Prognostic Value of Cellular Proliferation and

From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
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.
F
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 ,
MATERIALS AND METHODS
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.
0006-4971/95/8S12-0$3.00/0
367 1
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
MARTINET
3672
Table 1. Histologic Methods for Grading FL
Berard Method (used by the NLSG)
FSC
1 5 large noncleaved cells/follicular hpf
FM
r 5 but 115 large noncleaved cells/follicularhpf
FLC
215 large noncleaved cells/follicular hpf
Rappaport method
125% large cells/follicle
FSC (PDL)
24-49% large cells/follicle
FM (Mixed)
FLC (Histiocytic) 250% large cells/follicle
Lukes-Collins method
FSC
1 2 5 % large noncleaved cells/follicle
FLC
225% large noncleaved cells/follicle
Jaffe modification of the Berard method
FSC
1 5 large noncleaved cells/follicular hpf
FM
2 5 large noncleaved cells/follicular
hpf,
but
150% large cells
FLC
>50% large cells/follicular hpf (predominance
of large noncleaved and/or small noncleaved
cells)
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
analysis.
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.
B
AL
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/y.si.s. Quantitation of
Ki-67
antibody
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/y.si.s. 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
andPNA
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
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
HISTOLOGIC GRADE IN FOLLICULAR LYMPHOMA
Table 2. Clinical Characteristics of 106 PatientsWith F1
No. of Patients (%J
Characteristic
Age ( v )
<60
260
Sex
Male
Female
Stage
I
II
111
IV
Symptoms
A
27 (26)
13 (12)
23 (22)
43 (40)
85 (80)
21 (20)
B
LDH level
Normal
Elevated
Extranodal sites*
0
1-2
Bulk disease
210 cm
Karnofsky scoret
290
5 80
International indextS
Low risk
High risk
Response to treatment
Complete
Partiahone
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 .
+
RESULTS
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
3673
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).
DISCUSSION
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-
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
MARTIN ET AL
3674
9
7060V) 5050
40
40c
L 30.
30
L
g 3030
2,
50-
g
-E 3040-
Q)
i; 20-
8
10
-- A
0
2
4
Years
6
8
10
B
100 4,
0
2
6
4
10
8
Years
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
Patients
Mean PI +
SE (%l
P*
42
64
32.1 2 1.9
43.4 C 1.5
<'oool
85
21
8.6-59.5
t 1.4
48.3 t 2.0
.0°02
34.2-62.3
64
42
35.3 i 1.7
i 1.6
'0005
8.6-59.0
23.2-62.3
No. of
-m
...+.*;;
.E
> 60-
Classification
p=o.o22
........
..........+....** .............. *,
PI 240
,__.
i
Berard (NLSG) method
FSC F M
FLC
RappaporYJaffe methods
36.6
FSC FM
FLC
Lukes-Collins method
FSC
FLC
44.3
+
................ +
+
0
2
4
6
Years
Fig 3. OS of patients with FL by PI.
8
10
Abbreviation: SE, standard error.
* P Value based on the Student's t-test.
PI Range
(Oh)
8.6-59.5
10.2-62.3
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
HISTOLOGIC GRADE IN FOLLICULAR LYMPHOMA
3675
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
Bend
O0
90]y!..,
\
"r..,.+
pso.10
i.....
.."
..I.-.-"
Fsc
"1
+
FM 1
L"
-(.".-C
A
-0
2
4
6
8
10
6
8
10
YSam
Berard
l..).(*I.....+..l+>
......I..
90-
C
-
.........,
80.
70-
FSC+FM
.......I
..
it
+"*g,
....*..*....-
H..""
S
.= 60-
5
50-
=
40
U)
f
FLC
L
301
O 20
A
lo
0
0
2
6
4
8
10
0
Yeam
2
4
Ywm
RappapoRNaffe
Lukes-Cdlins
.- .
%+l
70
"r
I
0
2
FSC + FM
*......I"+ i.....,.
____._,_(
...*+..+.*+.. .............. H.....+
p=o.o082
I.
4
6
8
lo
10
0
c
0
2
4
6
8
10
Years
Fig 5. FFS of patients with FL according to the various classification systems.
10
0
2
4
6
8
10
YSarO
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,
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
MARTIN ET AL
3676
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
intent.
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
-5
.....
100-1
.-9
5
v)
90.
80706050-
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)
"
1
~
p=0.10
l _ c
L"-
0
2
)".,4 _ _ _ _ . _ I
4
Years
c-
6
\"Cl
8
10
Fig 6. FFS of patients with FL classified by both the Berard and
Jaffe methods (see text for explanation).
"l.
5 ao
L' .
2 70
2
60
50I"I"il
p:0014
-7
0
2
6
4
8
10
Years
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.
REFERENCES
1. Gallagher CJ, Gregory W,Jones AE, Stansfeld AG, Richards
MA, Dhaliwal HS, Malpas JS, Lister TA: Follicular lymphoma:
Prognostic factors for response and survival. J Clin Oncol 4:1470,
1986
2. McLaughlin P, Fuller LM, Velasquez WS, Butler JJ, Hagemeister FB, Sullivan-Halley JA, Dixon DO: Stage 111 follicular
lymphoma: Durable remissions with a combined chemotherapy-radiotherapy regimen. J Clin Oncol 5:867, 1987
3. Ersboll J, Schultz HB, Pedersen-Bjergaard J, Nissen NI: Follicular low-grade non-Hodgkin's lymphoma: Long-term outcome with
or without tumor progression. Eur J Haematol 42:155, 1989
4. Romaguera JE, McLaughlin P, North L, Dixon D, Silvermintz
KB, Garnsey LA, Velasquez WS, Hagemeister FB, Cabanillas F:
Multivariate analysis of prognostic factors in stage IV follicular lowgrade lymphoma: A risk model. J Clin Oncol 9:762, 1991
5. Soubeyran P, Eghbali H, Bonichon F, Trojani M, Richaud P,
Hoerni B: Low-grade follicular lymphomas: Analysis of prognosis
i n a series of 281 patients. Eur J Cancer 27:1606, 1991
6. Bastion Y , Berger F, Bryon P, Felman P, Ffrench M, Coiffier
B: Follicular lymphomas: Assessment of prognostic factors in 127
patients followed for I O years. Ann Oncol 2:123, 1991 (suppl 2)
7. Anderson JR, Vose JM, Bierman PJ, Weisenburger DD, Sanger
WG. Pierson J, Bast M, Armitage JO: Clinical features and prognosis
of follicular large-cell lymphoma: A report from the Nebraska
Lymphoma Study Group. J Clin Oncol 11:218, 1993
8. Coiffier B, Bastion Y, Berger F, Felman P, Bryon PA: Prognostic factors in follicular lymphomas. Semin Oncol 20:89, 1993 (suppl
5)
9. Metter GE, Nathwani BN, Burke JS, Winberg CD, Mann RB,
Barcos M, Kjeldsberg CR, Whitcomb CC, Dixon DO, Miller TP,
Jones SE: Morphological subclassification of follicular lymphoma:
Variability of diagnoses among hematopathologists, a collaborative
study between the Repository Center and Pathology Panel for
Lymphoma Clinical Studies. J Clin Oncol 3:25, 1985
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
HISTOLOGIC GRADE IN FOLLICULAR LYMPHOMA
10. Dardick I, Caldwell DR: Follicular center cell lymphoma:
Morphologic data relating to observer reproducibility. Cancer
58:2477, 1986
1 1 . Nathwani BN, Metter GE, Miller TP, Burke JS, Mann RB,
Barcos M, Kjeldsberg CR, Dixon DO, Winberg CD, Whitcomb CC,
Jones SE: What should be the morphologic criteria for the subdivision of follicular lymphomas? Blood 68:837, 1986
12. Bauer KD, Merkel DE, Winter JN, Marder RJ, Hauck W,
Wallemark CB, Williams TJ, Variakojis D: Prognostic implications
of ploidy and proliferative activity in diffuse large cell lymphomas.
Cancer Res 46:3173, 1986
13. Wooldridge TN, Grierson HL, Weisenburger DD, Armitage
JO, Sanger WC, Collins MM, Pierson JL, Pauza ME, Fordyce R,
Purtilo DT: Association of DNA content and proliferative activity
with clinical outcome in patients with diffuse mixed cell and large
cell non-Hodgkin’s lymphoma. Cancer Res 48:6608, 1988
14. Griffin NR, Howard MR, Quirke P, O’Brien CJ, Child JA,
Bird CC: Prognostic indicators in centroblastic-centrocytic
lymphoma. J Clin Pathol 41:866, 1988
15. Silvestrini R, Costa A, Giardini R, Boracchi P, Del Bin0
G, Marubini E, Rilke F: Prognostic implications of cell kinetics,
histopathology and pathologic stage in non-Hodgkin’s lymphomas.
Hematol Oncol 7:411, 1989
16. Rehn S, Glimelius B, Strang P, Sundstriim C, Tribukait B:
Prognostic significance offlow cytometry studies inB-cell nonHodgkin lymphoma. Hematol Oncol 8: 1, 1990
17. Lindh J, Lenner P, Osterman B, Roos G: Prognostic significance of serum lactic dehydrogenase levels and fraction of S-phase
cells in non-Hodgkin lymphomas. Eur J Haematol 50:258, 1993
18. Gerdes J, Lemke H, Biasch H, Wacker HH, Schwab U, Stein
H: Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol
133:1710, 1984
19. Schluter C, Duchrow M, Wohlenberg C, Becker MHG, Key
G, Had HD, Gerdes J: The cell proliferation-associated antigen of
antibody Ki-67: A very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J Cell Biol 123513, 1993
20. Gerdes J, Dallenbach F, Lennert K: Growth fractions in malignant non-Hodgkin’s lymphomas (NHL) as determined in situ with
the monoclonal antibody Ki-67. Hematol Oncol 2:365, 1984
21. Gerdes J, Stein H, Pileri S, Rivano MT, Gobbi M, Ralflciaer E,
Nielsen KM, Pallesen G, Bartels H, Palestro G, Delsol G: Prognostic
relevance of tumour-cell growth fraction in malignant non-Hodgkin‘s lymphomas. Lancet 2:448, 1987
22. Hall PA, Richards MA, Gregory WM. d’Ardenne AJ, Lister
TA, Stansfeld AG: The prognostic value of Ki67 immunostaining
in non-Hodgkin’s lymphoma. J Pathol 154: 223, 1988
23. Grogan TM, Lippman SM, Spier CM, Slymen DJ, Rybski
JA, Range1 CS, Richter LC, Miller T P Independent prognostic significanceof a nuclear proliferation antigen in diffuse large cell
lymphomas as determined by the monoclonal antibody Ki-67. Blood
71:1157, 1988
24. Miller TP, Grogan TM, Dahlberg S, Spier CM, Braziel RM,
Banks PM, Foucar K, Kjeldsberg CR, LevyN, Nathwani BN,
Schnitzer B, Tubbs RR, Gaynor ER, Fisher RI: Prognostic significance of the Ki-67-associated proliferative antigen in aggressive
non-Hodgkin’s lymphomas: A prospective Southwest Oncology
Group trial. Blood 83:1460, 1994
25. Schwartz BR, Pinkus G , Bacus S, Toder M, Weinberg DS:
Cell proliferation in non-Hodgkm’s lymphomas: Digital image analysis of Ki-67 antibody staining. Am J Pathol 134:327, 1989
26. Tominaga K, Yamaguchi Y, Nozawa Y, Abe M, Wakasa H:
Proliferation in non-Hodgkin’s lymphomas as determined by immu-
3677
nohistochemical double staining for Ki-67. Hematol Oncol 10:163,
1992
27. Sebo TJ, Roche PC, Witzig TE, Kurtin PJ: Proliferative activity in non-Hodgkin’s lymphomas: A comparison of the bromodeoxyuridine labeling index with PCNA immunostaining and quantitative
image analysis. Am J Clin Pathol 99:668, 1993
28. Swerdlow SH, Westermann CD, Pelstring RJ, Saboorian MH,
Williams ME: Growth fraction in centrocytic and follicular center
cell lymphomas: Assessment in paraffin sections with a proliferating
cell nuclear antigen antibody and morphometric correlates. Hum
Pathol 24540, 1993
29. Macartney JC, Camplejohn RS, Moms R, Hollowood K,
Clarke D, Timothy A: DNA flow cytometry of follicular non-Hodgkin’s lymphoma. J Clin Pathol 44:215, 1991
30. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project: A predictive model for aggressive non-Hodgkin’s
lymphoma. N Engl J Med 329:987, 1993
31. Mann RB, Berard CW: Criteria for the cytologic subclassification of follicular lymphomas: A proposed alternative method.
Hematol Oncol 1:187, 1983
32. Jaffe ES, Raffeld M, Medeiros U:Histopathologic subtypes
of indolent lymphomas: Caricatures of the mature B-cell system.
Semin Oncol 203, 1993 (suppl 5 )
33. Key G, Petersen JL, Becker MHG, Duchrow M, Schliiter C,
Askaa J, Gerdes J:New antiserum against Ki-67 antigen suitable
for double immunostaining of paraffin wax sections. J Clin Pathol
46:1080, 1993
34. Bacus JW, Grace LJ: Optical microscope system for standardized cell measurements and analyses. Appl Optics 26:3280, 1987
35. Dawson AE, Norton JA, Weinberg DS: Comparative assessment of proliferation and DNAcontent in breast carcinoma by image
analysis and flow cytometry. Am J Pathol 136:1115, 1990
36. Kennedy JC, El-Badawy N, DeRose PB, Cohen C: Comparson of cell proliferation in breast carcinoma using image analysis
(Ki-67) andflow cytometric systems. Anal Quant Cytol Histol
14:304, 1992
37. Kaplan GL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457, 1958
38. Pet0 R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard
SV, Mantel N, McPherson K, Pet0 J, Smith PG: Design and analysis
of randomized clinical trials requiring prolonged observation of each
patient. 11: Analysis and examples. Br J Cancer 35:1, 1977
39. Cox DR: Regression models and life tables. J R Stat SOC
34187, 1972
40. Bacus SS, Goldschmidt R, Chin D, Moran G, Weinberg D,
Bacus JW: Biological grading of breast cancer using antibodies to
proliferating cells and other markers. Am J Pathol 135:783, 1989
41. Schrape S , Jones DB, Wright DH: A comparison of three
methods for the determination of the growth fraction in non-Hodgkin’s lymphoma. Br J Cancer 55:283, 1987
42. Holte H, deLang Davies C, Beiske K, Stokke T, Morton PF,
Smeland EB, Hoie J, Kvaloy S: Ki67 and 4F2 antigen expression
as well as DNA synthesis at relapse/tumor progression in low-grade
B-cell lymphoma. Int I Cancer 44:975, 1989
43. Houmand A, Abrahamson B, Tinggaard Petersen N: Relevance of Ki-67 expression in the classification of non-Hodgkin‘s
lymphomas: A morphometric and double-immunostaining study.
Histopathology 20: 13, 1992
44. Cibull ML, Heryet A, Gatter KC, Mason DY: The utility
of Ki-67 immunostaining, nuclear organizer region counting, and
morphology in the assessment of follicular lymphomas. I Pathol
158:189, 1989
45. Weinberg DS: The role of cell cycle activity in the generation
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
3678
of morphologic heterogeneity in non-Hodgkin’s lymphoma. Am J
Pathol 135:759, 1989
46. Weiss LM, Strickler JG, Medeiros LJ, Gerdes J, Stein H,
Warnke RA: Proliferative rates of non-Hodgkin’s lymphomas as
assessed by Ki-67 antibody. Hum Pathol 18: 1155, 1987
47. Ellison DJ, Nathwani BN, Metter GE, Turner RR, Burke
JS, Miller TP, Mann RB, Barcos M, Kjeldsberg CR. Winberg CD,
Whitcomb CC: Mitotic counts in follicular lymphomas. Hum Pathol
18:502, 1987
48. Bartlett NL, Rizeq M, Dorfman RF, Halpern J, Horning SJ:
Follicular large-cell lymphoma: Intermediate or low grade? J Clin
Oncol 12:1349, 1994
49. Weisenburger DD: Pathological classification of non-Hodgkin’s lymphoma for epidemiological studies. Cancer Res 52:5456s,
1992 (suppl)
50. The Non-Hodgkin’s Lymphoma Pathologic Classification
Project: National Cancer Institute sponsored study of classifications
of non-Hodgkin’s lymphomas: Summary and description of a working formulation for clinical usage. Cancer 49:2112, 1982
51. Weisenburger D, Zahm S, WardM, Babbitt P, Holmes F,
Boysen C, Robel R, Saal R, Vaught J, Cantor K, Blair A: NonHodgkin’s lymphoma associated with the agricultural use of herbicides: Analysis by histologic type. Mod Pathol 3:105A, 1990
MARTIN ET AL
52. Osborne CK, Norton L, Young RC, Garvin AJ, Simon RM,
Berard CW, Hubbard S, DeVita VT: Nodular histiocytic lymphoma:
An aggressive nodular lymphoma with potential for prolonged disease-free survival. Blood 56:98, 1980
53. Glick JH, McFadden E, Costello W, Ezdinli E, Berard CW.
Bennett JM: Nodular histiocytic lymphoma: Factors influencing
prognosis and implications for aggressive chemotherapy. Cancer
49340, l982
54. Kantarjian HM, McLaughlin P, Fuller LM,Dixon DO, Osborne BM, Cabanillas FF: Follicular large cell lymphoma: Analysis
and prognostic factors in 62 patients. J Clin Oncol 2:811, 1984
55. Horning SJ, Weiss LM, Nevitt JB, Wamke RA: Clinical and
pathologic features of follicular large cell (nodular histiocytic)
lymphoma. Cancer 59: 1470, 1987
56. L6pez-Guillermo A, Montserrat E, Bosch F, Terol MJ, Campo
E, Rozman C: Applicability of the International Index for aggressive
lymphomas to patients with low-grade lymphoma. J Clin Oncol
12: 1343, 1994
57. Bastion Y, Coiffier B: Is the International Prognostic Index
for aggressive lymphoma patients useful for follicular lymphoma
patients? J Clin Oncol 12:1340, 1994
58. Aviles A: The International Index is not useful in the classification of low-grade lymphoma. J Clin Oncol 12:2766, 1994
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
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
Updated information and services can be found at:
http://www.bloodjournal.org/content/85/12/3671.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.