Prognostic Value of Cellular Proliferation and

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Prognostic Value of Cellular Proliferation and Histologic Grade in
Follicular Lymphoma
By Anita R. Martin, Dennis D. Weisenburger, Wing C. Chan, Elizabeth I. Ruby, James R. Anderson, Julie M. Vose,
Philip J. Bierman, Martin A. Bast, Donald T. Daley, and James 0.Armitage
The clinical usefulness ofhistologic grading in follicular
lymphoma (FL) is controversial and is furthercompromised
by the subjective nature and poor reproducibility of most
systems in current use. Therefore, we decided t o objectively
evaluate the importance of cellular proliferation in FL, along
with the current grading systems. We studied 106 patients
with FL whowereuniformly
staged and aggressively
treated. A proliferative index (PI) was determined quantitatively using an automatedimage analyzer and a new Ki-67
antibody thatstains archival paraffin
tissues. The cases were
also subclassified according t o t h eBerard, Rappaport, LukeCollins, and Jaffe methods, and survival analysis was performed. Patients with a low PI (C40461 had a significantly
longer overall survival
(OS)than thosewith a high PI (~40%).
but the PI did not predict failure-free survival (FFS). The
mean PI correlated well with the subgroups in each of the
various classifications. All four of the classification methods
were predictive ofOS, but only theBerard method appeared
t o predict FFS and suggest that a proportion of patients
with FL may be curable. In multivariate analysis, histologic
classification wasthe only independentpredictor ofOS (Berard method: relative risk, 3.1) and the International Prognostic Indexwas the only
independent predictor ofFFS (relative risk, 2.3).We conclude that the Berard method for
grading of FL is clinically useful and, along with theInternational Prognostic Index, should beincluded in future clinical
studies of FL. The measurement of cellular proliferation does
not appear t o add additional useful information in FL.
0 1995 by The American Society of Hematology.
the time of initial diagnosis by members of the Nebraska Lymphoma
Study Group (NLSG) between July 30, 1981 and December 20,
1991. Only patients whose initial diagnostic biopsy displayed at least
70% follicularity were included in the study. Patients were evaluated
before treatment with a complete history and physical examination,
conlplete blood count, chemistry profile, chest radiograph, computed
tomography of the abdomen and pelvis, and a bone marrow biopsy.
The Ann Arbor system was used to stage the patients. During the
early accrual period of this study, patients with localized disease
received involved-field irradiation as the primary treatment. Subsequently, all patients received as primary therapy one of four closely
related six-drug combination chemotherapy regimens including cyclophosphamide, doxorubicin or mitoxantrone, and procarbazine,
plus bleomycin, vincristine, and prednisone or dexamethasone (CAP/
BOP). The details of the four treatment plans have been previously
reported.' Of 130 eligible patients enrolled on this protocol, clinical
dataand adequate tissue for Ki-67 stains were available for 106
patients; of these 106 patients, 11 (10%) received involved-field
irradiation only asthe primary treatment for localized disease. A
complete response was defined as the absence of demonstrable disease after the completion of therapy and restaging.
Pathologic features. All 106 cases hadbeen previously subclassified using the Berard method" as follicular small cleaved cell
(FSC), mixed cell (FM), or large cell ( K C ) type for theNLSG
diagnosis of record. For the study, the cases were also subclassified
by two pathologists (A.R.M. and W.C.C.) using the Rappaport and
OLLICULAR LYMPHOMA (FLJ is generally considered
to be a low-grade malignancy characterized by an indolent clinical course and a prolonged survival. Although long
complete remissions can
be induced in some cases, only
a small
proportion of patients with FL are thought to be cured with
conventional the rap^."^ A number of clinicalfeatureshave
been associated with an adverse prognosis inFL, but the value
of pathologic subclassification of FL with regard to therapy
or prognosis is controversial.1-8 The usefulness of pathologic
subclassification of FL is further compromised by the lack of
uniform criteria for grading FL and poor diagnostic reproducibility due to the subjective nature of most grading system^.^"^
The level of proliferative activity, as determined by flow
cytometric or cell kinetic studies, has been shown to correlate
with tumor grade and have important prognostic implications
in non-Hodgkin's lymphoma (NHL).12"7The antibody Ki67, which defines a proliferation-associated nuclear antigen,l8.l9has been used in tissue sections of NHL to determine
proliferative activity2' and has recently been found to also
have prognostic v a l ~ e . Although
~ " ~ ~ a number of small series
have shown that proliferation increases with the number of
large cells in FL,25-28
only one study has shown the potential
utility of proliferation as an independent predictor of survival
in low-grade
However, the latter studyz9excluded cases
of follicular large cell lymphoma, and the patients were not
uniformly staged and aggressively treated.
Therefore, we decided to objectively evaluate the importance of proliferative activity in a large group of patients
with FL who were uniformly staged and aggressively treated.
We also evaluated the importance of histologic classification
of FL using four accepted methods (Rappaport, Lukes-Collins, Berard, and Jaffe). Finally, we evaluated the various
clinical factors of potential prognostic importance in FL,
including the recently developed International Prognostic Index for patients with diffuse aggressive NHL,30 and performed a multivariate analysis to determine which factors
were independent predictors of survival in F L ,
Patients. The patients included in this study are all adults who
were diagnosed with FL and were uniformly staged and treated at
Blood, Vol 85, No 12 (June 15). 1995: pp 3671-3678
From theDepartments of Pathology and Microbiology, Preventive
and Societal Medicine, and Internal Medicine, University of Nebraska Medical Center, Omaha, NE.
Submitted September 12, 1994; accepted January 31, 1995.
Supported in part by US Public Health Service Grant No.
CA36727 awarded by the National Cancer Institute, Department of
Health and Human Services.
Address reprint requests to Dennis D. Weisenburger.MD, Department of Pathology and Microbiology, University of Nebraska Medical Center, 600 S 42nd St, Omaha, NE 68198-3135.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
367 1
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Table 1. Histologic Methods for Grading FL
Berard Method (used by the NLSG)
1 5 large noncleaved cells/follicular hpf
r 5 but 115 large noncleaved cells/follicularhpf
215 large noncleaved cells/follicular hpf
Rappaport method
125% large cells/follicle
24-49% large cells/follicle
FM (Mixed)
FLC (Histiocytic) 250% large cells/follicle
Lukes-Collins method
1 2 5 % large noncleaved cells/follicle
225% large noncleaved cells/follicle
Jaffe modification of the Berard method
1 5 large noncleaved cells/follicular hpf
2 5 large noncleaved cells/follicular
150% large cells
>50% large cells/follicular hpf (predominance
of large noncleaved and/or small noncleaved
Abbreviations: hpf. high-power field (0.158mm'); PDL, poorlydifferentiated lymphocytic.
Lukes-Collins methods" and using a modified version of the Berard
method recently described by Jaffe et al" (Table I). The tissue
section withthe greatest number of large cells present within the
neoplastic follicles was used for this purpose and for immunostaining
as described below. In the early phase of the study. more patients
with the FLC type were enrolled than those with low-grade disease
(FSC or FM) hecause of the existence of a competing noncurative
treatment protocol for the latter group. However, since June of 1986.
all patients with FL who could tolerate aggressive therapy were
treated with CAP/BOP. Because of the small number of cases of
FSC type (S cases) identified using the Berard or Jaffe methods, the
FSC and FM cases were combined into onegroup for statistical
Ir,l,,lrrnosroini,l~ with Ki-67cmtibrxl~. Five-micron thick paraffin
sections of formalin-fixed tissue were deparaffinized and rehydrated.
To enhance antigen retrieval, sections were subjected to microwave
incubation in citrate buffer at pH 6.0 twice for S minutes at full power
and were then allowed to incubate in hot buffer for 10 minutes.
The slides were incubated with polyclonal rabbit antihuman Ki-67
antibody (A 047 diluted 150: Dako. Carpinteria, CA)," stained with
the avidin-biotin peroxidase complex technique, and developed with
H20Zand diaminobenzidine (Sigma Chemical. St Louis. MO). and
thenuclei were counterstained with 0.2% ethyl green in sodium
acetate buffer. pH 4.0.
Qrtnnrirrrriw irncrgr crm/ Quantitation of
staining was performed with the Cell Analysis Systems (CAS) 200
image analyzer7' using the CAS Quantitative Proliferation Software
(Becton Dickinson. Elmhurst, IL) to obtain the percent nuclear area
positive (PNA) and the cellular proliferation index (PI). These methods have been previously detailed and ~alidated.'~."."."' Fifteen
neoplastic follicular fields were analyzed in each case and the fields
were selected from follicles withthe greatest number of stained
cells. Positive staining was identified as granular nuclear stain (Fig
I). The PNA value is calculated as the immunostained nuclear area
divided by the total nuclear area which is ethyl green stained and is
expressed as a percentage. The P1 value is similar to the PNA except
that the immunostained nuclear area is first divided by the individual
immunostained nuclear size estimate and the total nuclear area is
divided by the overall nuclear size estimate, thus giving the percentage of positive cells.
Srcrristiccr/ cmcr/ Curves of overall survival (OS) and failurefree survival (FFS) were drawn for the various histologic subgroups.
by PI and PNA. and according to various clinical features of disease
using the method of Kaplan and Meier." The cutpoints for the PI
were determined by dividing the cases roughly into
quartiles around the median PI (39%) and median PNA (32%).
respectively. and evaluation of the curves of OS and FFS. Other
cutpoints considering the PI and PNA as continuous variables were
also evaluated. OS was defined as the time from initial therapy to
death from any cause or to last follow-up. FFS was defined a s the
time from the onset of initial therapy to the first occurrence of
progression, relapse after response, death from any cause, or to the
date of last follow-up if none of these events had occurred. Survival
curves were compared using the logrank testAxand all P values
reported are two-sided. Although the International Prognostic Index"'
describes four risk groups. ie, low, low intermediate. high intermediate, and high, we combined and compared the two low groups with
the two high groups because the number of patients in the high risk
group was small. A multivariate analysis to determine independent
predictors of survival was performed using the Cox proportional
hazards model for survival analysis."'
Fig 1. (A) Follicular lymphoma showing prominent delineation of the neoplastic follicles with the Ki-67 antibody stain. (B) Positive nuclear
staining marks the cells in the G,, S, GP,and M phases of the cell cycle (immunoperoxidasestains; A, x25; B, ~150.1
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Table 2. Clinical Characteristics of 106 PatientsWith F1
No. of Patients (%J
Age ( v )
27 (26)
13 (12)
23 (22)
43 (40)
85 (80)
21 (20)
LDH level
Extranodal sites*
Bulk disease
210 cm
Karnofsky scoret
5 80
International indextS
Low risk
High risk
Response to treatment
Not evaluable
79 (75)
27 (25)
63 (59)
43 (41)
21 ( 2 0 )
78 (76)
24 (241
88 (86)
14 (14)
Abbreviation: LDH, lactate dehydrogenase.
* Includes bone marrow, but not spleen.
t Data not available for all patients.
Inctudes the following factors: age
2 6 0 years; LDH > normal;
number of extranodal sites 2 2 ; stage III/IV; Karnofsky score 5 7 0 .
Clinical features. The clinical characteristics of the entire group are summarized in Table 2. The median age of
the patients was 61 years (range, 23 to 84 years) and the
median follow-up period was 46 months (range, 2 to 136
months). The predicted 5-year OS was 67% and the 5-year
FFS was 39% (Fig 2). By univariate analysis, the only clinical features that were predictive for OS were age (P= .098)
and the International Prognostic Index ( P = .074). The clinical features that were predictive for FFS included the stage
of disease ( P = .01 l), symptom status ( P = .034), number
of involved extranodal sites (P = .01l), Kamofsky score (P
= .022), and the International Prognostic Index (P = .014).
Pathologic features. The effect of the PI on OS is shown
in Fig 3. Patients with a low PI (<40%) had a significantly
longer OS than those with a high PI (240%; P = .022).
However, the PI did not predict for FFS, and the PNA was
not predictive of either OS or FFS. The number of cases by
histologic subgroup for each of the classifications is shown
in Table 3.When the FSC and FM categories were combined, the cases in this subgroup in our series were identical
when using either the Rappaport or Jaffe methods. Each of
the methods categorized the cases quite differently, with the
Berard methodhaving the most FLCcases and theRappaport
or Jaffe methods having the fewest FLC cases. The mean
PI correlated well with the subgroups in each of the classifications (Table 3).
All of the classification methods were predictive of OS
(Fig 4), but only the Berard method appeared to predict FFS
(Fig 5). Interestingly, using the Berard method, patients with
the FSC/FM type had a better OS, but poorer FFS, than
those with the FLC type (Figs 4A and 5A). The apparent
plateau in the FLC FFS curve (Fig 5A) suggests that a proportion of patients with this type may be cured. The predicted
8-year FFS of patients with FLC was 47% (95% confidence
interval [CI], 33% to 61%) versus only 17% for those with
FSC/FM (95% CI, 2% to 32%).
The superiority of the Berard method as compared with
the other methods for predicting FFS is shown in Fig 6.
Cases classified as FM by the Jaffe method but as FLC by
theBerard method, ie, those cases with 15 or more large
cells but less than 50% large cells per follicle, had the same
FFS as those classified as FLC by both methods (ie, cases
with 250% large cells per follicle). In contrast, the FFS of
cases classified as FM by both methods, ie, cases with 5 to
14 large cells per follicle, fails to plateauand is characterized
by a pattern of continuous relapse.
Multivariate analysis. A Cox proportional hazards
model for survival analysis wasused to determine which
prognostic factors were predictive of OS and FFS when
controlling for other factors found to be significant in the
univariate analysis. For OS, only the histologic classification
was an independent predictor. The Berard method was the
best predictor of OS with a relative risk of death of 3.1
for those with FLC as compared with those with FSC/FM,
whereas the Rappaport, Jaffe, and the Luke-Collins methods
gave relative risks of 2.4,2.4, and 2.1, respectively. Only
the International Prognostic Index was an independent predictor of FFS, with a relative risk of failure of 2.3 for the
high risk group (Fig 7).
The subjective nature and poor reproducibility of most
histologic grading systems of FL,9"' as well as ongoing controversy regarding the clinical value of such systems,"' led
us to study our cohort of patients with FL who were uniformly staged and aggressively treated. Because a number
of studies have indicated that proliferative activity is an important prognostic indicator in NHL,"-17. 2"2429 we decided
to evaluate this parameter in an objective and reproducible
manner. Image analysis is an ideal method for assessing
proliferation in FL because it is rapid, objective, and reprodUCible,25.27.'5.36.40and the analysis can be limited to the neoplastic follicles with the greatest number of large cells. In
our study, we used the automated, quantitative CAS image
analysis system25~z7~34-36
and a new Ki-67 antibody that works
in paraffin tissue,33thus allowing the study of archival tis-
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7060V) 5050
L 30.
g 3030
-E 3040-
i; 20-
-- A
100 4,
Fig 2.
OS (A) and FFS (B) of all patients with FL.
sues. Previous studies of NHL have used a Ki-67 antibody
that only works in snap-frozen tissue, thus limiting the number of cases available for study. However, one obstacle to
the use of archival tissues is the variation in Ki-67 antibody
staining of tissues that are fixed and processed at different
institutions, and a number of cases had to be excluded from
our study because of inadequate staining. The results that
we obtained for FL with the CAS system are similar to those
reported by Schwartz et a1': who also used the same system
and methods. However, our results are difficult to compare
with other reported results obtained by manual methods because only a small number of cases of FL have been so
studied and the Ki-67 immunohistochemical, cell counting,
and histologic classification methods used were quite heterogeneous. In four such s t ~ d i e s ? ~the
, ~ "manual
~ ~ results were
similar to our results, whereas in three other studies:0,22.44
two of which also included diffuse counterparts of FL,20,22
the results were somewhat lower. However, our study is the
first to evaluate Ki-67 proliferative activity in FL and relate
this parameter to survival.
Similar to the study of Macartney et al?9 which evaluated
the S-phase fraction of low-grade FL by DNA flow cytometry, we found that patients with FL having a low PI had a
significantly longer OS than those with a high PI (Fig 3).
However, in our study, the PI was not predictive of FFS and
was not an independent predictor of OS in the multivariate
analysis. As in other smaller st~dies:~-~*
we found that proliferation correlated with the histologic subgroups of FL in
each of the various classifications (Table 3), thus confirming
the association of cellular proliferation with large-cell cytology in NHL.4' In contrast, Weiss et a16 failed to find such
a correlation in FL when using a manual, semiquantitative
method to estimate Ki-67 proliferation rates. Interestingly,
two studies have failed to show a correlation between the
mitotic rate
and survival in
whereas a recent study
found that abundant mitoses predicted for better survival in
FLC NHL.48However, our study indicates that the Ki-67
antibody can be used to provide important information with
regard to the OS of patients with FL.
Because of the subjective nature and poor reproducibility
of methods for grading FL that require estimation of the
percentage of large cells:"'
we adopted the cell counting
method of Mann and Berard3' some years ago for use by
the NLSG. Nathwani et all' found that it was difficult for
hematopathologists to reproducibly separate favorable from
unfavorable FLs using subjective morphologic criteria alone
and suggested that the cell counting method is superior to
the estimation of percentages of large cells for grading FL.
Although reproducibility may also be a problem with the
Berard method: we have found this method to be less subjecTable 3. Association of PI With Histologic Subtypes of FL
Mean PI +
SE (%l
32.1 2 1.9
43.4 C 1.5
t 1.4
48.3 t 2.0
35.3 i 1.7
i 1.6
No. of
> 60-
..........+....** .............. *,
PI 240
Berard (NLSG) method
RappaporYJaffe methods
Lukes-Collins method
................ +
Fig 3. OS of patients with FL by PI.
Abbreviation: SE, standard error.
* P Value based on the Student's t-test.
PI Range
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tive and more reproducible than the other methods in our
daily practice. However, when the Berard method is used,
the proportion of cases in the various FL subgroups is different from that found by estimating percentages of large cells
(Table 3). In our study, which was biased toward the inclusion of FLC cases (see Materials and Methods), the majority
of FLs were classified as FLC type (60%) using the Berard
method, whereas lesser numbers of cases were classified as
such using the Lukes-Collins or RappapodJaffe methods
(40% and 20%, respectively). In a large and unselected survey of cases in the population-based registry of the NLSG,"9
we found that 16% of our cases of FL were classified as
FM 1
.= 60-
O 20
.- .
*......I"+ i.....,.
...*+..+.*+.. .............. H.....+
Fig 5. FFS of patients with FL according to the various classification systems.
Fig 4. OS of patients with FL according to the various olassification systems.
FSC, 37% as F M , and 47% as FLC type using the Berard
method. Our more recent data is as follows: FSC 25%, FM
36%, and FLC 39%. These findings are roughly the reverse
of what has been reported using more traditional metho d ~In .the~ only
~ study
~ ~ similar
to ours, Nathwani et al"
categorized FL according to the Berard method and found
the following: FSC 36%, FM 42%, and FLC 22%. However,
the cases in this latter study" were highly selected and the
distribution of cases may not be comparable to ours. The
possibility that an excess of FLC NHL occurs in Nebraska,
possibly because of heavy pesticide use,51is also a consideration and will be the subject of a future study. However,
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these differing findings may have particular relevance to the
comparisonand interpretation of clinical studiesthat use
different criteria for the selection and grading of FL.
In our study, we found that all of the classification methods were useful for predicting OS in FL (Fig 4). In fact,
histologic classification was the only independent predictor
of OS in the multivariate analysis, with the Berard method
being the best predictor of OS (relative risk, 3.1). The Berard
method also appeared to predict for FFS, whereas the other
grading methods were notpredictive of FFS (Fig 5). The
apparent plateau in the FLC FFS curve (Fig 5A) suggests
that a proportion of patients with this
type of FL may be
curable, as has been previously suggested by others using
the Rappaport method.52"s although longer follow-upof our
serieswill be necessary to confirmthisfinding. However,
using the Berard method, Bartlett et a148 have recently reported results similar to ours with regard to prolonged FFS
in aggressivelytreatedpatientswith
FLC NHL and have
suggested that such patients should be treated with curative
The superiority of the Berardmethod over the othermethods forpredicting FFS is further evidentin Fig 6. It is apparent from these curves that some cases classified as FM type
using the Jaffe method and other
methods, ie, those cases
with 15 or more large cells but less than 50% large cells per
follicle, have an FFS that is identical to those with 50% or
morelarge cellsper follicle. In otherwords,suchcases
should probably be placed for clinical purposes in the FLC
category, as with the Berard method. This same finding was
also recently reported by Bartlett et aI4* and indicates that
some cases classified as FM type using the other methods
may be curable. Further studies with longer follow-up will
also be necessary to confirm this finding. However, the results of our study suggest tous that theBerard method yields
more clinically relevant information than the other grading
methods for FL and that it should be used along with other
methods in future clinical studies.
In our study, we found that the International Prognostic
Index," which was developed as a predictive model for diffuse aggressive NHL, predicts for OS and FFS in FL. Others48,56.57
have also recently reported that the International
Prognostic Index is predictive of survival in FL, with one
Berardlarge caWJaHa mixed cell
(215 large cells. but < 50 % large cells)
Berard largeCnWJaffe large Cell
(D 50% large cells)
Berard rnlred dUJaffe mixed cell
(> 5 large cells b U c 15 large cells)
l _ c
)".,4 _ _ _ _ . _ I
Fig 6. FFS of patients with FL classified by both the Berard and
Jaffe methods (see text for explanation).
5 ao
L' .
2 70
Fig 7. FFS of patients with FL according to theInternational Prognostic Index.
exception.58 In our study, multivariate analysis showed that
the International Prognostic Index was the onlyindependent
predictor of FFS (relative risk, 2.3; Fig 7). The International
PrognosticIndex, or modificationsthereof, along with the
Berard method of grading, should be useful in the design of
future therapeutic trials of patients with €Xand in the selection of appropriate therapeutic approaches for individual patients.
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1995 85: 3671-3678
Prognostic value of cellular proliferation and histologic grade in
follicular lymphoma
AR Martin, DD Weisenburger, WC Chan, EI Ruby, JR Anderson, JM Vose, PJ Bierman, MA Bast,
DT Daley and JO Armitage
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