Leukemia & Lymphoma, 2014; Early Online: 1–7 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2014.951849

ORIGINAL ARTICLE: CLINICAL

High CD20ⴙ background cells predict a favorable outcome in classical Hodgkin lymphoma and antagonize CD68ⴙ macrophages Luigi Panico1, Valentina Tenneriello1, Fioravante Ronconi2,3, Marianna Lepore1, Nicola Cantore2, Antonietta Carmela Dell’Angelo1, Ludovica Ferbo1 & Felicetto Ferrara3 1Unit of Pathology and 2Unit of Hematology, Hospital S. G. Moscati, Avellino, Italy and 3Unit of Hematology, Hospital A.

Leuk Lymphoma Downloaded from informahealthcare.com by University of Otago on 12/30/14 For personal use only.

Cardarelli, Napoli, Italy

or IL7, CCL5, CCL17, CCL20 and CCL22, recruiting and activating lymphocytes [3,4]. Reactive cells in the microenvironment themselves synthesize specific cytokines and chemokines that help to maintain and amplify the inflammatory background; furthermore these cells give rise to a signaling network whose final effect is a pro-tumoral feedback to neoplastic cells. cHL carries an excellent prognosis, with 70–80% long-term remission following current therapies [5,6]; the remaining 20–30% of patients are not cured by conventional therapies and eventually die of the disease. Long-term survivors, however, experience therapy related morbidity: second malignancies and cardiovascular diseases predispose to reduced survival, respectively persisting beyond 10 years and 20 years of follow-up from initial diagnosis [7]. Patient stratification and therapeutic decision are based on clinical variables. The International Prognostic Score (IPS) is the standard system used for risk stratification of patients [8]; higher scores, according to seven risk factors, indicate increased risk. This system, originally designed for advanced disease, does not apply to patients with low-risk disease. The criteria of the European Organisation for Research and Treatment of Cancer (EORTC) are more suitable for stratifying patients with low-risk disease into favorable and unfavorable risk groups [9]. Gene expression profiling (GEP) studies have outlined the role of the microenvironment in determining the outcome of the disease [10–12]; discriminant genes are relevant as potential prognostic markers and also as potential therapeutic targets. IHC identification of these potential markers remains a crucial challenge. In a recent report, Steidl et al. [13] confirmed by immunohistochemistry the adverse prognostic role of tumor associated macrophages (TAMs) evidenced by GEP, in an independent cohort of 166 patients; they furthermore found that underexpression of germinal center B cell genes was related to treatment failure; conversely the IHC detection of a high background B cell count was significantly associated with advanced stage disease, with prolonged progression-free (PFS) and disease-specific

Abstract We studied by immunohistochemistry the background CD20 ⴙ cells in 131 cases of classical Hodgkin lymphoma (cHL). High CD20 ⴙ dispersed cells (CD20BG) showed a significant correlation with longer overall survival (OS) and a trend toward improved progression-free survival (PFS). At multivariate analysis high CD20BG was also an independent prognostic factor of improved PFS and OS. The prognostic role of CD20BG seems to be opposite with respect to tumor associated macrophages (TAMs) we studied previously in most cases of the series. We scored patients on the basis of the respective CD20BG and TAM count and found that the combination of low CD20BG and high TAMs was related to a significantly reduced PFS and OS at univariate and multivariate analysis. Microenvironment CD20 ⴙ cells seem to play a favorable prognostic role in cHL. Depletion of CD20 ⴙ cells together with an increase of TAMs identifies a group of patients with high-risk disease. Keywords: CD20, classical Hodgkin lymphoma, prognosis, immunohistochemistry, microenvironment

Introduction Classical Hodgkin lymphoma (cHL) is characterized by the presence of neoplastic cells (Hodgkin and Reed–Sternberg [HRS] cells) that constitute only a minority population in the affected lymph node (less than 1–10%) [1–3]. Malignant cells are outnumbered by non-neoplastic microenvironmental cells that include stromal and immune cells and account for the vast majority of the tumor bulk. The frequencies of these inflammatory cells, including macrophages, neutrophils, eosinophils, T and B cells, fibroblasts, plasma cells and mast cells, are variable between cHL subtypes [2,3]. The microenvironment milieu composition is partly due to the cytokines and chemokines secreted by HRS cells that act as chemotactic factors, such as interleukin 5 (IL5), IL8, IL9, CCL5 and CCL28, recruiting granulocytes, mast cells and macrophages,

Correspondence: Luigi Panico, Unit of Pathology, Hospital S. G. Moscati, Contrada Amoretta, 83100, Avellino, Italy. Tel:  390825203446. Fax:  390825203465. E-mail: [email protected] Received 4 June 2014; revised 15 July 2014; accepted 28 July 2014

1

Leuk Lymphoma Downloaded from informahealthcare.com by University of Otago on 12/30/14 For personal use only.

2

L. Panico et al.

survival. These latter results confirmed the GEP findings of Chetaille et al. [12] that showed a favorable outcome in cases of overexpression of B cell cluster genes. Greaves et al. [14] have studied the IHC expression of three microenvironmental markers: CD68, CD20 and the T-regulatory transcription factor FOXP3, in the cHL background, finding a prognostic role of high CD20 count that was associated with prolonged overall survival (OS). Tudor et al. [15] have recently studied different B cell populations in the cHL background and found a significant correlation between high CD20 positive cells, aggregated or isolated, and better survival. In a previous report concerning 121 of the 131 cases of the present series [16], we studied the macrophage density and vessel density (VD) using immunohistochemistry. We demonstrated an adverse significant prognostic role of TAMs in cHL with respect to OS, and a trend toward reduced PFS. TAM count was not an independent prognostic factor at multivariate analysis. VD was unrelated to prognosis. In the present series we studied by immunohistochemistry the expression of the B cell marker CD20 in bystander cells, aiming to analyze the relationships between microenvironmental cells, and to evaluate the correlations with clinicopathological features and the prognostic role of these markers in an independent group of patients, clinically well characterized and uniformly treated in a single institution.

Materials and methods Patients We retrospectively studied 131 consecutive cases of cHL diagnosed in the period 2003–2011 at the Hospital S. G. Moscati in Avellino, Italy. For all patients, clinical data and histological material were available. All patients were immunocompetent, and acquired immunodeficiency syndrome (AIDS) related cases were not included in the study. Histological diagnoses were reviewed according to the World Health Organization criteria. Patients were staged according to the Ann Arbor system. Baseline clinicopathological features were: sex, age, stage, presence of B symptoms or bulky disease, serum β2-microglobulin (B2M) and lactate dehydrogenase (LDH) levels, and histopathological type. Patients with high Ann Arbor stage (III–IV) were stratified into a high-risk group for IPS score  2; patients with lowstage disease (I–II) were classified as the early unfavorable risk group following the EORTC criteria. All patients were treated with curative intent with first-line ABVD (adriamycin, bleomycin, vinblastine and dacarbazine) or an ABVD-like regimen. Involved field radiotherapy was added in selected cases with limited disease or bulky mass.

Immunohistochemistry Primary antibodies were CD68 (KP1; Ventana, Tucson, AZ), CD34 (Qbend 10; Ventana) and CD20 (L26; Ventana), all ready to use. Four-micrometer tissue sections were stained using an automated immunostainer (Benchmark Ultra; Ventana), as previously described [16]. Briefly, the slides were incubated with primary antibody for 32 min at 37°C. Heat-induced anti-

gen retrieval was performed following the manufacturer’s recommendations. The results were evaluated by three researchers (L.P., V.T., M.L.), blinded as to outcome, using an Olympus BX43 light microscope equipped with a computer assisted digital camera (DP21). Slides were examined in a collegial visualization of the acquired image, projected on a computer screen. Areas of fibrosis and necrosis were not included. We acquired fields, each field corresponding to a counting frame on the computer screen. Within the frame, the cells were counted by a counting system included in the camera software. To analyze CD68 results as already described [16] we examined 20 frames at  400 magnification, each frame covering an area of 0.023 mm2, and the mean value of all frames was considered the TAM count; in the evaluation of CD34 we examined 10 fields at  200 magnification, each corresponding to an area of 0.09 mm2, and selected three hot spots, frames showing the most intense vascularization. CD20  non-aggregated background cell density (CD20BG) was assessed by the mean value of positive cells in three hot spot frames at  400 magnification. The frames were selected on the basis of the most intense IHC positivity and three frames were considered representative, because of the much higher cell density of CD20BG cells with respect to TAMs. We furthermore evaluated the number of CD20 aggregates (CD20A), scanning the whole section at low power ( 40) magnification, each frame corresponding to an area of 8.8 mm2. Aggregates were counted and the mean value per frame was considered the CD20A score.

Statistical analysis Patient characteristics were compared using χ2 and Fisher exact tests for categorical variables. Survival was analyzed by the Kaplan–Meier method and differences between survival curves were assessed using the log-rank test. PFS was defined as the time interval from diagnosis to disease progression, relapse or last contact. OS was defined as the time interval from diagnosis to death from any cause or last contact. Multivariate analysis was performed using Cox’s proportional hazards model. Median PFS and OS were not reached for surviving patients at a mean follow-up of 40 months (range 1–112 months). The correlation between continuous variables was evaluated by Spearman’s correlation test. A two-tailed p-value of  0.05 was considered statistically significant. A p-value between 0.05 and 0.1 was considered of borderline significance.

Results Patient characteristics The patients’ main characteristics are summarized in Table I. The age range was 12–84 years, median 33 and average 38.7 years; 87 patients (66.4%) were younger than 45 years and 74 (56.5%) were male. According to the Ann Arbor staging system, 15 cases were classified as stage I (11.5%), 67 (51.1%) as stage II, 24 (18.3%) as stage III and 25 (19.1%) as stage IV.

CD20 cells in classical Hodgkin lymphoma

3

Table I. Baseline clinicopathological characteristics and their prognostic role at univariate analysis for progression-free survival and overall survival. Cases

Leuk Lymphoma Downloaded from informahealthcare.com by University of Otago on 12/30/14 For personal use only.

Variable Age Range Average Median  45  45 Gender Male Female Stage (Ann Arbor) I II III IV Stage (early vs. advanced) Early Advanced Histological subtype Nodular sclerosis Mixed cellularity Lymphocyte rich Lymphocyte depleted B symptoms Yes No Bulky disease Yes No β2-Microglobulin levels* High ( 2.7 mg/L) Low ( 2.7 mg/L) Lactate dehydrogenase levels†  450 U/L  450 U/L IPS score‡ 0–2 2 EORTC score§ Early unfavorable Early favorable CD68  TAMs  30  30 CD34  vessel density Low High CD20BG  500  500 CD20A  10.3  10.3 Relapse Yes No Death Yes No

PFS (%)

Log-rank p-value

%

12–84 38.7 33 87 44

66.4% 33.6%

NS

96.6 72.7

0.000

74 57

56.5% 43.5%

NS

81.1 98.2

0.004

15 67 24 25

11.5% 51.1% 18.3% 19.1%

86.7 74.6 62.5 52.0

0.025

100 89.6 95.8 72.0

0.021

82 49

62.6% 37.4%

76.8 57.1

0.029

91.5 83.7

0.08¶

78 43 8 2

59.5% 32.8% 6.1% 1.5%

NS

52 79

39.7% 60.3%

NS

80.8 93.7

0.05

30 101

22.9% 77.1%

56.7 73.3

0.09¶

80.0 91.1

0.09¶

27 73

27.0% 73.0%

55.6 72.6

0.10¶

66.7 94.5

0.000

70 58

54.7% 45.3%

77.1 58.6

0.046

30 19

61.2% 38.8%

66.7 42.1

0.08¶

100 57.9

0.000

52 30

63.4% 36.6%

NS

86.5 100

0.04

76 55

58.0% 42.0%

0.08¶

92.1 83.6

0.043

62 69

47.3% 52.7%

53 78

40.5% 59.5%

68 63

51.9% 48.1%

40 91

30.5% 69.5%

15 116

11.5% 88.5%

73.7 63.5

OS (%)

Log-rank p-value

n

NS

NS

NS 60.4 75.6

NS

0.10¶

83.0 92.3

0.04

NS

83.8 93.7

0.06¶

PFS, progression-free survival; OS, overall survival; IPS, International Prognostic Score; EORTC, European Organisation for Research and Treatment of Cancer; TAMs, tumor associated macrophages; CD20BG, CD20  dispersed background cells; CD20A, CD20  aggregated background cells; NS, not significant. *n  100 (31 cases missing). †n  128 (3 cases missing). ‡Calculated in 49 advanced cases. §Calculated in 82 early cases. ¶Trend of borderline significance.

The most frequent histological type was nodular sclerosis (78 cases; 59.5%); mixed cellularity was diagnosed in 43 cases (32.8%), eight cases were lymphocyte-rich (6.1%) and two cases (1.5%) were lymphocyte depleted.

B symptoms were observed in 52 (39.7%) patients and a bulky mass was observed in 30 (22.9%) cases. B2M levels were elevated in 27 of 100 patients (27%) while high LDH levels were present in 58 of 128 patients (45.3%). An IPS score  2

Leuk Lymphoma Downloaded from informahealthcare.com by University of Otago on 12/30/14 For personal use only.

4

L. Panico et al.

was calculated in 19 of 49 (38.8%) patients with high-stage disease (III–IV); 52 of 82 (63.4%) patients with early-stage (I–II) disease were scored as unfavorable according to the EORTC. The ABVD regimen was administered in 111 (84.7%) cases and ABVD-like in 20 cases. Additional radiotherapy was required in 19 patients (14.6%). A good therapy response with complete remission was obtained in 102 (77.9%) patients. A partial remission was achieved in 12 cases (9.1%), while 17 (13%) patients experienced disease progression during first-line therapy. A failure of first-line therapy (recurrence after remission or disease progression) was observed in 40 patients (30.5%), including 11 cases of recurrence following complete remission and the above-cited 12 cases of partial remission, and 17 cases of progression during therapy; 15 patients (11.5%) died of disease or other causes.

Immunohistochemistry CD68 positive cells ranged from 3.3 to 93.1 (median 25.7; mean 28.2). In our previous report we set the cut-off at a value of 30 cells per frame at  400, according to the most significant difference in survival using the log-rank test. High TAM count was present in 55 (42.0%) cases. The VD ranged from 4.5 to 34.3 (median 16.5; mean 17) vessels; high VD was present in 69 cases (52.7%) using the median as cut-off. The CD20BG ranged from 36 to 2058 (median 645; mean 689). We considered different cut-off values (median, 250 cells, 500 cells and 1000 cells). The log-rank test revealed a cut-off of 500 cells to be the most effective for survival. Fiftythree cases (40.5%) showed fewer than 500 CD20 positive cells per frame at  400. The score of CD20A ranged from 1.5 to 27.5 per frame (median 10.3; mean 10.8). Likewise, we chose the median value as cut-off, because it was the most effective for survival using the log-rank test, in comparison with alternative values of 5, 15 and 20 or more aggregates; 68 (51.9%) cases showed a low CD20A count.

Correlation with baseline features High CD20BG count (Supplementary Table 1 to be found online at http://informahealthcare.com/doi/abs/10.3109/ 10428194.2014.951849) was directly correlated with male gender (p  0.004); a significant correlation was found between CD20BG and histological type (p  0.035) due to the incidence of high CD20 count in the lymphocyte-rich variant. A strong significant correlation was furthermore evidenced between high CD20BG count and low Ann Arbor stage of disease (p  0.000) or early disease (p  0.001), absence of B symptoms (p  0.004), favorable EORTC group (p  0.05) or IPS score  2 (p  0.025). A trend of borderline significance was observed between high CD20BG and absence of bulky disease (p  0.10). The presence of high CD20A number was significantly correlated with low Ann Arbor stage (p  0.034) and early disease (p  0.044); a trend was observed between high CD20A and absence of bulky disease (p  0.06) and low VD (p  0.09).

A significant correlation was found between high TAMs and age  45 (p  0.03), mixed cellularity histotype (p  0.017) and high B2M level (p  0.007). Low Ann Arbor stage was related to low TAM count (p  0.038), while a correlation was present between high TAMs and high VD (p  0.035). Except for the already cited trend between low VD and high CD20A, and the correlation between high TAMs and high VD, no significant relationship was evidenced between VD and other clinicopathological variables.

Survival The main clinicopathological features showed a significant correlation with outcome (Table I). A reduced PFS was observed in patients with high Ann Arbor stage (logrank p  0.025), advanced disease (log-rank p  0.029) and high LDH level (log-rank p  0.046). A trend toward a shorter PFS was present for high B2M (log-rank p  0.10), IPS  2 (log-rank p  0.08) and bulky disease (log-rank p  0.09). OS was significantly shorter in patients aged  45 (logrank p  0.000), in male gender (log rank p  0.004), in cases with B symptoms (log-rank p  0.05), in high Ann Arbor stages (log-rank p  0.021), in patients with IPS  2 (logrank p  0.000), in those with unfavorable EORTC score (log-rank p  0.04) and in cases with elevated B2M level (log-rank p  0.000). A trend was present for bulky (log-rank p  0.09) and advanced disease (log-rank p  0.08). Among the microenvironmental parameters, a prognostic role of high TAMs was confirmed, showing a correlation of borderline significance with PFS (log-rank p  0.08) and a significant correlation with OS (log-rank p  0.04). High CD20BG count showed a weaker, borderline correlation with improved PFS [Figure 1(A); logrank p  0.10] and a meaningful correlation with longer OS [Figure 1(B); log-rank p  0.04]. High CD20A count showed only a near significant correlation with prolonged OS (log-rank p  0.06). In the multivariate analysis we considered as covariates all the clinicopathological or microenvironmental parameters that were prognostically significant, or that showed a borderline correlation with respect to PFS and OS at univariate analysis. We alternatively introduced as covariates Ann Arbor stage or early versus advanced disease. IPS and EORTC scores were not included in the model because of the high number of missing cases. Table II reports the significant independent prognostic factors for PFS and/or OS. A high CD20BG count ( 500) was an independent prognostic factor of improved PFS (p  0.038; relative risk [RR] 0.46, 95% confidence interval [CI] 0.228–0.957); high TAMs showed a near significant trend toward a reduced PFS (p  0.084; RR 1.85, 95% CI 0.921–3.731). The multivariate analysis of OS revealed male sex (p  0.039; RR 3.01, 95% CI 1.059–8.606) and high B2M level (p  0.032; RR 4.46, 95% CI 1.13–17.53) as independent prognostic factors of reduced OS; age  45 showed a borderline (p  0.10) trend as an independent variable for reduced OS; conversely, high CD20BG count was an independent predictor of improved OS (p  0.013; RR 0.20, 95% CI 0.061–0.722).

Leuk Lymphoma Downloaded from informahealthcare.com by University of Otago on 12/30/14 For personal use only.

CD20 cells in classical Hodgkin lymphoma

5

Figure 1. Kaplan–Meier curves of progression-free survival (PFS) and overall survival (OS) in 131 cases of classical Hodgkin lymphoma. PFS (A) and OS (B) according to CD20  dispersed background cell count. Cases with high cell count showed a trend toward better PFS (log-rank p  0.10) and a significantly improved OS (log-rank p  0.04). PFS (C) and OS (D) according to combined count of CD20  background cells (CD20  BG) and CD68  tumor associated macrophages (TAMs), grouped in a microenvironmental score. Patients with low CD20  cells and high CD68 (score 4) showed a significantly reduced PFS (log-rank p  0.025) and OS (log-rank p  0.004).

Microenvironmental score Univariate analysis showed TAMs and CD20BG to be linked to disease outcome, with different effects. We examined the relationship between these two variables to understand whether their expressions were correlated or independent. The χ2 test and Spearman test showed that the two variables were not significantly related (data not shown). Because of their opposite impact on prognosis, we evaluated whether their combined expression could add further prognostic information. We therefore scored patients on the basis of matched values of TAM and CD20BG

counts. We assigned 1 point to the presence of each of the most favorable conditions (high CD20BG and low TAM count) and 2 points to each of the most unfavorable conditions (low CD20BG and high TAM count). Every patient was assigned 1 or 2 points on the basis of the CD20BG count and 1 or 2 points on the basis of the TAM count. The resulting sum of these two microenvironmental variables stratified patients into three groups showing a score 2 (high CD20BG and low TAMs), score 4 (low CD20BG and high TAMs) and score 3 (high CD20BG and high TAMs or low CD20BG and low TAMs). The correlation of the score

Table II. Multivariate statistical analysis: significant prognostic factors for overall survival and/or progressionfree survival. PFS Prognostic factor Gender Male Age  45 B2M  2.7 mg/L CD68  macrophages  30 CD20BG  500

Risk ratio

OS Risk ratio

95% CI

p-Value

NF

3.01

1.059–8.606

0.039

NF

3.37

0.79–14.38

0.10*

NS

4.46

1.13–17.53

0.032

95% CI

p-Value

1.85

0.921–3.731

0.084*

0.46

0.228–0.957

0.038

NS 0.20

0.061–0.722

0.013

PFS, progression-free survival; OS, overall survival; NF, variables not fitted in model because not significant at univariate analysis; CI, confidence interval; CD20BG, CD20  dispersed background cells. *Trend of borderline significance.

Leuk Lymphoma Downloaded from informahealthcare.com by University of Otago on 12/30/14 For personal use only.

6

L. Panico et al.

groups with clinicopathological parameters (data not shown) revealed a significant correlation between score 4 and high Ann Arbor stage (p  0.026) and advanced disease (p  0.004), as well as between score 4 and high B2M level (p  0.038). Univariate analysis [Figures 1(C) and 1(D)] revealed patients with score 4 to be a high-risk group, with both significantly reduced PFS (log-rank p  0.025) and OS (log-rank p  0.004). Introducing the score variable in the multivariate Cox model, in substitution of the single variables CD20BG and TAMs, the microenvironmental score proved to be an independent prognostic factor: a unique independent predictor of reduced PFS (p  0.008; RR 1.98, 95% CI 1.19–3.31), and the most powerful predictor of reduced OS (p  0.006; RR 3.65, 95% CI 1.45–9.18). Table III reports the significant independent prognostic factors for survival (PFS and/or OS) introducing the new microenvironmental covariate, showing different data with respect to the previous analysis, depending on the changed prognostic value of the examined variables. In the case of OS, male sex retained an independent prognostic role, age  45 acquired a more significant role (p  0.002) and bulky disease showed a borderline correlation (p  0.08).

Discussion Better understanding of the impact of bystander cells on the disease pathobiology has raised interest in the IHC typing of microenvironmental cells in cHL, in order to identify potential prognostic markers and therapeutic targets. Different biomarkers have been proposed; however, they need to be translated into clinical practice by validation of findings [17–19]. In recent years much interest has been shown in the role of TAMs. Steidl et al. first [13] validated the adverse prognostic role of TAMs using immunohistochemistry, previously evidenced by GEP results. In a previous article [16], including almost all of the cases in the present series (121/131), using immunohistochemistry we studied TAMs and angiogenesis in cHL and confirmed the unfavorable prognostic role of TAMs, as observed by Steidl et al. [13].

An increasing interest concerns the potential prognostic role of CD20-positive lymphocytes in a neoplastic background. A protective role of high B cell number was hypothesized by GEP studies that found [11–13] the expression of B cell cluster genes to be related to a favorable outcome. The IHC validation of these results demonstrated a prognostic role of reactive B cells [12,13]. In a recent study, Greaves and colleagues [14] used image analysis software to evaluate expression of the B cell marker CD20 in 10–20 high power fields (HPFs), together with two other microenvironmental markers, CD68 and FOXP3. High CD20-positive cases were related to a significantly better OS; the other biomarkers tested, CD68 and FOXP3, were not significantly related, and were more effective for survival at the univariate level, with better prognosis in the lowest CD68 density group and in the highest FOXP3 density group. Interestingly, a combined score of these two independent markers added further prognostic information and was effective for OS, diseasespecific survival and treatment failure. Tudor et al. [15] used immunohistochemistry to study the distribution of CD20A and non-aggregated CD20  B cells, together with other IHC markers of follicular, germinal center or plasma cell differentiation. The authors noted a significant reduction of CD20A in stage IV cases with respect to I–II stages. CD20A and dispersed CD20  cells were both related to an improved disease-specific survival; event-free survival was significantly better only in cases of high CD20A, but was not related to dispersed CD20  cells. Our results confirm a prognostic role of CD20  cells in the cHL microenvironment. We have demonstrated a significant correlation between CD20BG cells and prognostic clinicopathological variables. The survival analysis results confirm the prognostic role of baseline features, and validate our cohort as representative. High TAMs maintain their unfavorable prognostic role, with significantly reduced OS and borderline significance toward a reduced PFS. CD20BG count showed an opposite prognostic role with respect to TAMs, with a significantly better OS in high CD20  cases which also showed a trend toward a better PFS. CD20A count is a lesser prognostic indicator, showing only a trend between high CD20  count and improved OS.

Table III. Multivariate statistical analysis: significant prognostic factors for overall survival and/or progressionfree survival grouping CD20  background cells and tumor associated macrophages into a microenvironmental score. PFS Prognostic factor Gender Male Age  45 Bulky disease Yes Microenvironmental score 4

Risk ratio

OS Risk ratio

95% CI

p-Value

NF

3.26

1.13–9.41

0.028

NF

8.72

0.87–9.08

0.002

NS

2.82

0.87–9.08

0.08*

3.65

1.45–9.18

0.006

1.98

95% CI

1.19–3.31

p-Value

0.008

PFS, progression-free survival; OS, overall survival; NF, variables not fitted in model because not significant at univariate analysis; CI, confidence interval; NS, not significant. *Trend of borderline significance.

Leuk Lymphoma Downloaded from informahealthcare.com by University of Otago on 12/30/14 For personal use only.

CD20 cells in classical Hodgkin lymphoma At multivariate analysis a high density of CD20BG cells is an independent predictor of better PFS and OS survival, whereas high TAM count shows a trend toward a reduced PFS. High TAMs and high CD20  cells seem, therefore, to oppositely affect the outcome, despite the absence of an inverse reciprocal correlation. This observation led us to evaluate the impact on prognosis of the combination of the two microenvironmental variables, resulting in the identification of a group of patients at higher risk of relapse and reduced OS at univariate analysis. These patients showed high TAM count and low CD20BG cells. This high-risk phenotype was an independent predictor of poor outcome at multivariate analysis for either PFS or OS as well. These data could suggest an active role of CD20  B cells in determining the evolution of the disease, resulting in a protective action and favoring a good prognosis. In our series we found a reduction of dispersed and aggregated CD20  cells with increasing tumor stage; this finding, in conjunction with the improved survival of high dispersed CD20  cases, seems to indicate that the B cell background could exert an effective anti-tumoral action; the loss of this protective action is reflected in tumor progression, advanced stage and poor survival; alternatively it could be the consequence of the disease progression that destroys the lymphoid architecture and determines a reduction of survival. The specific anti-tumoral activity could be due to an immune antineoplastic response based on the synthesis of specific antibodies against neoplastic cells, on the secretion of cytokines and chemokines, a direct tumoricidal potential and tumor antigen presentation [20]. Our data demonstrate the absence of correlation between CD20  cells and TAM counts; however, an inverse functional relationship appears. We could hypothesize a CD20/TAM balance, with a resulting anti-tumoral effect if CD20  B cells prevail, or a protumoral action if TAMs prevail. The reported beneficial effects of rituximab on survival [21,22] could provide evidence against a protective role of CD20 B cells, although could demonstrate a pro-tumoral action abrogated by rituximab. An alternative explanation for this is the possible targeting of circulating clonotypic B cells, potential precursors of HRS cells [23,24]. Our findings imply a potential role of B cells in the cHL microenvironment, with improved survival; this role could be partly due to an antagonistic action toward TAMs. These results do not explain the mechanisms that rule the crosstalk between B lymphocytes and TAMs, however suggest a worse prognosis in cases characterized by low CD20  and high TAM count. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.

Supplementary material available online Supplementary Table 1 showing further results

7

References [1] Mani H, Jaffe ES. Hodgkin lymphoma: an update on its biology with newer insights into classification. Clin Lymphoma Myeloma 2009;9:206–216. [2] Pileri SA , Ascani S, Leoncini L, et al. Hodgkin’s lymphoma: the pathologist’s viewpoint. J Clin Pathol 2002;55:162–176. [3] Piccaluga PP, Agostinelli C, Gazzola A , et al. Pathobiology of Hodgkin lymphoma. Adv Hematol 2011;2011:920898. [4] Steidl C, Connors JM, Gascoyne RD. Molecular pathogenesis of Hodgkin’s lymphoma: increasing evidence of the importance of the microenvironment. J Clin Oncol 2011;29:1812–1826. [5] Canellos GP, Anderson JR, Propert KJ, et al. Chemotherapy of advanced Hodgkin’s disease with MOPP, ABVD, or MOPP alternating with ABVD. N Engl J Med 1992;327:1478–1484. [6] Canellos GP, Niedzwiecki D. Long term follow-up of Hodgkin’s disease trial. N Engl J Med 2002;346:1417–1418. [7] Castellino SM, Geiger AM, Mertens AC, et al. Morbidity and mortality in long-term survivors of Hodgkin lymphoma: a report from the childhood cancer survivor study. Blood 2011;117:1806–1816. [8] Hasenclever D, Dihel V. A prognostic score for advanced Hodgkin’s disease. N Engl J Med 1998;339:1506–1514. [9] Tubiana M, Henry-Amar H, Carde P, et al. Toward comprehensive management tailored to prognostic factors of patients with clinical stages I and II in Hodgkin’s disease. The EORTC lymphoma group controlled clinical trials: 1964–1987. Blood 1989;73:47–56. [10] Devilard E, Bertucci F, Trempat P, et al. Gene expression profiling defines molecular subtypes of classic Hodgkin’s disease. Oncogene 2002:21:3095–3102. [11] Sanchez-Aguilera A , Montalban C, de la Cueva P, et al. Tumor microenvironment and mitotic checkpoint are key factors in the outcome of classic Hodgkin lymphoma. Blood 2006;108:662–668. [12] Chetaille B, Bertucci F, Finetti P, et al. Molecular profiling of classic Hodgkin lymphoma tissue uncovers variations in the tumor microenvironment and correlations with EBV infection and outcome. Blood 2009;113:2765–2775. [13] Steidl C, Lee T, Shah SP, et al. Tumor-associated macrophages and survival in classic Hodgkin’s lymphoma. N Engl J Med 2010:362: 875–885. [14] Greaves P, Clear A , Coutinho R, et al. Expression of FOXP3, CD68 and CD20 at diagnosis in the microenvironment of classical Hodgkin lymphoma is predictive of outcome. J Clin Oncol 2012;31:256–262. [15] Tudor CS, Distel LV, Eckhardt J, et al. B cells in Hodgkin lymphoma are important actors rather than bystanders in the local immune reaction. Hum Pathol 2013;44:2475–2486. [16] Panico L, Ronconi F, Lepore M, et al. Prognostic role of tumor associated macrophages and angiogenesis in classical Hodgkin lymphoma. Leuk Lymphoma 2013;54:2418–2425. [17] Alvaro T, Lejeune M, Salvado MT, et al. Outcome in Hodgkin’s lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells. Clin Cancer Res 2005;11:1467–1473. [18] Kelley TW, Pohlman B, Elson P, et al. The ratio of FOXP3  regulatory T cells to Granzyme B cytotoxic T/NK cells predicts prognosis in classical Hodgkin lymphoma and is independent of bcl-2 and MAL expression. Am J Clin Pathol 2007:128:958–965. [19] Molin D, Edstrom A , Glimelius I, et al. Mast cell infiltration correlates with poor prognosis in Hodgkin’s lymphoma. Br J Hematol 2002;119:122–124. [20] Nelson BH. CD20  B cells: the other tumor-infiltrating lymphocytes. J Immunol 2010:185:4977–4982. [21] Younes A , Romaguera J, Hagemeister F, et al. A pilot study of rituximab in patients with recurrent, classic Hodgkin lymphoma. Cancer 2003;98:310–314. [22] Oki Y, Pro B, Fayad LE, et al. Phase 2 study of gemcitabine in combination with rituximab in patients with recurrent or refractory Hodgkin lymphoma. Cancer 2008;112:831–836. [23] Jones RJ, Gocke CD, Kasamon YL, et al. Circulating clonotypic B cells in classic Hodgkin lymphoma. Blood 2009;113:5920–5926. [24] Kasamon YL, Jacene HA, Gocke CD, et al. Phase 2 study of rituximabABVD in classical Hodgkin lymphoma. Blood 2012;119:4129–4132.

High CD20+ background cells predict a favorable outcome in classical Hodgkin lymphoma and antagonize CD68+ macrophages.

We studied by immunohistochemistry the background CD20 + cells in 131 cases of classical Hodgkin lymphoma (cHL). High CD20 + dispersed cells (CD20BG) ...
370KB Sizes 0 Downloads 4 Views