research paper
Histopathology, cell proliferation indices and clinical outcome in
304 patients with mantle cell lymphoma (MCL): a
clinicopathological study from the European MCL Network
Markus Tiemann,1* Carsten Schrader,1,2*
Wolfram Klapper,1 Martin H. Dreyling,3
Elias Campo,4 Andrew Norton,5
Francoise Berger,6 Philip Kluin,7 German
Ott,8 Stephano Pileri,9 Ennio Pedrinis,10
Alfred C. Feller,11 Hartmut Merz,11 Dirk
Janssen,1 Martin L. Hansmann,12 Han
van Krieken13 Peter Möller,14 Harald
Stein,15 Michael Unterhalt,3 Wolfgang
Hiddemann3 and Reza Parwaresch1
1
Department of Haematopathology and Lymph
Node Registry, 2Department of Haematology,
University Hospitals of Schleswig-Holstein,
Campus Kiel, Kiel, 3III. Division of Medicine,
University of Munich, Munich, Germany,
4
Department of Haematopathology, Barcelona,
Spain, 5Department of Histopathology, Barts and
the London NHS Trust, London, UK,
6
Department of Pathology, Centre Hospitalier
Lyon-Sud, Pierre Benite Cedex, France,
7
Department of Pathology, University of Leiden,
Leiden, The Netherlands, 8Department of
Pathology, University of Würzburg, Würzburg,
Germany, 9Haematopathology, University of
Bologna, Bologna, Italy, 10Department of
Pathology, Locarno, Switzerland, 11Department of
Pathology, University Hospitals of SchleswigHolstein, Campus Lübeck, Lübeck,
Summary
Mantle cell lymphoma (MCL) is a distinct lymphoma subtype with a
particularly poor clinical outcome. The clinical relevance of the
morphological characteristics of these tumours remains uncertain. The
European MCL Network reviewed 304 cases of MCL to determine
the prognostic significance of histopathological characteristics. Cytomorphological subtypes, growth pattern and markers of proliferation (mitotic
and Ki-67 indices) were analysed. In addition to the known cytological
subtypes, classical (87Æ5%), small cell (3Æ6%), pleomorphic (5Æ9%) and blastic
(2Æ6%), we identified new pleomorphic subgroups with mixtures of cells
(classical + pleomorphic type; 1Æ6%) or transitions (classical/pleomorphic type;
1Æ6%), which, however, did not differ significantly in overall survival time.
Exactly 80Æ5% of cases displayed a diffuse growth pattern, whereas 19Æ5%
of cases had a nodular growth pattern, which was associated with a slightly
more favourable prognosis. A high proliferation rate (mitotic or Ki-67
indices) was associated with shorter overall survival. Cut-off levels were
defined that allowed three subgroups with different proliferation rates to be
discriminated, which showed significantly different clinical outcomes
(P < 0Æ0001). Based on this large clinicopathological study of prospective
clinical trials, multivariate analysis confirmed the central prognostic role of
cell proliferation and its superiority to all other histomorphological and
clinical criteria.
Keywords: mantle cell lymphoma, cytology, growth pattern, Ki-67 index,
mitotic index.
12
Department
of Pathology, University of Frankfurt, Frankfurt,
Germany,
13
Department of Pathology, University
of Nijmegen, Nijmegen, The Netherlands,
14
Department of Pathology, University of Ulm,
Ulm, and 15Department of Pathology, University
of Berlin, Berlin, Germany
Received 23 May 2005; accepted for publication
17 July 2005
Correspondence: Dr C. Schrader, Department of
Haematology, University Hospitals of
Schleswig-Holstein, Campus Kiel, Chemnitzstr.
33, 24116 Kiel, Germany.
E-mail: [email protected]
*These authors contributed equally to this work.
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 29–38
doi:10.1111/j.1365-2141.2005.05716.x
M. Tiemann et al
Based on cytomorphological and histopathological observations, mantle cell lymphoma (MCL) was a well-accepted
lymphoma entity long before the characteristic chromosomal
translocation t(11;14) was described (Van Den Berghe et al,
1979; Medeiros et al, 1990; Williams et al, 1991). In addition
to the classical cytomorphology of MCL with small to
medium-sized cells containing medium-sized indented nuclei,
three cytological variants have been described: small cell [B-cell
chronic lymphocytic leukaemia (B-CLL)-like], pleomorphic
and blastic (Lardelli et al, 1990; Zoldan et al, 1996; Campo
et al, 1999). Although knowledge of these cytological variants
is useful for the diagnosis of MCL, their prognostic significance
is still uncertain (Lardelli et al, 1990; Fisher et al, 1995;
Argatoff et al, 1997; Decaudin et al, 1997). The term MCL
derives from the growth pattern of this lymphoma in the early
stages of the disease, when tumour cells surround residual
reactive germinal centres and replace the normal follicle mantle
(mantle zone pattern) (Weisenburger et al, 1982; Campo et al,
1999). Alternatively, MCL may show a nodular or diffuse
growth pattern (Zucca et al, 1994; Weisenburger & Armitage,
1996; Campo et al, 1999); however, there is still disagreement
as to the frequency and clinical relevance of these three
different growth patterns (Weisenburger et al, 1981; Lardelli
et al, 1990).
Generally, MCL is characterised by an aggressive clinical
course, a continuous relapse pattern after conventional chemotherapy and the poorest long-term outcome of all lymphoma subtypes (Lenz et al, 2004a). Recently, novel treatment
options, such as combined immuno-chemotherapy and myeloablative consolidation, have improved response rates and
progression-free survival, however, so far no survival plateau
indicating long-term remissions or potential cure has been
observed (Dreyling et al, 2004; Lenz et al, 2004b). In contrast,
a small subgroup of MCL patients shows a very indolent
course with an overall survival of up to 5–10 years (Campo
et al, 1999). Thus, reliable and routinely applicable prognostic
markers are urgently needed to identify the different prognostic patient subgroups and allow risk-adjusted therapeutic
approaches in MCL (Lenz et al, 2004b).
Several lines of evidence link a high cell proliferation rate in
MCL to an unfavourable course of the disease. Cell proliferation, assessed by counting of mitotic figures or expression of
Ki-67, has been previously proposed as a marker of poor
clinical outcome (Swerdlow et al, 1983; Argatoff et al, 1997;
Bosch et al, 1998; Raty et al, 2002; Schrader et al, 2004).
Additionally, gene expression profiling recently revealed that
the increased expression of the ‘proliferation signature’
representing an integrator of oncogenic events leading to the
higher expression of genes associated with cell proliferation
and cell cycle progression is a strong indicator of outcome in
MCL (Rosenwald et al, 2003). Some of the genes identified by
gene expression profiling, such as topoisomerase IIa (Rosenwald et al, 2003), have been confirmed in immunohistochemical
studies (Schrader et al, 2004). The clinical relevance of the
most widely used marker of cell proliferation, Ki-67, is still
30
being debated (Lardelli et al, 1990; Velders et al, 1996; Raty
et al, 2002).
The pathology panel of the European MCL Network analysed
MCL cases enrolled in eight clinical studies from six different
countries to determine the relevance of histopathological
parameters and cell proliferation to disease outcome. To our
knowledge, this is the largest clinicopathological study of
MCLs described so far.
Patients and methods
Patients
The study included biopsy specimens from 351 MCL patients
(92 females and 259 males; median age 64 years, range 27–86)
enrolled in three multi-centre prospective therapy trials and
five retrospective studies in the period between 1972 and 1994
(Tables I and II). Only cases with appropriate sample size and
quality were included after the histological diagnosis of MCL
had been confirmed by one of the participating pathologists
according to the criteria of the European Lymphoma Task
Force (ELTF) (Zucca et al, 1994) and the current World
Health Organisation (WHO) classification (Jaffe et al, 2001). If
the available material was insufficient, the case was classified as
not evaluable.
Review process
The review process was performed by two interactive panels of
five and six haematopathologists respectively. After all cases
were examined independently by each pathologist, a consensus
was considered to have been achieved if the subclassification
parameters assessed by at least four of five or five of six
pathologists matched (a < 0Æ05). Cases without consensus
were re-evaluated by all 11 pathologists on a multihead
microscope and the diagnosis made by the majority was
defined as the consensus diagnosis. On the basis of this
Table I. Country (centre) in which the study was performed, number
of patients enrolled in the study and slides available for review in the
present study.
Country or centre
Germany (trial 1988–1995)
Swiss (Locarno)
France (Lyon-Sud)
Great Britain (London)
Netherlands (Leiden)
Spain (Barcelona)
Germany (trial 1975–1980)
(Brittinger et al, 1984)
Germany (trial 1983–1988)
(Meusers et al, 1989)
Total
Number of
patients
Slides
available
90
35
48
66
41
34
87
56
20
27
64
39
26
71
84
48
485
351
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 29–38
Mantle Cell Lymphoma
Table II. Patient characteristics of all confirmed MCL (304 cases).
Table III. Cytological variants of mantle cell lymphoma.
Characteristics
Classical type
Small to medium-sized indented nuclei with moderately dispersed
chromatin
Scanty pale cytoplasm
Monotonous cell population
Occasional larger neoplastic cells infrequently interspersed
Small cell type
Small round or slightly indented nuclei with dense nuclear chro
matin
Mimics lymphocytic lymphoma/B-CLL with lack of prolymphocytes
and paraimmunoblasts
Pleomorphic type
Mixture of tumour cells with nuclei of different sizes (mediumlarge) and shapes mimics large B cell lymphoma
Nuclear chromatin plumed or pale
Moderate amount of cytoplasm
Blastic type
Monomorphic sheets of small to medium-sized blasts with fine
dispersed chromatin mimics lymphoblastic lymphoma/acute lym
phoblastic leukaemia
Narrow rim of cytoplasm
Frequent apoptotic bodies
Age (years)
<60
>60
Sex
Male
Female
Stage
1+2
3+4
Status (WHO)
0–1
‡2
LDH
Elevated
Normal
IPI
0–1
‡2
Bone marrow involvement
Yes
No
B symptoms
Yes
No
Extranodal involvement
Yes
No
n
123
178
230
71
23
267
233
43
56
140
15
75
207
81
107
155
161
16
LDH, lactate dehydrogenase; IPI, International Prognostic Index.
consensus diagnosis the diagnosis of MCL was confirmed or
the case was excluded.
(<50% nodular growth) according to Argatoff et al (1997) and
Zucca et al (1994).
Mitotic index
The mitotic index was analysed in 272 cases by counting the
number of mitotic figures/mm2. Hot spots of mitosis were
analysed separately.
Ki-67 index
Cytology
The cytology was classified as classical, small cell (B-CLL-like),
pleomorphic or blastic (Weisenburger & Armitage, 1996;
Campo et al, 1999) (Table III, Fig 1). In initial control
experiments, 20 cases with the confirmed diagnosis of MCL
were subclassified independently by each of the 11 panel
pathologists and a consensus cytology was defined. Subsequently, a second separate review was performed and the
results of the cytological diagnosis were compared. In this
blinded double evaluation, the cytological subtype (classical
MCL/small cell type versus blastoid/pleomorphic variant) of
the second review matched the consensus diagnosis in 85% of
cases (17 of 20) according to the above-mentioned criteria
(a < 0Æ05).
The immunohistochemical staining for Ki-67 (Ki-S5 or MiB-1)
was performed in the laboratories of 11 centres by either the
avidin–biotin complex (ABC) technique (Hsu et al, 1981) or
the alkaline phosphatase anti-alkaline phosphatase (APAAP)
method (Cordell et al, 1984), followed by counterstaining with
Mayer’s haematoxylin. The Ki-67 index was assessed in 187
cases by staining with specific monoclonal antibodies against
Ki-67 (Ki-S5 or MIB1) (Gerdes et al, 1984b; Kreipe et al,
1993). Neoplastic cells that were positive for Ki-67 were
counted by one pathologist in each centre in 10 consecutive
high power fields (400-fold magnification) using a haematological cell counter. By applying this method, a minimum of
500 neoplastic cells was assessed and the number of positive
cells was expressed as percentage of all tumour cells counted
(Ki-67 index). Proliferation hot spots were analysed separately.
Growth pattern
The growth pattern of the tumour was classified as nodular
with germinal centres (mantle zone pattern), predominantly
nodular (>50% nodular growth), or predominantly diffuse
Statistics
All statistical analyses were performed using the Statistical
Package for the Social Sciences (spss) software, version 11.
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 29–38
31
M. Tiemann et al
Fig 1. Cytological variants of MCL. (A) The classical type is characterised by small to medium-sized indented nuclei with moderately dispersed
chromatin. The cells have scanty pale cytoplasm. The cell population is monotonous with occasional larger neoplastic cells and a low proliferation rate
(Ki-67 index). Haematoxylin & eosin (H&E), ·630. (B) The small cell type has small round or slightly indented nuclei with dense nuclear chromatin
and mimics lymphocytic lymphoma/B-CLL with a lack of prolymphocytes and paraimmunoblasts. The proliferation rate is low. Typically, some
follicular dendritic cells are intermingled, in contrast to B-CLL. H&E, ·630. (C) The pleomorphic type shows a mixture of tumour cells with nuclei of
different sizes (medium-large) and shapes (mimicking large B-cell lymphoma). The nuclear chromatin can be plummed or pale. The cells have a
moderate amount of cytoplasm and a high proliferation rate. Often, the pleomorphic variant shows a mixture of blasts with indented and/or ovoid
nuclei with classical ‘centrocytes’ and a higher rate of mitosis and apoptosis. A continuous transition from the classical type is possible. H&E, ·630.
(D) The blastic type mimics lymphoblastic lymphoma/ALL with monomorphic sheets of small to medium-sized blasts with fine dispersed chromatin
and a narrow rim of cytoplasm. H&E, ·630. (E) The classical + pleomorphic type consists of a mixture of two cell populations, one with classical (C,
left side) and the other with pleomorphic (P, right side) cytology. H&E, ·400. (F) The classical/pleomorphic type consists of cells that display
cytomorphology resembling a continuous transition between the classical and pleomorphic type. H&E, ·630.
Overall survival time was calculated from the date of diagnosis
until death or loss to follow-up. Survival curves were estimated
according to the Kaplan–Meier method, and statistical comparisons were performed by log-rank test. P < 0Æ05 was
considered statistically significant. Univariate Cox regression
analysis was performed for all prognostic factors with respect
to overall survival. For 149 patients Ki-67 index, mitotic index,
age, performance status, lactate dehydrogenase (LDH), stage
and sex were available. They were used to compute a Cox
regression with likelihood ratio excluding stepwise parameters
with P > 0Æ1.
Results
Cytomorphology
Lymph node biopsy specimens from 351 MCL patients in eight
clinical trials were evaluated (Table I). Exactly 304 cases (87%)
were confirmed as MCL, 26 cases (7%) were classified as not
evaluable due to insufficient samples and in 21 cases (6%) a
different diagnosis was made. The cytological classification
revealed a classical cytomorphology (Fig 1A) in 87Æ5%
(n ¼ 266), a small cell type (Fig 1B) in 3Æ6% of cases
(n ¼ 11), a pleomorphic type (Fig 1C) in 5Æ9% (n ¼ 18) and
32
a blastic type (Fig 1D) in 2Æ6% (n ¼ 8). In one case no
cytological classification was performed.
In 10 of 18 cases of the pleomorphic type a mixture of
classical and pleomorphic areas was present. Based on these
observations we defined two new cytomorphological subtypes
of MCL: the classical + pleomorphic type (Fig 1E, n ¼ 5; 1Æ6%)
consisted of a mixture of two cell populations, one with a
classical and one with a pleomorphic cytology. In contrast, the
cytomorphology of the classical/pleomorphic type (Fig 1F,
n ¼ 5; 1Æ6%) displayed cells with a continuous transition
between classical and pleomorphic types.
Although this study included a large number of cases, the
large cell variants of MCL (pleomorphic and blastic) represented only a small minority of all cases. We therefore combined
the pleomorphic and blastic cases, including the intermediate
types (classical + pleomorphic type and classical/pleomorphic
type), in one group and the classical and small cell types in a
second group and compared the two groups in terms of
clinical outcome. No significant difference in overall survival
time was found between the two groups (P ¼ 0Æ1413, Fig 2A).
Nevertheless, some patients with classical or small cell type
survived for up to 11 years of follow-up, whereas no patients
suffering from pleomorphic or blastic variants were alive after
more than 5 years of follow-up (Fig 2A).
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 29–38
Mantle Cell Lymphoma
18Æ1% of cases (n ¼ 38) and a nodular growth pattern with
prominent residual germinal centres (mantle zone pattern) in
1Æ4% (n ¼ 3). Diffuse growth of the lymphoma cells was
observed in 80Æ5% (n ¼ 169) of cases. 94 cases could not be
evaluated, e.g. because of the small size of the biopsy specimen.
Cases showing nodular growth (nodular or mantle zone
pattern) with a diffuse component of <50% (n ¼ 41) had a
more favourable prognosis (median overall survival:
43 months) than cases that contained a diffusely growing
lymphoma component of more than 50% (median:
29 months, P ¼ 0Æ0074, Fig 2B). No statistically significant
correlation was found between the cytological subtype and the
growth pattern (data not shown).
Proliferation
Fig 2. Correlation of histopathological and cytomorphological features of MCL with overall survival. (A) Overall survival time of patients with classical or small cell MCL compared with patients with
pleomorphic and blastoid MCL. (B) Overall survival time of patients
with nodular MCL architecture compared with patients with diffuse
architecture.
Growth pattern
The architecture of the lymphoma infiltrates in the examined
lymph nodes displayed a predominantly nodular pattern in
The mitotic index was assessed in 273 cases and the Ki-67
index was assessed in 187 cases. The mitotic index ranged from
0 to 112 mitotic figures/mm2 with a mean of 15Æ4 and a
median of 11Æ0. The Ki-67 index ranged from 1% to 70% with
a mean of 16Æ8% and a median of 15Æ0%. As expected, we
found a strong correlation between the mitotic index and the
Ki-67 index (P < 0Æ0001, Fig 3A). Cases with classical or small
cell cytology showed a lower mitotic index (mean: 13Æ6%) and
Ki-67 index (mean: 15Æ3%) than cases with pleomorphic and
blastic cytology (28Æ9% and 28Æ8% respectively; P < 0Æ0001 for
Ki-67, Fig 3B). No correlation was found between the growth
pattern of the lymphoma and either the mitotic index
(P ¼ 0Æ8194) or the Ki-67 index (P ¼ 0Æ1107, Fig 3C).
To evaluate the mitotic index and the Ki-67 index as
prognostic markers in MCL, we performed a quartile-based
analysis of the study population. Significant differences
between the four quartile subgroups were found for both the
mitotic index (P < 0Æ0001) and the Ki-67 index (P ¼ 0Æ0292).
We defined cut-off levels of 25–50 mitotic figures/mm2 for the
mitotic index and 10%/40% for the Ki-67 index. The lower
cut-off level was chosen on the basis of values reported in the
literature (Velders et al, 1996). The higher cut-off level was
Fig 3. Correlation of the histopathological and cytomorphological features of MCL with markers of cell proliferation. (A) Correlation between Ki-67
index and mitotic index. (B) Correlation of the Ki-67 index with the cytomorphological subtype of MCL. (C) Correlation of the Ki-67 index with the
architecture.
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 29–38
33
M. Tiemann et al
Table IV. Ki-67 and mitotic indices compared – overall survival time
and P-values.
Ki-67 index
First quartile (0–6%)
Second quartile (7–14%)
Third quartile (15–19%)
Fourth quartile (20–100%)
Cut-off level (<10%)
Cut-off level (10–40%)
Cut-off level (>40%)
Hot spots
No hot spots
Mitotic index
First quartile (0–3/mm2)
Second quartile (4–10/mm2)
Third quartile (11–19Æ5/mm2)
Fourth quartile (>19Æ5/mm2)
Cut-off level <25/mm2)
Cut-off level (25–49/mm2)
Cut-off level (‡50/mm2)
Hot spots
No hot spots
n
Median overall
survival (months)
47
47
46
47
48
131
8
40
147
42
37
27
23
42
30
15
28
33
0Æ0292
69
68
68
68
225
34
14
89
184
50
33
37
24
38
21
17
42
32
<0Æ0001
P-value
<0Æ0001
0Æ7193
<0Æ0001
0Æ3834
chosen on the basis of the data published by Gerdes et al
(1984a), who demonstrated that high-grade lymphomas
showed Ki-67 indices of more than 40%. The survival curves
of the resulting three subgroups were compared by log-rank
test (Table IV). As shown in Fig 4(A), cases with a high mitotic
index of more than 50 mitotic figures/mm2 had a significantly
worse clinical outcome (median overall survival: 17 months,
n ¼ 14) than cases with a moderate mitotic index between 25
and 50 mitotic figures/mm2 (21 months, n ¼ 34) or cases with
a low mitotic index below 25/mm2 (38 months, n ¼ 225,
P ¼ 0Æ0019). Similarly a high Ki-67 index of more than 40%
(n ¼ 7) resulted in significantly shorter overall survival
(median: 15 months) compared with cases with a moderate
Ki-67 index between 10% and 40% (median: 30 months,
n ¼ 131) and cases with a low Ki-67 index of <10% (median:
42 months, n ¼ 48, P ¼ <0Æ0001, Fig 4B). The presence of
proliferation hot spots, as measured by the mitotic or the Ki-67
index, had no impact on overall survival (Table IV).
A univariate Cox regression analysis of all prognostic factors
with respect to overall survival was performed in 304 MCL
patients. Ki-67 index (P < 0Æ0001), mitotic index (P < 0Æ0001),
growth pattern (P ¼ 0Æ0074), age (P ¼ 0Æ0020), bone marrow
infiltration (P ¼ 0Æ0110), performance status (P ¼ 0Æ0440),
LDH (P ¼ 0Æ0305), stage (0Æ0048), B symptoms (P ¼ 0Æ0343)
and extranodal involvement (P ¼ 0Æ0041) resulted in significant differences in overall survival time. Interestingly the
International Prognostic Index (IPI) was not significantly
related to overall survival time (P ¼ 0Æ6497).
Because too many data were missing, a multivariate Cox
regression analysis was not performed with all clinical and
34
Fig 4. Correlation of mitotic index and Ki-67 index with overall survival in MCL. (A) Overall survival time of patients with various mitotic
indices. (B) Overall survival time of patients with various Ki-67
indices.
histological parameters. In the first analysis of 122 patients,
Ki-67 index, mitotic index, growth pattern and cytology were
available. In this analysis only the mitotic index was significant
(P ¼ 0Æ0025). In the second analysis of 53 patients the two
proliferation markers were analysed in relation to the IPI. The
IPI also had no clinical impact in this smaller group and only
the mitotic index was significant (P ¼ 0Æ0066). In the third
multivariate analysis of 149 patients, Ki-67 index, mitotic
index, age, performance status, LDH, stage and sex were
available and were used to compute a Cox regression with
likelihood ratio and a stepwise conditional approach excluded
parameters with P > 0Æ1. Only the parameters Ki-67 index,
mitotic index and age were of significant influence in this
model (Table V).
Discussion
Mantle cell lymphoma was originally recognised by Tolksdorf
et al (1980) on the basis of its cytomorphological features,
which in some respect resemble those of centrocytes. Additional cytological variants, such as blastic (Ott et al, 1997, 1998;
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 29–38
Mantle Cell Lymphoma
Table V. Univariate Cox regression analysis of all prognostic factors with respect to overall survival in 304 MCL patients. For 149 patients the Ki-67
index, mitotic index, age, performance status, LDH, stage and sex were available and were used to compute a Cox regression with likelihood ratio
excluding stepwise parameters with a P-value >0Æ1. Only the parameters Ki-67 index, mitotic index and age were of significant influence in this model
(n.i. ¼ not included in the computation of the model because too many data were missing).
P value
Characteristics
Reference level
Univariate
Ki-67 index
Mitotic index
Growth pattern
Cytology
International Prognostic Index
Age
Bone marrow infiltration
Performance status (WHO)
LDH
Stage
B symptoms
Sex
Extranodal involvement
<10% vs. 10–40% vs.>40%
<25 vs. 25–50 vs. >50
Mantle zone + nodular versus diffuse
Classical + small cell versus blastic + pleo + variants
0–1 vs. ‡2
<60 years vs. >60 years
Yes versus no
0 + 1 vs. ‡2
Normal versus elevated
1 + 2 vs. 3 + 4
Yes versus no
Male versus female
Yes versus no
n
n
n
n
n
n
n
n
n
n
n
n
n
¼
¼
¼
¼
¼
¼
¼
¼
¼
¼
¼
¼
¼
187, P < 0Æ0001
271, P < 0Æ0001
208, P ¼ 0Æ0074
284, P ¼ 0Æ1413
90, P ¼ 0Æ3629
304, P ¼ 0Æ0020
288, P ¼ 0Æ011
276, P ¼ 0Æ044
196, P ¼ 0Æ0305
290, P ¼ 0Æ0048
262, P ¼ 0Æ0343
304, P ¼ 0Æ2749
177, P ¼ 0Æ0041
Multivariate (n ¼ 149)
P¼
P¼
n.i.
n.i.
n.i.
P¼
n.i.
P¼
P¼
P¼
n.i.
P¼
n.i.
0Æ0812
0Æ0009
0Æ0191
0Æ8900
0Æ1602
0Æ2347
0Æ8346
WHO, World Health Organization; LDH, lactate dehydrogenase
Campo et al, 1999; Laszlo & Matolcsy, 1999), pleomorphic
(Zucca et al, 1994; Campo et al, 1999) and small cell type
(Weisenburger & Armitage, 1996; Decaudin et al, 1997;
Campo et al, 1999) have been described. Several authors have
not distinguished between the pleomorphic and blastic subtypes
and used the term blastoid to summarise both types (Jaffe et al,
2001). The current study included an exceptionally large
number of cases that were all enrolled in controlled studies,
and was carried out by a panel of highly experienced
haematopathologists. It confirmed the existence of the cytological subtypes classical, small cell, pleomorphic and blastic.
Furthermore, we described two new cytomorphological subtypes of MCL. The classical + pleomorphic type shows a
mixture of cells of classical and pleomorphic cytology, whereas
the classical/pleomorphic type consists of cells of intermediate
differentiation between classical and pleomorphic cytology. The
clinical relevance of the cytomorphological variants is still
being debated. The blastic subtype has been described as having
a more aggressive clinical course (Lardelli et al, 1990; Zucca
et al, 1994; Fisher et al, 1995; Weisenburger & Armitage, 1996;
Decaudin et al, 1997; Ott et al, 1997; Campo et al, 1999).
However, in the present study there was no significant
difference in overall survival between classic MCL and the
small cell type versus the pleomorphic and blastic variants,
respectively, although the latter subtype displayed a significantly higher mitotic and Ki-67 index. This discrepancy might
be explained by the relatively low number of pleomorphic and
blastic subtypes in our series, which altogether represented
<10% of all cases. Similar to previous reports (Bosch et al,
1998; Raty et al, 2002), the vast majority of MCL in our series
displayed a classical cytology. As our series represents a large
number of multicentre studies with various inclusion criteria,
the abundance of the cytological subtypes in our series might
reflect a more realistic prevalence of these subtypes. Consequently, variant subtypes of MCL with small cell, pleomorphic
and blastic cytomorphology should be considered to be rare
and their histological classification can pose a diagnostic
problem. The reproducibility of cytological classifications is a
problem in routine diagnostic work. Univariate and multivariate analysis did not show cytological subtypes to be a
prognostic marker, but knowledge of these cytological variants
is important for the differential diagnosis of this disease.
Mantle cell lymphoma shows three characteristic growth
patterns: mantle zone, nodular and diffuse (Weisenburger
et al, 1981; Pittaluga et al, 1995; Campo et al, 1999). It has
been suggested that the growth pattern represents a progression of the disease, with the lymphoma initially growing in the
mantle zone and then progressing to nodular and finally
diffuse growth (Swerdlow et al, 1983; Pittaluga et al, 1995;
Majlis et al, 1997). The clinical relevance of the different
growth patterns has not yet been determined. In some studies
lymphomas with a mantle zone pattern showed a significantly
better prognosis (Lardelli et al, 1990; Majlis et al, 1997),
whereas other studies did not find any differences in overall
survival time between the different growth patterns (Argatoff
et al, 1997). As in our series a mantle zone pattern was rare
(n ¼ 3) and the number of cases was too small for a separate
statistical analysis, we grouped those cases with the pure
nodular cases and compared them to the cases that showed a
diffuse component. Patients with a predominantly nodular
growth pattern had a more favourable prognosis than patients
with a growth pattern showing a diffuse component
(P ¼ 0Æ0074). The growth pattern thus represents a readily
assessable prognostic marker.
In previous studies, Ki-67 labelling in classical MCL was low,
whereas (blastic or pleomorphic) variants have been suggested
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 131, 29–38
35
M. Tiemann et al
to show high indices (Lardelli et al, 1990; Ott et al, 1994, 1997;
Jares et al, 1996; Campo et al, 1999). The present, extensive
study of various prospective trials confirmed these data. The
majority of cases with a classical cytology had a low Ki-67
index of <10%. Nevertheless, some classical subtypes displayed
a high proliferation rate of more than 40%. In contrast to
previous reports, our study applied different antigen retrieval
methods, but a standardised counting method (Lardelli et al,
1990; Velders et al, 1996).
Several reports have suggested that an increased mitotic
index is an important prognostic parameter in MCL (Lardelli
et al, 1990; Argatoff et al, 1997; Bosch et al, 1998). Recently,
gene expression profiling indicated that upregulation of genes
associated with proliferation and cell cycle progression can be
detected in a subgroup of MCL with poor prognosis.
Accordingly, in our study the subgroup of MCL with high
Ki-67 or mitotic index had a significantly worse clinical
outcome (median overall survival 15 months vs. 42 months in
the cases with low proliferation). Interestingly, a subset of
MCL with high cell proliferation but classical cytology was
identified that also displayed a poor clinical outcome (Pinyol
et al, 1998; Campo et al, 1999).
In the current study, we employed two well-established
methods to evaluate the proliferation of malignant cells in
paraffin-embedded tissue. The mitotic index and the Ki-67
index can be assessed during routine diagnostic procedures.
Both proliferation indices showed a good correlation with
clinical outcome. However, the mitotic index was found to be
superior in the multivariate analysis (Table V). In our study no
inter- or intra-observer variability was tested. However, this
issue will be of importance if proliferation indices are to be
assessed for use in risk-adapted therapy.
In summary, our study included an exceptionally large
number of MCL cases that were analysed by a large number of
highly experienced haematopathologists to generate reliable
markers for histological subtyping of MCL. We conclude that
small cell, pleomorphic and blastic subtypes of MCL are
relatively rare, but knowledge of these subtypes is necessary for
the histological diagnosis. Assessment of proliferation can be
used to predict the clinical course of the disease and seems to
be superior to the growth pattern. Future studies will focus on
the establishment of a combined biological and clinical risk
score for MCL that is superior to the IPI, defined for diffuse
large cell lymphomas and the follicular lymphoma IPI (SolalCeligny et al, 2004). Additionally, we need to re-evaluate
whether the Ki-67 and mitotic indices remain important
prognostic markers in patients treated with combined
immuno-chemotherapy or myeloablative consolidation followed by autologous stem cell transplantation.
Acknowledgements
This work was supported in part by Biomed II grant BMH4CT-96-1539 and European Framework 5 grant QLG1-CT2000-00687.
36
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