histológica! grade and, consequently, with worse prognosis (22, 23). Likewise, in acutelymphoblastic leukemia, high RNA content correlates with anadverse prognosis (40-42). Our experience with relapsing LCLparallels these previous observations: within this one histolog-ical category, the natural history of the disease is most aggressive for high RNA-content LCLs.

In contrast, the pattern with previously untreated LCLshowed a striking deviation from the expected worse prognosiswith higher RI. In these patients, who received courses ofintensive combination chemotherapy every 3 weeks, an intermediate RI was associated with the highest potential for cure,and patients with very high RI had virtually the same outcomeas those with very low RI. Thus, LCLs with a high fraction ofquiescent cells with low RI may be the least sensitive to thisschedule of chemotherapy. Since low-grade lymphomas have acharacteristically low RI (22, 23), it is possible that a low RIidentifies a group of LCL patients who have a background of

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NUCLEIC ACID FLOW CYTOMETRY IN LARGE CELL LYMPHOMA

indolent lymphoma and who are thus less likely to be curedwith currently available therapy.

In the present study, the presence of aneuploidy was anadverse prognostic factor for newly diagnosed patients. Aneuploidy has been associated with adverse outcome in a numberof other malignancies (17), and in lymphoma aneuploidy isnoted with increasing frequency as the histológica! grade increases (22,23). Our findings contrast with the reports of Bauerel al. (12). who did not note any predictive role of DI, andWooldridge et al. (43), who found patients had better responseand survival rates with aneuploid LCLs than with diploid. Thedifferent findings in our series and Bauer's may be partly due

to technical differences. In contrast to our use of AO on freshlybiopsied tissue, they used PI or DAPI (4,6-diamidine-2-phenyl-indole dihydrochloride) to stain deparaffinized tissue. Whilegenerally reliable (44, 45), the resolution of Go-i DNA peaks ofdeparaffinized tissue is not as good as with fresh tissue. WhileAO gives slightly poorer resolution of DNA peaks than manyDNA dyes, good concordance has been shown between AO andPI studies of fresh tissue (46), and the simultaneous quantita-tion of RNA provides not only the independently importantRNA data, but also often permits separation on the basis ofRNA content of different cell populations with similar oridentical DNA contents. Both Bauer's and Wooldridge's series

were retrospective while ours was prospective, but this difference does not readily explain the different results. While furtherstudies of both archival material and freshly biopsied specimenswill be needed to be certain of the impact of aneuploidy, ourcurrent data indicate that aneuploidy is an adverse prognosticfactor in LCL.

The impact of (S+G2M)% on outcome in LCL appears to betwofold. First, there is a significant survival advantage for low(S+G:M)% LCLs, even when the analysis is restricted toDLCL. This finding is largely in accord with the findings ofBauer et al. (12) and Wooldridge et al. (43), although theiremphasis was on the adverse outcome of patients with veryhigh (S+G:M)% (>20), which included 42% of Wooldridge'spatients, but only 15% of Bauer's. Our second and perhaps

more provocative finding concerning (S+G:M)% was .that patients with high (S+G2M)% (>18)may have the highest curativepotential, based on the apparent 60% FFS plateau observed inthese patients (Fig. 4). While this observation is based onrelatively short follow-up and is not statistically significant,almost a third of the patients in the high (S+G2M)% grouphave been followed for over 24 months and are therefore pastthe time of highest risk for relapse. If continued follow-upconfirms this impression, it will contradict the observation thathighly proliferarne lymphomas carry an adverse prognosis (47).Thus our data may indicate that chemotherapy has a particularly profound effect on the clinical course of highly prolifera-tive LCLs.

Patients with relapsing LCL are generally incurable withcurrent therapy. It is not surprising, then, that the prognosticimplications of the FCM findings for relapsing patients weredifferent than for newly diagnosed patients. No relapsing patients with a substantial chance of cure were identified. In theabsence of potentially curative therapy, the FCM features thatcorrelated with longer survival were those that correlate with amore indolent natural history: low RNA index and low(S+G2M)%.

The prognostic utility of AO FCM was compared with otherknown clinical features of prognostic significance through adetailed analysis of untreated patients. For LCL. a number ofprognostically important clinical features have been well de

fined, including serum LDH level, extranodal involvement, andtumor burden, (8-11). The multivariate analysis of clinical andFCM features identified RNA index as the most importantfactor in the current series. Serum LDH level and (S+G2M)%also conveyed significant independent prognostic information.While this analysis requires confirmation in a larger group ofpatients, it indicates that the biological data provided by AOFCM not only gives insight into cell kinetics, but also appearsto be the most valuable predictor of survival for patients withLCL.

ACKNOWLEDGMENTS

We thank Sabrina Harris for her expert secretarial assistance withthe preparation of the manuscript and Peggy Daniels for her help withthe statistical analyses.

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1988;48:6614-6619. Cancer Res   Peter McLaughlin, Barbara M. Osborne, Dennis Johnston, et al.   Nucleic Acid Flow Cytometry in Large Cell Lymphoma

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