Leukemia (1999) 13, 1448–1455  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu CD44 expression predicts disease outcome in localized large B lymphoma P Drillenburg1, VJM Wielenga1, MHH Kramer2, JHJM van Krieken2, HC Kluin-Nelemans3,4, J Hermans4,5, S Heisterkamp6, EM Noordijk4,7, PM Kluin2,4 and ST Pals1

Departments of 1Pathology and 6Clinical Epidemiology and Biostatistics, Academic Medical Center, Amsterdam; Departments of 2Pathology, 3Hematology, 5Medical Statistics and 7Clinical Oncology, Leiden University Medical Center, Leiden; and 4Comprehensive Cancer Center West (CCCW), Leiden, The Netherlands

Diffuse large B cell non-Hodgkin’s lymphomas (DLCL) form a and on aggressive lymphomas.17,20–23 Clinical heterogeneous group of tumors with diverse morphology, clini- studies in non-Hodgkin’s lymphomas have demonstrated that cal features, treatment response and prognosis. The biological expression of CD44s epitopes is associated with disseminated variables underlying this heterogeneity are unknown. In the 12,14,24,25 present study, we explored the value of the homing disease and tumor-related death. A causal role of CD44, a putative determinant of lymphoma dissemi- CD44s in determining this unfavorable disease outcome was nation, in predicting prognosis in DLCL. Expression of the suggested by experiments in nude mice, showing an enhanc- standard form of CD44 (CD44s) and of CD44 isoforms contain- ing effect of CD44s on lymphoma growth and dissemi- ing v6 (CD44v6) on tumor cells was assessed by immu- nation.15 More recently, CD44 splice variants containing exon nohistochemistry in a cohort of 276 DLCL patients from a popu- v6 were shown to be expressed in a subgroup of high-grade lation based lymphoma registry. We observed that CD44s as 21 well as CD44v6 expression correlated with tumor dissemination lymphomas with poor prognosis. In rodents, homologues of in patients with primary nodal DLCL. Importantly, in patients these variants confer metastatic potential on carcinoma and with localized nodal disease, CD44s was a strong prognosti- lymphoma cell lines.13,26 Together, these data indicate that cator predicting tumor related death independent of the other CD44s and CD44v6 are important mediators of lymphoma parameters of the International Prognostic Index (IPI). Incorpor- dissemination that could be of use as molecular markers to ation of CD44s in the IPI parameter ‘stage’, increased the prog- nostic value of this parameter in nodal DLCL. Our data identify identify subgroups of high-risk DLCL patients. Here, we have CD44 as a biological prognosticator, which can be used to ‘fine- explored this hypothesis by studying CD44s and CD44v6 tune’ the IPI for nodal DLCL. expression in tumor tissue of patients with DLCL from a popu- Keywords: CD44; ; B cell lymphoma; prognosis lation-based NHL registry; the results indicate that CD44 can be considered as a major prognosticator in patients with localized DLCL. Introduction

Diffuse large B cell lymphomas (DLCL) constitute 30 to 40% Materials and methods of adult non-Hodgkin’s lymphomas and are biologically related to (activated) mature B cells.1–3 In the recently pro- Patients and materials posed Revised European American Lymphoma (REAL) classi- fication these tumors are categorized as one single group, Between 1981 and 1989, 1168 patients with a histologically since previous attempts to subclassify them on morphological proven diagnosis of NHL were included in the population- grounds, were largely irreproducible.2 However, there is con- based non-Hodgkin’s lymphoma registry program of the Com- sensus that DLCL represent a diverse group of , with prehensive Cancer Center West (CCCW) in The Nether- heterogeneous genetic background, clinical features, treat- lands.4,27–30 The region of the CCCW comprises 1.6 million ment response, and prognosis.1–6 Although prognosis in DLCL inhabitants and 15 hospitals. Only newly diagnosed NHL can be predicted on the basis of clinical characteristics,6 the were included. All tumors were reviewed by a panel of hema- genetic and molecular basis underlying the heterogeneity in topathologists. Patients were staged according to the Ann disease aggressiveness and tumor progression, as well as in Arbor classification31 with modifications for extranodal lym- response to therapy remain to be elucidated. phoma.32 For adequate staging at least a chest radiograph Normal recirculating lymphocytes express cell-surface and/or CT scan, a CT scan of the abdomen or lymphangio- of the CD44 family. These molecules have a gram supplemented by isotope spleen and liver scan in the wide tissue distribution and function in several important bio- early years of registration, and a bone marrow biopsy had to logical processes including lymphocyte homing and acti- be performed. In addition to clinical stage, the presence or vation,7–10 hematopoiesis,7–11 and tumor progression and met- absence of lymphoma at various anatomical sites was astasis.7,12–16 Due to extensive and post- recorded. Patients were treated according to the preference of translational modification by N- and O-linked sugars, as well the local specialist in internal medicine, and this treatment as by (GAG) side chains,13,17–19 CD44 is was considered ‘adequate’ for large cell NHL, when doxorub- highly diverse. On lymphocytes, the short 80–90 kDa stan- icin-containing polychemotherapy was administered with or dard (hematopoietic) form of CD44 (CD44s) is most abundant, without radiotherapy, whereas for stage I patients, radio- while larger variants predominate on some normal and neo- therapy alone was also considered as adequate. All tissue plastic epithelial cells and are also expressed on activated specimens were obtained prior to treatment. Clinical para- meters as well as treatment modalities and outcome were recorded. The follow-up was annually updated up to 1996. In the CCCW records 494 B cell lymphomas were diag- Correspondence: ST Pals, Department of Pathology, Academic Medi- 33 cal Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; nosed according to the Kiel classification as diffuse centro- Fax: 31 20 6960389 blastic-centrocytic (diff CbCc), diffuse centroblastic (diff Cb), Received 23 February 1999; accepted 10 May 1999 immunoblastic (Ib) and mucosa-associated lymphoid tissue CD44 in large B cell lymphoma P Drillenburg et al 1449 lymphomas of high malignancy (MALT high), compatible with Austria) against the epitope QWFGNRWHEGYRQT on exon large B cell NHL as recently defined by the REAL-classi- CD44v6.35 After incubation with the primary mAb, the sec- fication.2 From 310 patients, tissue blocks containing tumor tions were incubated with biotinylated rabbit anti-mouse anti- were available and slides of paraffin embedded tumor tissue bodies (DAKO, Glostrup, Denmark) in PBS containing 10% were made. From 276 patients, interpretable staining of tumor normal human serum, and with streptavidin-biotin peroxidase tissue for CD44s and CD44v6 was obtained. For 236 of these complex (DAKO) each for 30 min. HRP activity was detected patients, all prognostic parameters of the International Prog- by incubation in 1 mg/ml 3,3-diaminobenzidine tetrahydro- nostic Index (IPI) were available (Table 1). Demographics, and chloride (Sigma, St Louis, MO, USA) and 0.015% H2O2 in stage distribution of these 236 patients was not different than 50 mM Tris-HCL (pH = 7.6). Slides were counterstained with those of the total group of 494 B cell lymphomas in the CCCW Mayer’s hematoxylin. Normal tonsil tissue was used as a posi- registry (data not shown). tive control in each staining procedure. Furthermore, within According to our previous reports4,27–29 at presentation the tumors, non-neoplastic lymphocytes and macrophages three patterns of lymphoma localization were defined: (1) ‘pri- expressing high levels of CD44s were always present and mary nodal’ NHL with presentation in lymph node, Waldey- served as internal standard and control. To facilitate identifi- er’s ring or spleen (n = 155); (2) ‘primary extranodal’ NHL with cation of the neoplastic lymphoid cells, serial sections were presentation at other sites without or with regional lymph routinely stained for CD20 (CD20, DAKO) and CD3 (CD3, node involvement (n = 91); (3) other NHL with disseminated DAKO). On the basis of the estimated percentage of positively disease at presentation and no possibility of determining the staining tumor cells, CD44s and CD44v6 expression was primary site (n = 30); the latter were called ‘extensive’. Both scored as follows: 0 (negative/low) = less than 10% positive in primary nodal and extranodal disease, bone marrow tumor cells; 1 (intermediate) 10–50% tumor cells positive; 2 involvement was accepted in cases with a positive staging (high) more than 50% positive tumor cells. This scoring system biopsy. All parameters of the IPI were available in these three is based on our experience from previous studies.24,36 Intensity subgroups for 137, 75 and 24 patients, respectively. of the stainings was also estimated, but subdivision of the groups based on staining intensity proved of no additional prognostic value (data not shown). All slides were read by two Immunohistochemistry independent observers (P Drillenburg and ST Pals); discrep- ancies were solved by consensus. During the whole pro- Tissues were fixed in buffered formalin and routinely embed- cedure, the observers were blinded for clinical data/disease ded in paraffin. Sections of 5 ␮m were cut and put on APES- outcome. coated slides. After deparaffinization in xylene and rehy- dration in alcohol endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide in methanol for Statistical analysis 20 min. Prior to staining with monoclonal (mAb) VFF18 (see below), the tissue sections were incubated in a The correlation of CD44s and CD44v6 with biological and microwave oven at 100°C for 10 min in citrate buffer (0.01 M, clinical parameters was estimated with the Spearman corre- pH = 6). The slides were washed in PBS, pre-incubated for lation coefficient. Survival curves were plotted following the 15 min with 10% normal goat serum, and then incubated for Kaplan–Meier method and tested for statistical significance 1 h with the primary mAb. The primary monoclonal anti- using the log- test. Overall survival (OS) was calculated bodies used were Hermes-334 against an epitope on the con- from the date of diagnosis until death (all causes) or last fol- stant part of CD44 (kindly provided by Dr S Jalkanen, Turku low-up. For patients who reached complete remission (CR), University, Turku, Finland), and VFF18 (Bender, Vienna, disease-free survival was estimated from the date of CR until

Table 1 Patient characteristics

Total n = 276 (%) Nodal n = 155 (%) Extranodal n = 91 (%) Extensive n = 30 (%)

Male/female 131/145 82/73 38/53 11/19 Age (range) 20–93 21–91 20–93 29–86 Age (mean) 65 64 67 65 Ann Arbor Stage I 88 (32) 39 (25) 49 (54) 0 Stage II 73 (26) 53 (34) 20 (22) 0 Stage III 38 (14) 38 (25) 0 0 Stage IV 77 (28) 25 (16) 22 (24) 30 (100) IPI Low 97 (35) 57 (37) 40 (44) 0 Low/intermediate 62 (23) 46 (30) 14 (15) 2 (7) High/intermediate 39 (14) 23 (15) 10 (11) 6 (20) High 38 (14) 11 (7) 11 (12) 16 (53) Not evaluable 40 (14) 18 (11) 16 (18) 6 (20) No therapy and surgery only 36 (13) 11 (7) 23 (25) 2 (7) Radiotherapy only 67 (25) 38 (25) 28 (31) 1 (3) Chemotherapy and +/− radiotherapy Without doxorubicin 48 (17) 24 (15) 11 (12) 13 (43) Containing doxorubicin 125 (45) 82 (53) 29 (32) 14 (47) CD44 in large B cell lymphoma P Drillenburg et al 1450 first relapse or last contact, if disease free. For the multivariate tumor dissemination (P = 0.21). Also, CD44v6 expression analysis and calculation of the hazard ratios and its confi- showed a significant correlation to tumor stage in nodal, but dence intervals Cox proportional hazard model was used. In not in extranodal tumors (P = 0.04). Bone marrow involve- Cox regression, the forward stepwise selection method was ment did not correlate with expression of CD44s or CD44v6 used, the results were controlled with backward stepwise (P = 0.28 and P = 0.72, respectively). Table 2 shows the selection. relation between CD44 expression and the clinical parameters of the International Prognostic Index (IPI). With the exception of the parameter ‘stage’, neither of the IPI parameters corre- Results lated with CD44 expression.

CD44s and CD44v6 expression in diffuse large B cell lymphoma (DLCL) CD44 predicts prognosis in localized DLCL

CD44s expression in DLCL was highly variable, ranging from We next explored the prognostic value of CD44s and negative to strongly positive, which confirms a previous study CD44v6. For the whole cohort of patients (n = 276), CD44s from our laboratory.24 Intermediate or high expression of and CD44v6 did not predict overall survival (P = 0.17 and CD44s was present in 13% and 63% of the tumors, respect- 0.96, respectively). Interestingly, however, in patients with ively; 24% of the tumors were CD44s negative. Unlike localized (stage I) disease (n = 88), CD44s proved to be a CD44s, CD44v6 was only expressed in a minority of the highly significant unfavorable prognosticator (P = 0.0076) tumors: whereas 84% showed no detectable staining for (Figure 2a). No statistically significant prognostic value was CD44v6, intermediate or high expression was present in 10% present in stage II or IV disease (Figure 2b, c) although there and 6% of the tumors, respectively. was a similar trend in stage II patients. The stage III patient Subclassification of DLCL on the basis of morphology (Kiel group was too small to be analyzed. classification) or primary tumor localization did not reveal sig- Although a strong prognostic effect of CD44s was evident nificant differences in distribution of CD44s or CD44v6 in the whole stage I group, this was largely accounted for by between the groups (data not shown). the effect of CD44s in the patient subgroup (n = 39) with a primary nodal lymphoma (P = 0.0014) (Figure 2d). If stage I extranodal lymphomas were analyzed separately, CD44s had Correlation of CD44s and CD44v6 with clinical risk no significant prognostic value (P = 0.79). Hence, CD44s was factors a strong prognosticator in patients with localized nodal DLCL. In a multivariate comparison with the clinical parameters of In view of the strong evidence for a role of CD44 in lympho- the International Prognostic Index (IPI), CD44s was the major cyte homing and lymphoma dissemination, we analyzed the prognosticator in this patient group (RR = 5.07; 95% CI: 1.12– relation between CD44 expression and tumor dissemination 22.90). These data were also found for the 26 patients with in our study group. In the total group (n = 276), no correlation nodal stage I disease who were treated with doxorubicin- between CD44s expression and tumor dissemination was containing polychemotherapy with of without radiotherapy, found (P = 0.20). However, in the tumors with a primary nodal or radiotherapy alone (Figure 3a). localization (n = 155), CD44s expression significantly corre- It is important to note that the proportion of patients who lated to clinical stage (Figure 1a) (P = 0.003) and as an alterna- received doxorubicin-containing chemotherapy and/or radio- tive measure of dissemination, to the total number of anato- therapy did not differ significantly between the CD44s positive mical sites involved by tumor (Figure 1b) (P = 0.018). In and negative groups (Table 3). Also the percentage of patients extranodal lymphomas (n = 91), CD44s did not correlate with who entered complete remission was similar (Table 3). How-

Figure 1 CD44s expression in nodal DLCL is related to tumor dissemination. (a) Relation between CD44s expression and Ann Arbor stage; (b) Relation between CD44s expression and the number of anatomical sites involved by tumor. Since intermediate (1+) and high (2+) expression of CD44s were both positively correlated with tumor stage/dissemination and since the survival curves for these patient groups were identical, both groups have been combined (+/++). CD44 in large B cell lymphoma P Drillenburg et al 1451 Table 2 Expression of CD44 related to the clinical parameters of the International Prognostic Index (n = 236)

n = 236 n = 137 (nodal)

CD44s(%) CD44v6(%) CD44s(%) CD44v6(%)

n −+/++ P −+/++ Pn−+/++ P −+/++ P

Age classes р60 years 80 24 76 NS 81 19 NS 48 29 71 NS 81 19 NS Ͼ60 156 22 78 85 15 89 19 81 84 16 LDH Normal 114 21 79 NS 83 17 NS 60 30 70 NS 87 13 NS Abnormal 122 24 76 84 16 77 17 83 81 19 Karnofsky у80% 174 23 77 NS 85 15 NS 113 23 77 NS 85 15 NS Ͻ80% 62 21 79 79 21 24 21 79 75 25 Ann Arbor stage I + II 140 26 74 NS 86 14 NS 81 31 69 0.005 88 12 NS III + IV 96 18 82 80 20 56 11 89 77 23 Extranodal sites р1 197 22 78 NS 83 17 NS 137 23 77 83 17 Ͼ1 392674 8713 0

−, (negative/low), less than 10% positive tumor cells; +/++, (intermediate/high) more than 10% positive.

ever, the relapse rate for CD44s positive lymphomas (58%) Discussion was significantly increased in comparison to the negative lym- phomas (10%, P = 0.018) (Table 3). Of the relapses 86% took The heterogeneity in outcomes in aggressive non-Hodgkin’s place outside the region originally involved by tumor. In line 6 with the high relapse rate in CD44s positive patients, CD44s lymphoma has prompted the development of the IPI, a clini- expression was also an unfavorable prognosticator for disease- cal prognostic factor model, which identifies patients with dif- free survival (P = 0.03) (Figure 3b). The number of patients ferent likelihoods of being cured of their disease. However, with a relapse was too small to analyze the impact of treat- there is consensus that the clinical prognostic features incor- porated in the IPI are, in large part, surrogate variables, with a ment on relapse rates in CD44s positive and negative cases. remote relation to the biological variables determining disease heterogeneity.6,37 Identification of these unknown biological variables is of great importance since they may improve prog- Incorporation of CD44 in the IPI parameter ‘stage’ nostic factor models and, in addition, serve as targets for ther- improves its prognostic power in nodal lymphoma apy in specific subsets of patients. In our present study, we demonstrate that CD44s, a biological determinant of lym- phoma dissemination, predicts outcome in patients with In view of the evidence that CD44s is instrumental in localized DLCL and that the IPI parameter ‘stage’ can be mediating lymphoma dissemination, we adopted the working improved by incorporating CD44s as an additional parameter. hypothesis that the adverse prognostic effect of CD44s in clini- Previous studies from our own and other laboratories have cally localized tumors might result from occult dissemination. related CD44 expression to tumor dissemination in non-Hodg- If so, CD44s might be used as a tool to predict outcome. As kin’s lymphoma.12,14,24,25 This association is confirmed in our the International Prognostic Index is currently widely used to present population-based study group. In patients presenting predict prognosis in DLCL, we addressed this hypothesis by with a primary nodal DLCL lymphoma, but not in those with exploring whether the prognostic power of the IPI parameter extranodal disease, both CD44s and CD44v6 were correlated ‘stage’ might be improved by including CD44s as an with tumor spread (Figure 1). We currently have no expla- additional parameter in primary nodal lymphomas. Hence, we nation for the differential effect of CD44 in nodal vs extrano- replaced the parameter Ann Arbor stage ϾII disease by Ann dal lymphoma. It should be noted, however, that nodal and Arbor stage ϾII disease or positivity for CD44s. This redefined extranodal lymphomas show important genetic, functional parameter (‘stage/CD44s’) was compared in multivariate and phenotypic differences suggesting that they represent dis- analysis with the conventional parameters of the IPI, ie age tinct biological entities.2,27,38,39 These differences include dif- Ͼ60, elevated LDH, Karnofsky performance status Ͻ80 and ferential expression of adhesion/homing receptors other than Ann Arbor stage ϾII. Upon regression analysis (Table 4), the CD44, for example of L-, which may co-determine parameter ‘stage/CD44s’ (and not the conventional IPI para- tumor spread.40 meter ‘stage’) was selected, indicating that inclusion of CD44s The key finding of our present study is that CD44s is a increases the prognostic power of the IPI parameter ‘stage’. strong independent prognosticator in localized nodal DLCL As is shown in Figure 4, incorporation of CD44s into the IPI (Figure 2). Whereas patients with localized (stage I) disease parameter ‘stage’ clearly improves the separation between the and a CD44s negative tumor have a favorable prognosis, the risk groups (Figure 4a vs b). prognosis of those with CD44s positive tumors is poor, even CD44 in large B cell lymphoma P Drillenburg et al 1452

Figure 2 CD44s predicts outcome in patients with localized DLCL. Overall survival for patients with CD44s positive (1+ and 2+) and negative (−) DLCL subdivided according to Ann Arbor stage. (a) Stage I, whole study group; (b) stage II, whole study group; (c) stage IV, whole study group: (d) stage I, primarily nodal lymphomas. The stage III group was too small to be analyzed.

at stage I. The observation that CD44s expression is both cor- patients with localized disease. As the IPI is currently widely related with lymphoma dissemination in the entire group and accepted as a major tool for the identification of ‘high’ or ‘low’ predictive of relapse of disease in patients with localized dis- risk in DLCL, this was analyzed by including CD44s in the IPI ease, matches with studies indicating an important physiologi- parameter ‘stage’. Indeed, we observed that the IPI parameter cal role of CD44s in lymphocyte trafficking. In this trafficking ‘stage’ can be improved by including all CD44s positive process, CD44s mediates lymphocyte binding to high endo- patients in the ‘high risk for stage’ group. In a multivariate thelial venules,8,10,34 lymphocyte rolling41,42 and migration to comparison with the other IPI parameters, the modified para- inflammatory sites.43,44 Presumably, CD44s expression in meter ‘stage/CD44s’ incorporating CD44s, and not the con- localized DLCL reflects a high propensity to disseminate ventional IPI parameter ‘stage’, was selected (Table 4). Fur- and/or the presence of occult dissemination, a scenario which thermore, by using this modified staging parameter a superior is supported by the fact that CD44s promotes the dissemi- separation of the distinct IPI risk groups was obtained nation of human lymphoma cells xenografted into nude (Figure 4a vs b). mice.15 A similar scenario has also been proposed to explain We considered the possibility that more effective treatment the prognostic value of CD44 splice variants containing exon of CD44s negative lymphomas might explain the favorable v6 in colorectal and breast cancer.36,45 It should be noted, outcome, however, there were no differences in therapy however, that other biological functions of CD44, specifically between the CD44s negative and positive groups (Table 3). its recently described role in counteracting ,46 may The percentage of patients entering a complete remission was also contribute to the less aggressive biological behavior of also identical in the CD44s positive and negative groups, by CD44 negative tumors. contrast, relapses were much more common in patients with In view of the strong evidence that CD44 is instrumental in CD44s positive tumors (Table 3). As the vast majority of these mediating lymphoma dissemination, we argued that CD44s relapses occurred outside the anatomical area of initial tumor might be used as an additional prognostic parameter in involvement, this high relapse rate in CD44s positive tumors CD44 in large B cell lymphoma P Drillenburg et al 1453

Figure 3 Cumulative survival for adequately treated patients with CD44s positive (1+ and 2+) and negative (−) stage I, nodal DLCL. (a) Overall survival (OS); (b) disease-free survival (DFS).

Table 3 CD44s expression in stage I, nodal DLCL in relation to therapy, complete remission and relapse in ‘adequately’ treated patients.

n Chemotherapya +/− Radiotherapy only Complete Relapse radiotherapy n = 9n= 17 remission rate

% P %P %P %P

CD44s − 11 45 55 91 10 NS NS NS 0.018 +/++ 15 27 73 80 58 aDoxorubicin-containing polychemotherapy. −, (negative/low) less than 10% positive tumor cells; +/++, intermediate/high more than 10% positive.

Table 4 Factors independently prognostic of overall survival in including this novel therapeutic modality. Meanwhile, our = nodal DLCL (n 137) current study clearly shows that stage I DLCL patients with a CD44 negative tumor have an excellent prognosis. Factor Relative 95% CI P value Step of Our current findings extend earlier studies from our own risk selection and Jalkanen’s laboratory.14,24 We previously reported that B- DLCL with low CD44s expression have a favorable prognosis. Karnofsky у80% 1.0 In the current much larger study, this finding is confirmed and, Ͻ80% 1.87 1.15–3.06 0.012 1 moreover, the prognostic effect of CD44s is shown to be inde- Stage/CD44sa pendent of other IPI parameters.24 Jalkanen and colleagues14 negative 1.0 reported CD44s to have independent prognostic value, how- positive 2.62 1.31–5.27 0.007 2 ever, their study and our present study differ at important Age р60 years 1.0 points. Whereas our present study focusses on a single clinico- Ͼ60 years 2.08 1.32–3.28 0.002 3 pathological entity, Jalkanen’s study group was hetero- LDH geneous, containing lymphomas of different subtype and normal 1.0 grade. Furthermore, in their study from the pre-IPI era, not all abnormal 1.55 1.02–2.37 0.042 4 clinical parameters of IPI were included in the analysis and, instead of overall survival rates, survival rates corrected for aSee text for definition. intercurrent death and death of unkown cause, were used as the end point. Although these differences make a direct com- is consistent with a role of CD44s in (occult) lymphoma dis- parison between the two studies difficult, both studies under- semination. Analysis of these patients with a relapse for the score the prognostic value of CD44s. impact of therapy was not possible due to the small number In our present study CD44v6 expression was not a signifi- of patients. Since recent data indicate that the most adequate cant prognosticator. In contrast to our data, Stauder and col- therapy for stage I patients is a combination of three cycles leagues21 reported CD44v6 to be a better prognosticator than of polychemotherapy and radiotherapy, instead of radio- or CD44s. This discrepancy might be due to differences in the chemotherapy alone,47 it will be very interesting to see composition of the study groups. Unlike in our study group, whether the impact of CD44s holds in a prospective study which contains only B-DLCL, the ‘high-grade’ B-NHL group CD44 in large B cell lymphoma P Drillenburg et al 1454

Figure 4 Incorporation of CD44s into the IPI (see text) improves the separation between the risk groups for nodal DLCL (a) conventional IPI risk groups; (b) IPI risk groups in which the staging parameter has been modified by incorporating CD44s. L, denotes low risk; LI, low intermediate risk; HI, high intermediate risk; and H, high risk.

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