Bcl-2 protein expression is the strongest independent prognostic factor of survival in primary cutaneous large B-cell lymphomas

doi:10.1182/blood-2003-08-2726Prepublished online January 15, 2004;

Laroche, Martine Bagot and Janine WechslerBeatrice Vergier, Pierre Dechelotte, Elisabeth Cassagnau, Philippe Courville, Philippe Saiag, LilianeLaurent Machet, Marie-Francoise Avril, Sophie Dalac, Philippe Bernard, Agnes Carlotti, Eric Esteve, Florent Grange, Tony Petrella, Marie Beylot-Barry, Pascal Joly, Michel D'Incan, Michele Delaunay, of survival in Primary Cutaneous Large B-Cell LymphomasBcl-2 protein expression is the strongest independent prognostic factor

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1

Bcl-2 protein expression is the strongest independent prognostic factor of

survival in Primary Cutaneous Large B-Cell Lymphomas

(Short title: bcl-2 expression in Primary Cutaneous Large B-cell Lymphomas)

F Grange, T Petrella, M Beylot-Barry, P Joly, M D’Incan, M Delaunay, L Machet, MF Avril, S Dalac, P

Bernard, A Carlotti, E Esteve, B Vergier, P Dechelotte, E Cassagnau, P Courville, P Saiag, L Laroche, M

Bagot, J Wechsler (French Study Group on Cutaneous Lymphomas) .

From the Department of Dermatology, Hôpital Pasteur, Colmar, France; Centre de Pathologie and Department of Pathology, Hôpital du Bocage, Dijon; Department of Dermatology, Hôpital Henri-Mondor, Créteil; Department of Dermatology, Hôpital du Haut Lévêque, Pessac; Department of Dermatology, Hôpital Charles Nicolle, Rouen; Department of Dermatology, Hôtel Dieu, Clermont-Ferrand; Department of Dermatology and Cancerology, Hôpital Pellegrin, Bordeaux; Department of Dermatology, Hôpital Trousseau, Tours; Department of Dermatology, Institut Gustave Roussy, Villejuif; Department of Dermatology, Hôpital du Bocage, Dijon; Department of Dermatology, Hôpital Robert Debré, Reims; Department of Pathology, Hôpital Tarnier, Paris; Department of Dermatology, Hôpital Porte Madeleine, Orléans; Department of Pathology, Hôpital du Haut Lévêque, Pessac; Department of Pathology, Hôtel Dieu, Clermont-Ferrand; Department of Pathology, Hôtel-Dieu, Nantes; Department of Pathology, Hôpital Charles Nicolle, Rouen; Department of Dermatology, Hôpital Ambroise Paré, Boulogne; Department of Dermatology, Hôpital Avicenne, Bobigny; Department of Pathology, Hôpital Henri-Mondor, Créteil, France.

Corresponding author:

Florent Grange, Department of Dermatology, Hôpital Pasteur, 39 avenue de la Liberté 68024 Colmar

Cedex, France.

Phone number: 33 (0)3 89 80 41 58. Fax number: 33 (0)3 89 12 47 69e-mail: [email protected]

3800 wordsScientific heading: Clinical Observations, Interventions, and Therapeutic Trials.

Acknowledgements: We thank the following clinicians ,biologists, pathologists and epidemiologists who

actively participated in the study: MH Delfau-Larue, JP Merlio, B Audhuy, A Carlotti, B Dreno, J Bosq,

Anne Colson , A Durlach, JC Guillaume, G Hedelin, F Maître, C Michel, A de Muret, B Schubert, P

Souteyrand, A de Muret, L Vaillant, T Clerici, MC Tortel. We also thank Sylvie Espin and Marie-

Antoinette Lignier (Centre de Pathologie de Dijon) for their technical assistance.

Blood First Edition Paper, prepublished online January 15, 2004; DOI 10.1182/blood-2003-08-2726

Copyright (c) 2004 American Society of Hematology




2

Abstract

Bcl-2 protein expression has been associated with poor prognosis in patients with non-cutaneous diffuse large B-cell lymphomas. In primary cutaneous large B-cell lymphomas, the location on the leg, the round-cell morphology defined as the predominance of centroblasts and immunoblasts over large centrocytes, and multiple skin lesions were identified as adverse prognostic factors. The prognostic value of bcl-2 protein expression has not been studied in large series of patients.We evaluated 80 primary cutaneous large B-cell lymphomas collected by the French Study Group on Cutaneous Lymphoma. The prognostic value of age, sex, number of lesions, cutaneous extent, location, LDH level, B symptoms, morphology and bcl-2 protein expression was studied. The overall 5-year specific survival rate was 65%. In univariate analysis, advanced age, multiple skin lesions (n=48), location on the leg (n=25), round-cell morphology (n=32) and bcl-2 expression (n=39) were significantly related to death from lymphoma. In multivariate analysis, bcl-2 expression (p=.0003), multiple skin lesions (p=.001) and age remained independent prognostic factors. Five-year specific survival rates in bcl-2+ and bcl-2- patients were 41% and 89% respectively (p<.0001)A new prognostic classification of primary cutaneous B-cell lymphoma should be based primarily on bcl-2 protein expression rather than the location of skin lesions.




3

Introduction

Primary cutaneous B-cell lymphomas (PCBCL) comprise approximately 20% of cutaneous lymphomas

(1,2). Whereas most PCBCL have an indolent clinical course, a variable proportion of patients with these

lymphomas eventually die of their disease (2-16).

PCBCL presenting with skin tumors of the leg were first recognized as having a poorer prognosis (4,8).

These tumors of the leg were consistently composed of a majority of large B-cells (centroblasts, large

centrocytes and immunoblasts in variable proportions). The term primary cutaneous large B-cell

lymphoma of the leg (PCLBCL-leg) was used (2,8). In the European Organization for Research and

Treatment of Cancer (EORTC) (2) classification for primary cutaneous lymphomas, these PCLBCL-leg

were classified as a separate clinical entity of intermediate prognosis. Using the Revised European-

American (REAL) classification, they were classified as diffuse large B-cell lymphomas (17).

Patients with skin tumors at other sites (mostly the head or the trunk) that were composed of various

proportions of small centrocytes, large centrocytes, centroblasts and rarely immunoblasts appeared to

have a more favorable prognosis (2,4,5,11,15). In the EORTC classification they were classified as

primary cutaneous follicle center-cell lymphomas (PCFCCL) and considered as indolent lymphomas

irrespective of their cytologic features. These PCFCCL included both small cell and large-cell

lymphomas. According to the REAL classification, they were classified as either follicle center-cell

lymphomas or diffuse large B-cell lymphomas.

Although the EORTC classification highly contributed to a more uniform diagnosis and management of

patients with primary cutaneous lymphomas, this subdivision of PCBCL has been much disputed (5,18-

24). The controversy particularly concerned PCBCL with a predominance of large cells (Primary

Cutaneous Large B-cell Lymphomas, PCLBCL) since they were divided into two different categories

(PCLBCL-leg and PCFCCL), primarily on the basis of the site of presentation (leg versus other sites)

(2,25). Indeed, it was found that PCLBCL-leg differed from PCFCCL not only by anatomical location

and overall prognosis, but also by other characteristics including a higher age of onset and a more




4

frequent bcl-2 protein expression (8,26). Therefore, it could be suspected that factors other than location

might dictate the prognosis of PCLBCL.

In a previous study (13), we attempted to identify independent prognostic factors in a large series of

PCLBCL, including both PCLBCL-leg and PCFCCL as defined in the EORTC classification. Bcl-2

protein expression was not included in this prognostic analysis. We found that a round-cell morphology,

the location on the leg and multiple skin lesions were independent predictive parameters for survival. The

round-cell morphology was defined as the predominance of centroblasts and/or immunoblasts and

opposed to the cleaved-cell morphology in which large centrocytes predominated. This morphologic

distinction appeared to be the strongest prognostic factor. However, its reproducibility among

pathologists was insufficient.

With the exception of this study, prognostic analyses of patients with PCLBCL were performed only in

small groups of patients (12,16,27,28).

Bcl-2 protein is an antiapoptotic protein whose overexpression has been found to be associated with a

poor prognosis in non-cutaneous diffuse large B-cell lymphomas (29-32). The prognostic value of bcl-2

protein expression has not been studied previously in a large series of patients with PCLBCL.

In the present multicenter study, we analysed the prognostic value of bcl-2 protein expression together

with other potential prognostic factors in patients with primary cutaneous large B-cell lymphomas.

Patients and methods

Inclusion criteria

Clinical and histologic data on all patients included in the registry of the French Study Group on

Cutaneous Lymphomas (FSGCL) for a diagnosis of B-cell lymphoma were reviewed. Cases were

selected for analysis if they met the following criteria: (1) diagnosis of cutaneous B-cell lymphoma

between 1 January, 1984 and 30 September, 2001; (2) absence of extracutaneous disease detected by a




5

comprehensive staging procedure at diagnosis; (3) histologic and immunophenotypic features showing a

majority (i.e. > 50%) of large cells among neoplastic B-cells.

Staging procedure at diagnosis included in all cases physical examination, routine laboratory tests, chest

radiograph or thoracic computed tomographic (CT) scan, abdominal ultrasound tomography or

abdominal CT scan, bone marrow aspirate (7.5% of cases), bone marrow biopsy (25%) or both (67.5%).

Six cases were excluded from the study due to incomplete staging procedure at diagnosis, most of whom

being old patients who had no bone marrow examination. Fourteen cases were excluded because of a

positive initial staging. One case was excluded because he had a previous history of nodal B-cell

lymphoma. Seven cases were excluded since no material was available for the histologic review.

Eighty patients with a PCLBCL were included in the study. Thirty-three of them had been included in a

previous study (13).

According to the criteria of the Revised European-American Classification of Lymphoid Neoplasms

(Harris) and the World Health Organization (33) classification all 80 cases were classified as diffuse

large B-cell lymphomas.

Histologic review and bcl-2 study

Skin biopsies were reviewed by a panel of three expert pathologists (TP, PC, JW) from different centers,

without previous knowledge of the clinical data.

For each case, hematoxylin eosin slides and CD3 and CD20 stainings were studied. Histologic

subclassification was essentially based on the relative proportions of immunoblasts, centroblasts (large

noncleaved cells) and large centrocytes (large cleaved cells), including multilobated cells, as previously

described (13). Cases including more than 50% large B-cells with round nuclei (i.e., centroblasts and

immunoblasts) were classified as round-cell lymphomas. Cases which showed a predominance of large

cleaved cells were classified as cleaved-cell lymphomas. When a disagreement was observed within the

pathology panel, final classification was obtained by consensus using a multiheaded microscope.

Bcl-2 protein expression was studied in all cases using formalin-fixed and/or Bouin’s liquid-fixed,




6

paraffin-embedded sections, deparaffinized and stained with an appropriate monoclonal antibody (clone

124, DAKO, Copenhagen, Denmark). In order to avoid technical-dependant variability in bcl2 staining,

bcl2 immunostaining was performed in the same laboratory (TP) and at the same time. Positive and

negative controls were done using reactive lymph node and bcl-2-positive nodal follicular lymphoma.

Keeping in mind that the small reactive lymphocytes generally express bcl-2 protein, the small cells were

not counted, with the help of CD20 and CD3 markers when necessary. An exact quantification of the

proportion of neoplastic large cells which showed an unequivocal bcl-2 positivity was performed by

consensus between the three pathologists. Bcl-2 staining was finally considered positive if this

proportion exceeded 50%.

Since bcl-2 protein expression was the variable of interest in prognostic analysis, the reproducibility of

this measure was studied. Four pathologists who had not taken part in the expert panel were asked to

review separately bcl-2 slides in all cases. Results were compared with those obtained by the expert

panel.

Data collection

Variables analyzed for prognostic value included demographic characteristics, bcl-2 protein expression

and all factors which were significantly associated with survival in previous multivariate studies on

PCBCL (13,14). These variables were: age at diagnosis; sex; anatomic site (head and neck, arm, anterior

aspect of the trunk, posterior aspect of the trunk including the buttock, or leg); number of skin lesions;

cutaneous extent (referred to as “localized” when either one or multiple skin lesions were restricted to

one anatomic site, and “disseminated” when several anatomic sites or several limbs were involved);

duration of skin lesions before diagnosis; serum lactate dehydrogenase (LDH) level; histologic group

(round-cell versus cleaved-cell morphology) and bcl-2 protein expression. In addition, patients were

classified according to the number of risk factors of the International Prognostic Index (IPI) using age

(≤60 vs. > 60), LDH level (normal vs. elevated) and cutaneous extent (localized vs. disseminated). The

number of extranodal sites was not usable since patients with extracutaneous disease were not included




7

in the study. Performance status was not used because it had not been registered in a large number of

patients at the beginning of the study period.

Follow-up data

The endpoint was 30 March, 2002. Follow-up information recorded until the endpoint included therapy,

achievement of a complete response, relapse, nodal or visceral progression of the disease, final status,

and date and cause of death. The follow-up time ranged from 3 to 167 months (median, 35 months;

mean, 43 months). Status at the endpoint was known for 77 patients (96%). Only three patients (4%)

were lost to follow-up before 30 March, 2002. For these patients, the follow-up time was 4, 5 and 7

years, respectively.

Statistical analysis

Comparison between subgroups of patients were performed using the usual Chi 2 test or Fisher’s exact

test for categorical variables and Student’s t test or Mann-Whitney test for continuous variables. Specific

survival duration was calculated from diagnosis to date of disease-related death or censoring. Patients

whose deaths were unrelated to lymphoma were considered censored. Survival rates were estimated in

the entire study group and in subgroups of patients according to bcl-2 protein expression. Survival curves

were computed using the method of Kaplan and Meier (34).

Prognostic factors were evaluated by specific survival univariate and multivariate analyses using a Cox

proportional-hazards model (35). Factors significant at the 0.2 level in univariate analysis were included

in stepwise regression multivariate analyses.

Results

Clinical and histological characteristics of the patients at diagnosis and follow-up data

Eighty patients were included in the study. Their clinical characteristics and follow-up data are

summarized in Table 1. Forty three (54%) were male and 37 (46%) were female. Age ranged from 28 to




8

98 years (mean: 62; median: 64). Clinically, patients presented with cutaneous nodules or tumors (90%)

or deeply infiltrated plaques (10%). Thirty- two patients (40%) had only one lesion, 36 (45%) had two

lesions and 12 (15%) more than two lesions. Thirty-one patients (39%) had lesions on the trunk, 28

(35%) on the head, 25 (31%) on the leg and 6 (7.5%) on the arm.

A high LDH level was present in 8 cases (10%). Four patients (5%) had B symptoms. The number of IPI

risk factors was 0 in 28 cases (35%), 1 in 43 cases (54%), 2 in 7 cases (9%) and 3 in 2 cases (2%).

After histological review, 48 cases (60%) were classified as cleaved-cell PCLBCL and 32 cases (60%)

were classified as round-cell PCLBCL. The results of bcl-2 protein expression assessment are shown in

Table 2. Thirty-nine cases (49%) exhibited a high expression of bcl-2 protein (≥ 50%) and were

considered positive.

The initial treatment consisted of local radiotherapy in 41 cases (51%), a systemic polychemotherapy in

20 cases (25%) and the association of both therapies in 10 cases (12.5%). The last 9 patients (11.5%)

underwent excision alone, interferon or simple observation.

Among 66 patients (82.5%) who achieved a complete response, 38 (58%) had no relapse, whereas 28

(42%) experienced one or several relapses. Twenty-one of the 80 patients (26%) developed an

extracutaneous disease. Time until extracutaneous dissemination ranged from 2 to 63 months (mean: 20

months, median: 16 months). In 4 of 21 patients, extracutaneous progression occurred within 6 months

after the histological diagnosis of cutaneous lymphoma. Progression was restricted to the lymph nodes in

all four cases. These patients had experienced multiple cutaneous tumors for 2 to 12 months (mean: 6

months) prior to the histological diagnosis of cutaneous lymphoma and 5 to 15 months (mean: 10

months) prior to the diagnosis of nodal progression.

Of the 21 patients who developed extracutaneous disease, the progression was restricted to the lymph

nodes in 7 cases. The remaining14 cases had a visceral disease either associated with lymph node

involvement (7 cases) or not (7 cases). The central nervous system (CNS) was the most frequent site of

visceral dissemination (4 cases). CNS involvement was observed in 2 male and 2 female patients over 65

years of age, with a bcl-2 positive (3 cases) or negative (1 case) PCLBCL that was primarily located




9

either on the lower leg (2 cases) or at other sites (2 cases). Other locations of visceral dissemination

included the liver (2 cases), the testis (2 cases), the bones (1 case), the lung (1 case), the pancreas (1

case), the kidney (1 case), the breast (1 case), the pelvis (1 case) and the brachial plexus (1 case).

Twenty-five of the 80 patients (31%) died of lymphoma, whereas 5 patients (6%) died of unrelated

disease. Death from lymphoma occurred in 18 of 25 patients within 3 years after diagnosis. The 3-year

and 5-year disease-specific survival rates were 75% and 65%, respectively.

Uni- and multivariate analyses

Univariate analysis showed that the following variables were related to death from lymphoma: advanced

age (p < 0.0001); the number of IPI risk factors (p < 0.0001); the location on the leg (p < 0.0001); the

presence of more than one skin lesion at diagnosis (p = 0.007); round-cell morphology (p< 0.0001) and

positive bcl-2 protein expression (p < 0.0001). The most predictive cutoff point for age was 70. Sex,

duration of lesions before diagnosis, LDH level, extent of skin lesions and initial therapy (either

including a polychemotherapy or not) had no significant effect on death from lymphoma. Multivariate

analysis of disease-specific survival using all candidate variables identified bcl-2 protein expression (p =

0.0003), multiple skin lesions at diagnosis (p=0.001) and age > 70 (p = 0.004) as independent factors

associated with a poor prognosis (Table 3). When the number of IPI risk factors was forced in the model

as a unique parameter (0 vs. ≥ 1), bcl-2 expression remained the strongest predictive factor of survival

(p=0.0001, RR = 8.4) independently of the number of IPI risk factors (p=0.02, RR = 6.7) and the number

of skin lesions (p = 0.0006, RR = 4.9). Similar results of univariate and multivariate analyses were

obtained when the four patients who underwent an extracutaneous dissemination within 6 months after

diagnosis were excluded from the study. Multivariate analysis also showed similar results when therapy

was forced in the model. Bcl-2 protein expression was the strongest prognostic factor of survival both in

the group of patients whose therapy included a polychemotherapy and in those who were given

radiotherapy or other treatments.




10

Characteristics of the patients and survival rates according to bcl-2 protein expression

Since bcl-2 protein expression was the strongest prognostic factor, the main features at diagnosis and in

follow-up data according to this variable were analysed (Table 1). Patients with a bcl-2 positive PCLBCL

differed from patients with a bcl-2 negative lymphoma by a higher age of onset, a female predominance,

a shorter duration of skin lesions before diagnosis consistent with more rapidly growing tumors, a higher

number of IPI risk factors, and more frequent location on the leg and round-cell morphology. Bcl-2

expression and morphology were in high concordance, with a kappa coefficient of 0.72. This coefficient

was only 0.50 when concordance between bcl-2 expression and location on the leg was studied. Initial

therapy was not significantly different between patients with bcl-2 negative and bcl-2 positive PCLBCL.

However, there was a tendency for multi-agent chemotherapies to be used more frequently in bcl-2

positive cases, possibly due to a more aggressive presentation and/or a more frequent location on the leg.

Despite this tendency, patients with a bcl-2 positive lymphoma had a poorer prognosis, with a 5-year

specific survival rate of 41%, versus 89% in patients with a bcl-2 negative PCLBCL. Kaplan Meier

disease-specific survival curves in both groups are shown in Fig.1.

The reproducibility study of bcl-2 protein expression assessment showed the following results: 67 of the

80 cases (84%) were classified consistently as either bcl-2 positive or negative by all of the four

pathologists, in accordance with the consensus of the panel review. In 9 cases (11%), all but one of the

four pathologists agreed with the classification. In only four cases (5%) two or more pathologists

disagreed with the others and with the consensus of the panel review.

Survival rates according to bcl-2 expression and number of skin lesions

The presence of multiple skin lesions and bcl-2 protein overexpression were statistically unrelated

(Table1). Since these two variables were the strongest independent prognostic factors (Table 3), we

studied survival in the three at-risk groups defined by the presence of none (group 1 = 18 cases), one




11

(group 2 = 37 cases) or both (group 3 = 25 cases) of these factors. The 5-year specific survival rates in

groups 1, 2 and 3 were 93%, 82% and 18%, respectively.

Discussion

In the present report, we have studied bcl-2 protein expression and prognostic factors in a series of 80

patients with PCLBCL. We found that bcl-2 was expressed by ≥ 50% of tumor cells in about 50% of

these lymphomas. Univariate and multivariate analyses showed that bcl-2 protein expression was the

strongest predictive factor of death from lymphoma. This result has not been demonstrated previously in

PCLBCL. It extends previous observations of the negative prognostic value of bcl-2 overexpression in

systemic and nodal lymphomas.

Although primary cutaneous lymphomas were theoretically defined in the EORTC classification by the

absence of extracutaneous progression within 6 months after diagnosis, we adopted in the present study

the pragmatic, widely-used definition based on a complete negative staging at diagnosis (5, 7, 10, 11, 13,

28, 36). This definition was recently recommended for two major reasons: 1 - aggressive lymphomas that

arise in the skin may show dissemination before a period of 6 months; 2 - patients need to be treated at

presentation, thus a clear-cut diagnosis must be established immediately, not 6 months afterwards (5). In

our series, 4 of 80 patients had extracutaneous progression (restricted to lymph nodes) within 6 months

after diagnosis. Since these patients had experienced multiple cutaneous tumors for an average of 10

months prior to nodal progression, it seems likely that they had a primary cutaneous disease rather than

secondary cutaneous involvement by a nodal lymphoma. When these patients were excluded from the

prognostic analysis, similar results were observed. Therefore, it can be assumed that adverse prognostic

factors identified in the present study, including bcl-2 protein expression, do not reflect undiagnosed

associated systemic diseases and would apply to primary cutaneous lymphomas, either defined

restrictively or not.




12

Among 14 patients who underwent a visceral progression of their disease, four (29%) had a CNS

localization. It is noteworthy that no patient in our series had an intravascular large cell lymphoma, an

entity in which CNS involvement is a common feature. CNS involvement was recently reported in 4 of

160 patients with a primary cutaneous B-cell lymphoma (36) and briefly mentioned in 2 of 4 cases in

another study (28). No clinical or pathological feature predictive of a secondary CNS involvement can be

identified from these few cases. Clinicians may therefore be aware of this rare site of progression when

following patients with PCLBCL.

We identified bcl-2 protein expression as the main negative prognostic factor in PCLBCL. The five-year

specific survival rate was 41% in patients with a bcl-2 positive PCLBCL versus 89% in patients with a

bcl-2 negative PCLBCL. After adjustment for other prognostic factors, the relative risk for death

associated with bcl-2 overexpression was 7.6 (95% confidence interval, 2 to 28) (Table 2).

Previous studies of the prognostic value of bcl-2 protein expression have been performed only in patients

with non-cutaneous diffuse large B-cell lymphomas. Whereas some of these studies, including less than

65 patients, led to negative results (37-40), more powerful analyses of more than 150 patients found that

bcl-2 protein overexpression was independently related to relapse (29), disease-free survival (30,31) or

overall survival (32). In view of recent in vitro studies in human leukemia cell lines (41,42), it has been

suspected that a high bcl-2 expression may confer resistance to several antineoplastic agents (30,39,40).

In our study, the high prognostic value of bcl-2 expression was independent of therapy (i.e.

polychemotherapy versus other treatments), suggesting that mechanisms other than drug resistance may

explain the poor prognosis related to bcl-2 overexpression in patients with PCLBCL.

The use of prognostic parameters defined by immunohistochemistry may be limited in clinical practice

by the lack of a clear definition and poor reproducibility. Variable percentages of bcl-2 positive tumor

cells have been used to define bcl-2 overexpression (37,38,40). However, major studies in non-cutaneous




13

diffuse large B-cell lymphomas consistently classified cases as bcl-2 expressing lymphomas if the

protein was detected in more than 50% (31,32) or 60% (30) of tumor cells. In our patients with a

PCLBCL, the 50% cut-off had a high clinical pertinence and a strong independent prognostic value. In

80% of cases the percentage of cells expressing the bcl-2 protein was either less than 20% or greater than

80% (Table 1). Therefore, the assessment of bcl-2 positivity could seem easy in most cases. However, its

reproducibility had not previously been evaluated in cutaneous lymphomas. When it was tested in our

series, we found that 95% of cases were classified by all (84%) or all but one (11%) of four pathologists

in accordance with the expert consensus. These data indicate that interpretation of bcl-2 immunostaining

in PCLBCL is reproducible and therefore reliable.

In the present study, tumors were not investigated for the presence of the t(14;18) translocation.

Increased expression of bcl-2 protein has been detected in lymphomas with t(14; 18). After this

translocation, the BCL2 oncogene, located on chromosome 18q, is subject to regulatory elements of the

immunoglobulin heavy-chain gene, which leads to constitutive activation of the gene (43). However, it

has been demonstrated that the increased bcl-2 expression in a series of PCLBCL was not associated with

the t(14; 18) (26). Pathological mechanisms other than the t(14; 18) may increase the expression of bcl-2,

as has been hypothesized in non-cutaneous lymphomas (29,30).

Beside bcl-2 protein expression, other prognostic factors in PCLBCL must be taken into account. In a

previous study, we found that the round cell morphology, the location on the leg and multiple skin

lesions were independent adverse prognostic factors. When bcl-2 protein expression was included in the

present study, neither morphology nor location of skin tumors gave any additional prognostic

information. There was a high concordance between bcl-2 protein expression and round-cell morphology

(κ = 0.72) (Fig. 2). However, bcl-2 protein expression had the strongest prognostic value. Furthermore,

we found previously that the reproducibility of the morphologic distinction between round and cleaved-




14

cell PCLBCL was insufficient (13). It seems therefore preferable, in clinical practice, to use the primary

distinction between bcl-2 negative and bcl-2 positive PCLBCL.

It has been previously suggested that the subdivision of PCLBCL according to location (leg versus other

sites) was quite simple and reproducible (2). However, it may be difficult, using this topographic

distinction, to classify cases with multiple skin tumors located both on the leg and at other sites, or those

with lesions at borderline sites like the buttock. In view of this and of the absence of independent

prognostic value of location after taking into account other factors including bcl-2, we think that the

primary subdivision of PCLBCL according to anatomic location, which has been previously proposed,

should be reconsidered.

In the present study, the number of skin lesions remained a strong independent prognostic factor. This

result is in accordance with our previous study on PCLBCL (13) and with more general studies which

underlined the prognostic value of variables related to tumor burden in cutaneous or non-cutaneous

lymphomas (11,44,45). Beside bcl-2 protein expression, we found the number of skin lesions to be of

major importance. Only patients with a bcl-2 positive PCLBCL and multiple skin tumors had a very poor

prognosis, with a 5-year specific survival rate of 18%. In contrast, 5-year survival rates of patients with

none or only one of these two adverse prognostic factors were 93% and 82% respectively.

The IPI including age, LDH level, performance status, clinical stage and number of extranodal sites was

defined as the most effective tool for predicting outcome in patients with non-cutaneous aggressive

lymphomas (46). Although the IPI is not directly transposable to primary cutaneous lymphomas, an

adaptation of this index using extent of skin lesions for clinical staging, as in the present study, and

excluding the (inappropriate) number of extranodal sites, could be validated and used in further studies

on cutaneous lymphomas. However, the IPI only reflects factors linked to the patient’s characteristics

and to the disease’s extension and does not encompass molecular abnormalities of tumor cells which may

play a critical role in determining outcome. In our patients, the prognostic value of bcl-2 protein




15

expression was found to be independent of age, LDH level, extent of skin lesions and a clinical index

combining these factors. Although this analysis did not include the performance status, it extends

previous observations of the independent prognostic value of bcl-2 expression and IPI risk factors in non-

cutaneous diffuse large B-cell lymphomas (31).

In conclusion, we have identified bcl-2 protein expression as the strongest prognostic factor in PCLBCL

and found bcl-2 immunostaining to be quite reproducible in these lymphomas, allowing its use in routine

examination. Therefore, further classifications of primary cutaneous B-cell lymphomas should be based

primarily on this biological parameter. Additional prognostic factors, i.e., age and number of skin lesions

should be taken into account for the choice of treatment in patients with PCLBCL. On the basis of these

prognostic parameters, further therapeutic trials should be performed. Combinations of anthracyclin

containing polychemotherapies and rituximab were found to be more effective than chemotherapies

alone in elderly patients with non-cutaneous diffuse large B-cell lymphomas (47), particularly in cases

with bcl-2 overexpression (48). Such combined therapies should be evaluated for patients with PCLBCL

and adverse prognostic factors, as identified in the present study.




16

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Table 1: Main findings at diagnosis and follow-up data in the entire series

and according to bcl-2 protein expression

Total(%)

Bcl-2 negative(%)

Bcl-2 positive(%)

p-value*

Number of cases 80 41 39

AgeMean 62 52 73

<0.0001

Median 64 48 76Range 28-98 28-92 44-98

SexMale 43 (54) 29 (71) 14 (36)

0.002

Female 37 (46) 12 (29) 25 (64)

Mean duration before diagnosis(months)

19 25 13 0.02

Number of lesions0.5

1 32 (40) 18 (44) 14 (36)

> 1 48 (60) 23 (56) 25 (64)

Site <0.0001leg 25 (31) 3 (7) 22 (56)

Other sites 55 (69) 38 (93) 17 (44)

Extent 0.5Localized 69 (86) 36 (88) 33 (85)

Disseminated 11 (14) 5 (12) 6 (15)

LDH 0.15normal 72 (90) 35 (95) 33 (85)

high 8 (10) 2 (5) 6 (15)

Number of IPI risk factors <0.00010 28 (35) 24 (58) 4 (10)

1 43 (54) 15 (37) 28 (72)

2 7 (9) 2 (5) 5 (13)

3 2 (2) 0 (0) 2 (5)

Morphology <0.0001Cleaved-cell 48 (60) 39 (95) 9 (23)

Round-cell 32 (40) 2 (5) 30 (77)

Initial therapy 0.1Radiotherapy (RT) 41 (51) 24 (58) 17 (44)

Chemotherapy ° 21 (26) 6 (15) 15 (38)

RT + Chemotherapy° 10 (13) 5 (12) 5 (13)

Other 8 (10) 6 (15) 2 (5)

Complete response 0.02Yes 66 (82.5) 38 (93) 28 (72)No 14 (17.5) 3 (7) 11 (28)

Relapse # <0.003No 38 (58) 26 (68) 12 (43)

Yes 28 (42) 12 (32) 16 (57)

Extracutaneous progression <0.01No 59 (74) 36 (88) 23 (59)

Yes 21 (26) 5 (12) 16 (41)




Status <0.0001Alive, disease free 48 (60) 36 (88) 12 (31)

Alive with disease 2 (3) 1 (2.5) 1 (2.5)

Died of lymphoma 25 (31) 3 (7) 22 (56)

Died, other cause 5 (6) 1 (2.5) 4 (10.5)

Specific survival rates <0.00013-year 75% 94% 55%5-year 65% 89% 41%

* indicates differences between bcl-2 negative and bcl-2 positive PCLBCL. For 5-year survival rates, p values were calculated from comparison of survival curves.° consisted in a multiagent chemotherapy in all cases but one. # relapses were considered only in patients who achieved a complete response (66 cases).




Table 2: Bcl-2 protein expression in 80 patients with a

primary cutaneous large B-cell lymphoma.

% of positive tumor cells Number of cases % of cases051020

111681

45%

253040

131

6%

50606570809095100

231146121

49%




Table 3: Results from multivariate analysis in 80 patients with a

primary cutaneous large B-cell lymphoma.

RR 95% CI p-value

Bcl-2 0.0003• < 50% 1• ≥ 50% 7.6 2 - 28

Number of lesions 0.001• single 1• multiple 4.5 1.6 - 12

Age 0.004• < 70 1• > 70 3.8 1.4 - 10

RR= relative risk; CI= confidence interval




Fig.1: Kaplan-Meier specific survival curves according to bcl-2 protein expression

B(p < 0.0001)

0

0,25

0,5

0,75

1

0 12 24 36 48 60months

surv

ival bcl-2 - (n=41)

bcl-2 + (n=39)




Fig 2. Bcl-2 staining (X 400). (A) Case with a round-cell morphology and high bcl-2 expression; (B)

Case with a cleaved-cell morphology and negative bcl-2 staining.

A

B







Bcl-2 protein expression is the strongest independent prognostic factor of survival in primary cutaneous large B-cell lymphomas

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