Leukemia & Lymphoma, July 2014; 55(7): 1596–1601 © 2014 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2013.854889
ORIGINAL ARTICLE: CLINICAL
Peripheral blood CD4/CD19 cell ratio is an independent prognostic factor in classical Hodgkin lymphoma Francesco Gaudio, Tommasina Perrone, Anna Mestice, Paola Curci, Annamaria Giordano, Mario Delia, Domenico Pastore & Giorgina Specchia
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Hematology Section, Department of Emergency and Organ Transplantation, University of Bari Medical School, Bari, Italy
Despite numerous studies regarding their immunophenotypes and secreted cytokines and chemokines, the pathogenic roles of these Hodgkin lymphoma-infiltrating lymphocytes remain poorly understood [2,3]. Several lines of evidence suggest that the reactive cells are attracted by chemokines produced by the HRS cells [4–7]. In return, these infiltrating cells produce cytokines that may contribute to the proliferation and survival of the HRS cells [8,9]. Even though cHL is curable, approximately 30% of all patients relapse and eventually die of progressive disease or complications of therapy [10–12]. Several clinical and laboratory features have been used to predict survival so as to adjust therapy according to the predicted risk of relapse [13,14]. Absolute lymphocyte/monocyte ratio at diagnosis and tumor-infiltrating lymphocytes have been implicated in the prognosis of cHL [15–17]. Recent gene-expression profiling studies in cHL have demonstrated that tumor-infiltrating macrophage cells also predict clinical outcomes [18]. Furthermore, a low lymphocyte count, defined by an International Prognostic Score (IPS) as ⬍ 600 cells/μL or ⬍ 8% of the white blood cell count (WBC), is a negative prognostic factor for survival in cHL [13,14]. However, most models, including the IPS, fail to identify a sizeable fraction of patients whose chance of cure is less than 50%. Therefore, we decided to determine whether features directly related to the biology of cHL can predict clinical outcome. We studied the role of the peripheral blood B, T and natural killer (NK) cell count at diagnosis as a simple biomarker combining an estimate of host immune homeostasis and the tumor microenvironment in clinical outcome in patients with cHL.
Abstract Classical Hodgkin lymphoma (cHL) is characterized by the presence of tumoral cells in a rich background of T and B cells, macrophages and other inflammatory cells. The contribution of these non-tumoral cells to the pathogenesis of HL is still poorly understood. In our study we evaluated the prognostic significance of peripheral blood B, T and natural killer (NK) cells at diagnosis in 118 immunocompetent patients with cHL treated at our institution between January 2006 and December 2010. Fifty-four (46%) were male and 64 (54%) female. Median age at diagnosis was 33 years (range 15–82), and 71 patients (60%) presented an advanced stage (IIB–IV), 54 (46%) had bulky disease and 55 (47%) presented B symptoms. At the end of treatment, 94 patients (80%) had a complete response (CR) and 24 (20%) had a partial response. After a median follow-up of 54 months, 18 patients (15%) had relapsed. The variables that had a negative impact on progression-free survival (PFS) at univariate analysis were advanced stage, bone marrow involvement, International Prognostic Score (IPS) ⱖ 3, positive interim positron emission tomography (int-PET), NK cells ⬍ 200/μL, CD19 cells ⬍ 85/μL, CD3/CD19 ratio ⱖ 13 and CD4/CD19 ratio ⱖ 10. At multivariate analysis, advanced stage, positive int-PET and CD4/CD19 ratio ⱖ 10 were independent prognostic factors of PFS. New biological markers could be predictive of the response to treatment and survival in cHL. A CD4/CD19 ratio ⱖ 10 seems to be associated with a worse outcome. Keywords: Lymphocytes, Hodgkin lymphoma, prognosis
Introduction The characteristic morphological appearance of classical Hodgkin lymphoma (cHL) is a minority of neoplastic Hodgkin and Reed–Sternberg (HRS) cells surrounded by a vast majority of reactive infiltrating cells. The infiltrating cells are T cells, B cells, eosinophils, neutrophils, plasma cells, histiocytes and fibroblasts [1].
Materials and methods Patients This retrospective study was focused on 118 patients with newly diagnosed cHL treated at Bari University Hospital (Italy) between 2006 and 2010. Histologic diagnoses were
Correspondence: Francesco Gaudio, Department of Emergency and Organ Transplantation, Ematologia con Trapianto, University of Bari Medical School, Piazza G. Cesare 11, Bari 70124, Italy. Tel ⫹ 39-0805594145. Fax ⫹ 39-0805593454. E-mail: [email protected] Received 24 June 2013; accepted 6 October 2013
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CD4/CD19 cell ratio in Hodgkin lymphoma 1597 established according to the World Health Organization (WHO) classification [19]. Patients diagnosed with nodular lymphocyte predominant Hodgkin lymphoma, treated only with radiation or palliative care, or positive for human immunodeficiency virus (HIV) were excluded. No patient refused authorization to use their medical records for research. No patients were lost to follow-up. Approval for the observational study was obtained from the Institutional Review Board and the research was conducted in accordance with the Declaration of Helsinki. Clinical characteristics at diagnosis are listed in Table I; 54 (46%) were male and 64 (54%) female. Median age at diagnosis was 33 years (range 15–82), and 71 patients (60%) had an advanced stage (IIB–IV), three (3%) had bone marrow involvement, 54 (46%) had bulky disease, and 55 (47%) presented B symptoms, 30 (25%) spleen involvement and 31 (26%) extranodal disease. The histology was nodular sclerosis in 100 (85%) and mixed cellularity in 18 (15%). The treatment policy differed during the study period according to the Ann Arbor clinical stage. All patients were treated with the ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) regimen; 51 patients (42%) received involved field radiotherapy, 34 with early stage and 17 with advanced stage disease. Median follow-up was 54 months. Two patients died of causes not related to lymphoma and six patients died secondary to lymphoma progression.
Staging and routine laboratory evaluations All patients were clinically staged according to the Ann Arbor system, with history, complete physical examination, blood counts, biochemical profile, chest X-radiography, computed tomography of the chest, abdomen and pelvis, and unilateral bone marrow biopsy. Hemoglobin
Table I. Baseline patient characteristics at diagnosis. n Age, years, median (range) Gender Male Female B symptoms Advanced stage Histology Nodular sclerosis Mixed cellularity Bulky disease Bone marrow involvement Spleen involvement Extranodal disease Radiotherapy IPS risk factors Age ⬎ 45 Albumin ⬍ 4 g/dL ALC ⬍ 600/μL Hemoglobin ⱕ 10.5 g/dL WBC ⬎ 15 ⫻ 109/L Stage IV IPS ⱖ 3 Int-PET Positive
%
118 33 (15–82) 54 64 55 71
46 54 47 60
100 18 54 3 30 31 51
85 15 46 3 25 26 43
41 55 11 14 12 25 30
35 47 9 12 10 21 25
15
13
IPS, International Prognostic Score; ALC, absolute lymphocyte count; WBC, white blood cell count; Int-PET, interim positron emission tomography.
concentration, white blood cell count and differential, erythrocyte sedimentation rate (ESR), serum albumin and serum lactate dehydrogenase (LDH) levels were measured using standard assays.
Flow cytometry methods Samples were processed and analyzed by flow cytometry. A panel of antibodies against T (CD3⫹, CD4⫹, CD8⫹), B (CD19⫹) and NK cells (CD3⫺/CD56⫹) was used for each sample. Fluorescein isothiocyanate- and phycoerythrinconjugated monoclonal antibodies were used. Cells were incubated with antibody for 30 min at 4°C, washed with phosphate-buffered saline, and resuspended in 1 mL of 1% paraformaldehyde in phosphate-buffered saline for flow cytometry. A minimum of 10 000 cells was analyzed on each sample tube. An isotype control was used to gate lymphocytes by size and to set the negative fluorescence markers.
Response evaluation Tumor responses were assessed at the end of treatment and were classified as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD) according to the International Workshop Criteria [20].
Statistical analysis Definitions of response criteria and progression-free survival (PFS) were based on the guidelines from the International Harmonization Project for lymphoma [20]. PFS was defined as the time from cHL diagnosis to the time of progression, relapse from complete response, death as a result of any cause or last follow-up. The estimation of actuarial PFS was performed by the Kaplan–Meier method. The identification of prognostic factors at univariate analysis was based on the log-rank test. Independent prognostic factors were identified using a Cox’s proportional hazards model. χ2 tests were used to determine relationships between categorical variables. All p-values represented were two-sided and statistical significance was set at p ⬍ 0.05. The choice of the best cut-off value for the continuous variables, to assess survival, was based on their utility as a marker for the clinically relevant binary outcome of response/no response to first-line chemotherapy using the receiver operating characteristic curve (ROC) and area under the curve (AUC).
Results During the study period, 118 newly diagnosed cases of cHL were treated with the ABVD regimen. After two cycles of chemotherapy, all patients underwent interim positron emission tomography (int-PET) scanning. Clinical characteristics are shown in Table I. Radiotherapy was administered in 51 patients (43%), 34/47 (72%) with early stage and 17/71 (23%) with advanced stage disease. At the end of treatment, 94 patients (80%) were in CR, and 24 patients (20%) in PR; 18 patients (15%) relapsed after a median follow-up of 54 months (range: 12–62 months). PFS at 60 months was 70%.
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Cut-off values for NK cells, CD19 cells, CD3/CD19 ratio, CD4/CD19 ratio NK cells ⱖ 200 cells/μL had an AUC of 0.61 with a sensitivity of 70% and specificity of 54% [Figure 1(a)]. CD19 ⱖ 85 cells/μL had an AUC of 0.63 with a sensitivity of 63% and specificity of 48% [Figure 1(b)]. A CD3/CD19 ratio ⱖ 13 had an AUC of 0.62 with a sensitivity of 63% and a specificity of 45% [Figure 1(c)]. A CD4/CD19 ratio ⱖ 10 had an AUC of 0.60 with a sensitivity of 58% and a specificity of 22% [Figure 1(d)].
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Response to treatment and outcome: prognostic analysis The variables that were significantly negatively correlated at univariate analysis (Table II) with response to treatment were advanced stage (p ⫽ 0.031), positive int-PET (p ⬍ 0.001), bone marrow involvement (p ⫽ 0.043), WBC ⬎ 15 ⫻ 103/L (p ⫽ 0.021), NK cells ⬍ 200/μL (p ⫽ 0.049), CD19 ⬍ 85/μL (p ⫽ 0.039), CD3/CD19 ratio ⱖ 13 (p ⫽ 0.007) and CD4/CD19 ratio ⱖ 10 (p ⫽ 0.003).
Survival Patients with CD19 cells ⱖ 85/μL, NK cells ⱖ 200/μL, CD3/ CD19 ratio ⬍ 13 and CD4/CD19 ratio ⬍ 10 experienced superior PFS compared with patients with CD19 cells ⬍ 85/μL (30-month PFS rates of 76% vs. 58%; p ⫽ 0.04 [Figure 2(a)]), NK cells ⬍ 200/μL (30-month PFS rates of 78% vs. 56%; p ⫽ 0.019 [Figure 2(b)]), CD3/CD19 ratio ⱖ 13 (30-month PFS rates of 76% vs. 58%; p ⫽ 0.003 [Figure 2(c)]) and CD4/CD19 ratio ⱖ 10 (60-month PFS rates of 76% vs. 48%; p ⫽ 0.002 [Figure 2(d)]), respectively. The variables that had a negative impact on PFS at univariate analysis were advanced stage (p ⫽ 0.003), bone marrow involvement (p ⫽ 0.001), IPS score ⱖ 3 (p ⫽ 0.032), positive int-PET (p ⬍ 0.001), NK cells ⬍ 200/μL (p ⫽ 0.019), CD19 ⬍ 85/μL (p ⫽ 0.043), CD3/CD19 ratio ⱖ 13 (p ⫽ 0.036) and CD4/CD19 ratio ⱖ 10 (p ⫽ 0.002). At multivariate analysis, advanced stage (p ⫽ 0.017), positive
Figure 1. Receiver operating characteristic curve (ROC) and area under the curve (AUC) for natural killer cells (a), CD19 cells (b), CD3/CD19 ratio (c), CD4/CD10 ratio (d).
CD4/CD19 cell ratio in Hodgkin lymphoma 1599 Table II. Response rate. Patient characteristics Advanced vs. early stage Int-PET positive vs. negative Bone marrow involvement vs. not WBC ⱖ 15 vs. ⬍ 15 ⫻ 103/μL NK cells ⬍ 200 vs. ⱖ 200/μL CD19 ⬍ 85 vs. ⱖ 85/μL CD3/CD19 ⱖ 13 vs. ⬍ 13 CD4/CD19 ⱖ 10 vs. ⬍ 10
Discussion Response rate (%)
p-Value
65 vs. 83 50 vs. 93 33 vs. 81 59 vs. 83 70 vs. 85 71 vs. 86 52 vs. 88 54 vs. 80
0.031 ⬍ 0.001 0.043 0.021 0.049 0.039 0.007 0.003
Int-PET, interim positron emission tomography; WBC, white blood cell count; NK, natural killer.
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int-PET (p ⬍ 0.001) and CD4/CD19 ⱖ 10 (p ⫽ 0.008) were independent prognostic factors of PFS (Table III).
CD4/CD19 ratio and int-PET stratified groups Patients were stratified into four groups: group 1 included patients with a negative int-PET and CD4/CD19 ratio ⬍ 10; group 2 included positive int-PET and CD4/CD19 ratio ⬍ 10; group 3 included negative int-PET and CD4/CD19 ratio ⱖ 10; and group 4 included positive int-PET and CD4/CD19 ratio ⱖ 10. The 30-month PFS rates (Figure 3) for each group were: 76% (group 1); 10% (group 2); 69% (group 3); and 50% (group 4), p ⬍ 0.0001.
cHL is curable by chemotherapy, combined modality therapy (chemotherapy plus radiotherapy) or radiotherapy alone. The ABVD regimen is considered standard care, producing a high rate of durable complete responses [21–23]. However, even after this treatment, 5–10% of early and 20–40% of advanced stage patients either progress during treatment or relapse following an initial response [10,12,23]. Pretreatment identification of patients with a high likelihood of failing to benefit from standard treatment is of paramount importance. Several investigators have attempted to identify such patients by formulating prognostic models based on clinical and routine laboratory parameters [13,14]. None of the described models, however, has proven capable of selecting a sizeable group of patients with a sufficiently low PFS to necessitate their inclusion in protocols using first-line intensified treatment. In addition, the clinical variables included in these models do not provide insight into the biology of cHL or guidance for its rational treatment [13,14]. Some markers based on the presumed pathogenesis of cHL have been reported to have a prognostic value for patients with cHL, but there is no consensus on biologic markers that add prognostic value to the clinical parameters [24–32].
Figure 2. Progression-free survival based on CD19 cells (a), natural killer cells (b), CD3/CD19 ratio (c), CD4/CD19 ratio (d).
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F. Gaudio et al. Table III. Univariate and multivariate analysis for progression-free survival. Univariate analysis
Multivariate analysis
Patient characteristics
HR
95% CI
p-Value
HR
95% CI
p-Value
Advanced stage Bone marrow involvement IPS ⱖ 3 Int-PET positive NK cells ⬍ 200/μL CD19 ⬍ 85/μL CD3/CD19 ratio ⱖ 13 CD4/CD19 ratio ⱖ 10
3.3 2.5 1.8 3.5 1.5 2.2 2.8 3.8
1.5–6.3 1.9–5.8 0.6–2.3 0.4–2.6 0.5–4.2 1.7–5.4 1.4–5.9 1.8–7.5
0.003 0.001 0.032 ⬍ 0.001 0.019 0.043 0.003 0.002
1.5 — — 3.2 — — — 2.3
1.4–5 — — 1.3–5.1 — — — 1.5–4.8
0.017 — — ⬍ 0.001 — — — 0.008
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IPS, International Prognostic Score; Int-PET, interim positron emission tomography; NK, natural killer.
In patients with advanced-stage HL, the early response after two courses of ABVD chemotherapy, when evaluated with int-PET scan, shows important prognostic significance [33–34]. It is considered to be similar to a test of chemosensitivity, and overrides all conventional prognostic factors, including IPS score. Advanced-stage patients after two courses of ABVD are projected to achieve a 2-year PFS of ⬎ 90%, while a 2-year PFS of ⬍ 10% is expected in int-PET⫹ patients. cHL is characterized by a minority of tumor cells surrounded by a large amount of non-neoplastic cells [1,2]. A better knowledge of the biology of the disease, particularly the interaction between HRS cells and the surrounding cells, is essential in order to improve its diagnosis and treatment. Although the composition of the background infiltrate is heterogeneous, most cells are CD4 ⫹ lymphocytes. Recent studies of HL have shown that infiltrating lymphocytes in cHL lymph nodes are predominantly CD4⫹, CD25 ⫹ regulatory T cells, which have suppressive functions and induce a profoundly immunosuppressive environment [35–37]. In this retrospective study of 118 newly diagnosed cases of cHL, we have shown that there is important prognostic information to be gained from evaluating peripheral blood
B, T and NK cell count at diagnosis and, in particular, the CD3/CD19 and CD4/CD19 ratios. Using the χ2 test, we compared the response rate in various groups of patients defined by known prognostic factors, the peripheral blood B, T and NK cell count, CD3/CD19 and CD4/CD19 ratios. We found that in univariate analysis, advanced stage, positive int-PET, bone marrow involvement, WBC ⬎ 15 ⫻ 103/L, NK cells ⬍ 200/μL, CD19 ⬍ 85/μL, CD3/ CD19 ratio ⱖ 13 and CD4/CD19 ratio ⱖ 10 negatively influenced the response to treatment (Table III). At multivariate analysis, int-PET positivity was the strongest prognostic factor for PFS (p ⬍ 0.001), followed by CD4/CD19 ⱖ 10 (p ⫽ 0.008) and advanced stage (p ⫽ 0.017). We confirm earlier findings of a high positive predictive value of fluorodeoxyglucose (FDG)-PET for HL [32,17]. We categorized the patients into four groups based on positive versus negative int-PET and CD4/CD19 ratio ⬍ 10 versus CD4/CD19 ratio ⱖ 10. Patients in the group with negative int-PET and CD4/CD19 ratio ⬍ 10 experienced superior PFS in comparison with any other group. Early interim FDG-PET, CD4/CD19 ratio and advanced stage identify a group with a marker of high tumor burden, low host immunity and/or immunosuppressive microenvironment, where a vast majority are destined to have short-term progression or relapse. This could be important in the selection of patients for early treatment intensification. To our knowledge, the present study is the first to show the important prognostic value of the peripheral blood B, T and NK cell count at diagnosis. This retrospective study provides evidence that new biological markers taking into account the surrounding microenvironment could be predictive of response to treatment and survival in patients with cHL. These results warrant further studies in a larger group of patients with cHL. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
References Figure 3. Superior progression-free survival observed in patients in group 1 compared with other groups. Group 1 ⫽ negative interim positron emission tomography (PET) scan and CD4/CD19 cell ratio ⬍ 10; group 2 ⫽ positive interim PET scan and CD4/CD19 cell ratio ⬍ 10; group 3 ⫽ negative interim PET scan and CD4/CD19 cell ratio ⱖ 10; and group 4 ⫽ positive interim PET scan and CD4/CD19 cell ratio ⱖ 10.
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CD4/CD19 cell ratio in Hodgkin lymphoma 1601 the characteristic T-cell infiltrate in Hodgkin’s lymphoma. Am J Pathol 1999;154:1685–1691. [5] Teruya-Feldstein J, Tosato G, Jaffe ES. The role of chemokines in Hodgkin’s disease. Leuk Lymphoma 2000;38:363–371. [6] Peh SC, Kim LH, Poppema S. TARC, a CC chemokine, is frequently expressed in classic Hodgkin’s lymphoma but not in NLP Hodgkin’s lymphoma, T-cell-rich B-cell lymphoma, and most cases of anaplastic large cell lymphoma. Am J Surg Pathol 2001;25:925–929. [7] Hedvat CV, Jaffe ES, Qin J, et al. Macrophage-derived chemokine expression in classical Hodgkin’s lymphoma: application of tissue microarrays. Mod Pathol 2001;14:1270–1276. [8] Gruss HJ, Pinto A , Duyster J, et al. Hodgkin’s disease: a tumor with disturbed immunological pathways. Immunol Today 1997;18:156–163. [9] Atayar C, Poppema S, Visser L, et al. Cytokine gene expression profile distinguishes CD4⫹/CD57 ⫹ T cells of the nodular lymphocyte predominance type of Hodgkin’s lymphoma from their tonsillar counterparts. J Pathol 2006;208:423–430. [10] Diehl V, Stein H, Hummel M, et al. Hodgkin’s lymphoma: biology and treatment strategies for primary refractory and relapsed disease. Hematology Am Soc Hematol Educ Program 2003:225–247. [11] Hahn T, Benekli M, Wong C, et al. A prognostic model for prolonged event-free survival after autologous or allogeneic blood or marrow transplantation for relapsed and refractory Hodgkin’s disease. Bone Marrow Transplant 2005;6:557–566. [12] Josting A , Rueffer U, Franfklin J, et al. Prognostic factors and treatment outcome in primary progressive Hodgkin lymphoma: a report from the German Hodgkin Lymphoma Study Group. Blood 2000;96:1280–1286. [13] Hasenclever D, Diehl V. A prognostic score for advanced Hodgkin’s disease: International Prognostic Factors Project on advanced Hodgkin’s disease. N Engl J Med 1998;339:1506–1514. [14] Bierman PJ, Lynch JC, Bociek RG, et al. The International Prognostic Factors Project score for advanced Hodgkin’s disease is useful for predicting outcome of autologous hematopoietic stem cell transplantation. Ann Oncol 2002;9:1370–1377. [15] Porrata LF, Ristow KM, Habermann TM, et al. Absolute lymphocyte/monocyte ratio at diagnosis and interim positronemission tomography predict survival in classical Hodgkin lymphoma. J Cancer Ther 2013;4:452–459. [16] Schreck S, Friebel D, Buettner M, et al. Prognostic impact of tumour-infiltrating Th2 and regulatory T cells in classical Hodgkin lymphoma. Hematol Oncol 2009;27:31–39. [17] Alvaro-Naranjo T, Lejeune M, Salvado-Usach MT, et al. Tumorinfiltrating cells as a prognostic factor in Hodgkin’s lymphoma: a quantitative tissue microarray study in a large retrospective cohort of 267 patients. Leuk Lymphoma 2005;46:1581–1591. [18] 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. [19] Swerdlow SH, Campo E, Harris NL, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon: IARC Press ; 2008. [20] Cheson BD, Pfistner B, Juweid ME, et al. Revised response criteria for malignant lymphoma. J Clin Oncol 2007;25:579–586. [21] Brusamolino E, Bacigalupo A , Barosi G, et al. Classical Hodgkin’s lymphoma in adults: guidelines of the Italian Society of Hematology,
the Italian Society of Experimental Hematology, and the Italian Group for Bone Marrow Transplantation on initial work-up, management, and follow-up. Haematologica 2009;94:550–565. [22] Santoro A , Bonadonna G, Bonfante V, et al. Alternating drug combinations in the treatment of advanced Hodgkin’s disease. N Engl J Med 1982;306:770–775. [23] 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. [24] Colonna P, Jais JP, Desablens B, et al. Mediastinal tumor size and response to chemotherapy are the only prognostic factors in supradiaphragmatic Hodgkin’s disease treated by ABVD plus radiotherapy: ten-year results of the Paris-Ouest-France 81/12 trial, including 262 patients. J Clin Oncol 1996;14:1928–1935. [25] Sup SJ, Alemany CA , Pohlman B, et al. Expression of bcl2 in classical Hodgkin’s lymphoma: an independent predictor of poor outcome. J Clin Oncol 2005;23:3773–3779. [26] Nadali G, Tavecchia L, Zanolin E, et al. Serum level of the soluble form of the CD30 molecule identifies patients with Hodgkin’s disease at high risk of unfavorable outcome. Blood 1998;91: 3011–3016. [27] Reynolds GM, Billingham LJ, Gray LJ, et al. Interleukin 6 expression by Hodgkin/Reed-Sternberg cells is associated with the presence of B symptoms and failure to achieve complete remission in patients with advanced Hodgkin’s disease. Br J Haematol 2002;118: 195–201. [28] Glimelius I, Edstrom A , Amini RM, et al. IL-9 expression contributes to the cellular composition in Hodgkin lymphoma. Eur J Haematol 2006;4:278–283. [29] Spector N, Milito CB, Biasoli I, et al. The prognostic value of the expression of bcl2, p53 and LMP1 in patients with Hodgkin’s lymphoma. Leuk Lymphoma 2005;9:1301–1306. [30] Tzankov A , Krugmann J, Fend F, et al. Prognostic significance of CD20 expression in classical Hodgkin lymhoma: a clinicopathological study of 119 cases. Clin Cancer Res 2003;9:1381–1386. [31] Portlock CS, Donnelly GB, Qin J, et al. Adverse prognostic significance of CD20 positive Reed-Sternberg cells in classical Hodgkin’s disease. Br J Haematol 2004;125:701–708. [32] Molin D, Edstrom A , Glimelius I, et al. Mast cell infiltration correlates with poor prognosis in Hodgkin’s lymphoma. Br J Haematol 2002;119:122–124. [33] 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. [34] Marshall N, Christie L, Munro L, et al. Immunosuppressive regulatory T-cells are abundant in the reactive lymphocytes of Hodgkin lymphoma. Blood 2004;103:1755–1762. [35] Hutchings M, Loft A , Hansen M, et al. FDG-PET after two cycles of chemotherapy predicts treatment failure and progression free survival in Hodgkin Lymphoma. Blood 2006;107:52–59. [36] Zinzani PL, Tani M, Fanti S, et al. Early positron emission tomography (PET) restaging: a predictive final response in Hodgkin lymphoma patients. Ann Oncol 2006;17:1296–1300. [37] Gallamini A , Hutchings M, Rigacci L, et al. Early interim 2-[18F]-fluoro-2-deoxy-d-glucose positron emission tomography is prognostically superior to International Prognostic Score in advancedstage Hodgkin’s lymphoma: a report from joint Italian-Danish study. J Clin Oncol 2007;25:3746–3752.
ORIGINAL ARTICLE: CLINICAL
Peripheral blood CD4/CD19 cell ratio is an independent prognostic factor in classical Hodgkin lymphoma Francesco Gaudio, Tommasina Perrone, Anna Mestice, Paola Curci, Annamaria Giordano, Mario Delia, Domenico Pastore & Giorgina Specchia
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Hematology Section, Department of Emergency and Organ Transplantation, University of Bari Medical School, Bari, Italy
Despite numerous studies regarding their immunophenotypes and secreted cytokines and chemokines, the pathogenic roles of these Hodgkin lymphoma-infiltrating lymphocytes remain poorly understood [2,3]. Several lines of evidence suggest that the reactive cells are attracted by chemokines produced by the HRS cells [4–7]. In return, these infiltrating cells produce cytokines that may contribute to the proliferation and survival of the HRS cells [8,9]. Even though cHL is curable, approximately 30% of all patients relapse and eventually die of progressive disease or complications of therapy [10–12]. Several clinical and laboratory features have been used to predict survival so as to adjust therapy according to the predicted risk of relapse [13,14]. Absolute lymphocyte/monocyte ratio at diagnosis and tumor-infiltrating lymphocytes have been implicated in the prognosis of cHL [15–17]. Recent gene-expression profiling studies in cHL have demonstrated that tumor-infiltrating macrophage cells also predict clinical outcomes [18]. Furthermore, a low lymphocyte count, defined by an International Prognostic Score (IPS) as ⬍ 600 cells/μL or ⬍ 8% of the white blood cell count (WBC), is a negative prognostic factor for survival in cHL [13,14]. However, most models, including the IPS, fail to identify a sizeable fraction of patients whose chance of cure is less than 50%. Therefore, we decided to determine whether features directly related to the biology of cHL can predict clinical outcome. We studied the role of the peripheral blood B, T and natural killer (NK) cell count at diagnosis as a simple biomarker combining an estimate of host immune homeostasis and the tumor microenvironment in clinical outcome in patients with cHL.
Abstract Classical Hodgkin lymphoma (cHL) is characterized by the presence of tumoral cells in a rich background of T and B cells, macrophages and other inflammatory cells. The contribution of these non-tumoral cells to the pathogenesis of HL is still poorly understood. In our study we evaluated the prognostic significance of peripheral blood B, T and natural killer (NK) cells at diagnosis in 118 immunocompetent patients with cHL treated at our institution between January 2006 and December 2010. Fifty-four (46%) were male and 64 (54%) female. Median age at diagnosis was 33 years (range 15–82), and 71 patients (60%) presented an advanced stage (IIB–IV), 54 (46%) had bulky disease and 55 (47%) presented B symptoms. At the end of treatment, 94 patients (80%) had a complete response (CR) and 24 (20%) had a partial response. After a median follow-up of 54 months, 18 patients (15%) had relapsed. The variables that had a negative impact on progression-free survival (PFS) at univariate analysis were advanced stage, bone marrow involvement, International Prognostic Score (IPS) ⱖ 3, positive interim positron emission tomography (int-PET), NK cells ⬍ 200/μL, CD19 cells ⬍ 85/μL, CD3/CD19 ratio ⱖ 13 and CD4/CD19 ratio ⱖ 10. At multivariate analysis, advanced stage, positive int-PET and CD4/CD19 ratio ⱖ 10 were independent prognostic factors of PFS. New biological markers could be predictive of the response to treatment and survival in cHL. A CD4/CD19 ratio ⱖ 10 seems to be associated with a worse outcome. Keywords: Lymphocytes, Hodgkin lymphoma, prognosis
Introduction The characteristic morphological appearance of classical Hodgkin lymphoma (cHL) is a minority of neoplastic Hodgkin and Reed–Sternberg (HRS) cells surrounded by a vast majority of reactive infiltrating cells. The infiltrating cells are T cells, B cells, eosinophils, neutrophils, plasma cells, histiocytes and fibroblasts [1].
Materials and methods Patients This retrospective study was focused on 118 patients with newly diagnosed cHL treated at Bari University Hospital (Italy) between 2006 and 2010. Histologic diagnoses were
Correspondence: Francesco Gaudio, Department of Emergency and Organ Transplantation, Ematologia con Trapianto, University of Bari Medical School, Piazza G. Cesare 11, Bari 70124, Italy. Tel ⫹ 39-0805594145. Fax ⫹ 39-0805593454. E-mail: [email protected] Received 24 June 2013; accepted 6 October 2013
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CD4/CD19 cell ratio in Hodgkin lymphoma 1597 established according to the World Health Organization (WHO) classification [19]. Patients diagnosed with nodular lymphocyte predominant Hodgkin lymphoma, treated only with radiation or palliative care, or positive for human immunodeficiency virus (HIV) were excluded. No patient refused authorization to use their medical records for research. No patients were lost to follow-up. Approval for the observational study was obtained from the Institutional Review Board and the research was conducted in accordance with the Declaration of Helsinki. Clinical characteristics at diagnosis are listed in Table I; 54 (46%) were male and 64 (54%) female. Median age at diagnosis was 33 years (range 15–82), and 71 patients (60%) had an advanced stage (IIB–IV), three (3%) had bone marrow involvement, 54 (46%) had bulky disease, and 55 (47%) presented B symptoms, 30 (25%) spleen involvement and 31 (26%) extranodal disease. The histology was nodular sclerosis in 100 (85%) and mixed cellularity in 18 (15%). The treatment policy differed during the study period according to the Ann Arbor clinical stage. All patients were treated with the ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) regimen; 51 patients (42%) received involved field radiotherapy, 34 with early stage and 17 with advanced stage disease. Median follow-up was 54 months. Two patients died of causes not related to lymphoma and six patients died secondary to lymphoma progression.
Staging and routine laboratory evaluations All patients were clinically staged according to the Ann Arbor system, with history, complete physical examination, blood counts, biochemical profile, chest X-radiography, computed tomography of the chest, abdomen and pelvis, and unilateral bone marrow biopsy. Hemoglobin
Table I. Baseline patient characteristics at diagnosis. n Age, years, median (range) Gender Male Female B symptoms Advanced stage Histology Nodular sclerosis Mixed cellularity Bulky disease Bone marrow involvement Spleen involvement Extranodal disease Radiotherapy IPS risk factors Age ⬎ 45 Albumin ⬍ 4 g/dL ALC ⬍ 600/μL Hemoglobin ⱕ 10.5 g/dL WBC ⬎ 15 ⫻ 109/L Stage IV IPS ⱖ 3 Int-PET Positive
%
118 33 (15–82) 54 64 55 71
46 54 47 60
100 18 54 3 30 31 51
85 15 46 3 25 26 43
41 55 11 14 12 25 30
35 47 9 12 10 21 25
15
13
IPS, International Prognostic Score; ALC, absolute lymphocyte count; WBC, white blood cell count; Int-PET, interim positron emission tomography.
concentration, white blood cell count and differential, erythrocyte sedimentation rate (ESR), serum albumin and serum lactate dehydrogenase (LDH) levels were measured using standard assays.
Flow cytometry methods Samples were processed and analyzed by flow cytometry. A panel of antibodies against T (CD3⫹, CD4⫹, CD8⫹), B (CD19⫹) and NK cells (CD3⫺/CD56⫹) was used for each sample. Fluorescein isothiocyanate- and phycoerythrinconjugated monoclonal antibodies were used. Cells were incubated with antibody for 30 min at 4°C, washed with phosphate-buffered saline, and resuspended in 1 mL of 1% paraformaldehyde in phosphate-buffered saline for flow cytometry. A minimum of 10 000 cells was analyzed on each sample tube. An isotype control was used to gate lymphocytes by size and to set the negative fluorescence markers.
Response evaluation Tumor responses were assessed at the end of treatment and were classified as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD) according to the International Workshop Criteria [20].
Statistical analysis Definitions of response criteria and progression-free survival (PFS) were based on the guidelines from the International Harmonization Project for lymphoma [20]. PFS was defined as the time from cHL diagnosis to the time of progression, relapse from complete response, death as a result of any cause or last follow-up. The estimation of actuarial PFS was performed by the Kaplan–Meier method. The identification of prognostic factors at univariate analysis was based on the log-rank test. Independent prognostic factors were identified using a Cox’s proportional hazards model. χ2 tests were used to determine relationships between categorical variables. All p-values represented were two-sided and statistical significance was set at p ⬍ 0.05. The choice of the best cut-off value for the continuous variables, to assess survival, was based on their utility as a marker for the clinically relevant binary outcome of response/no response to first-line chemotherapy using the receiver operating characteristic curve (ROC) and area under the curve (AUC).
Results During the study period, 118 newly diagnosed cases of cHL were treated with the ABVD regimen. After two cycles of chemotherapy, all patients underwent interim positron emission tomography (int-PET) scanning. Clinical characteristics are shown in Table I. Radiotherapy was administered in 51 patients (43%), 34/47 (72%) with early stage and 17/71 (23%) with advanced stage disease. At the end of treatment, 94 patients (80%) were in CR, and 24 patients (20%) in PR; 18 patients (15%) relapsed after a median follow-up of 54 months (range: 12–62 months). PFS at 60 months was 70%.
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Cut-off values for NK cells, CD19 cells, CD3/CD19 ratio, CD4/CD19 ratio NK cells ⱖ 200 cells/μL had an AUC of 0.61 with a sensitivity of 70% and specificity of 54% [Figure 1(a)]. CD19 ⱖ 85 cells/μL had an AUC of 0.63 with a sensitivity of 63% and specificity of 48% [Figure 1(b)]. A CD3/CD19 ratio ⱖ 13 had an AUC of 0.62 with a sensitivity of 63% and a specificity of 45% [Figure 1(c)]. A CD4/CD19 ratio ⱖ 10 had an AUC of 0.60 with a sensitivity of 58% and a specificity of 22% [Figure 1(d)].
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Response to treatment and outcome: prognostic analysis The variables that were significantly negatively correlated at univariate analysis (Table II) with response to treatment were advanced stage (p ⫽ 0.031), positive int-PET (p ⬍ 0.001), bone marrow involvement (p ⫽ 0.043), WBC ⬎ 15 ⫻ 103/L (p ⫽ 0.021), NK cells ⬍ 200/μL (p ⫽ 0.049), CD19 ⬍ 85/μL (p ⫽ 0.039), CD3/CD19 ratio ⱖ 13 (p ⫽ 0.007) and CD4/CD19 ratio ⱖ 10 (p ⫽ 0.003).
Survival Patients with CD19 cells ⱖ 85/μL, NK cells ⱖ 200/μL, CD3/ CD19 ratio ⬍ 13 and CD4/CD19 ratio ⬍ 10 experienced superior PFS compared with patients with CD19 cells ⬍ 85/μL (30-month PFS rates of 76% vs. 58%; p ⫽ 0.04 [Figure 2(a)]), NK cells ⬍ 200/μL (30-month PFS rates of 78% vs. 56%; p ⫽ 0.019 [Figure 2(b)]), CD3/CD19 ratio ⱖ 13 (30-month PFS rates of 76% vs. 58%; p ⫽ 0.003 [Figure 2(c)]) and CD4/CD19 ratio ⱖ 10 (60-month PFS rates of 76% vs. 48%; p ⫽ 0.002 [Figure 2(d)]), respectively. The variables that had a negative impact on PFS at univariate analysis were advanced stage (p ⫽ 0.003), bone marrow involvement (p ⫽ 0.001), IPS score ⱖ 3 (p ⫽ 0.032), positive int-PET (p ⬍ 0.001), NK cells ⬍ 200/μL (p ⫽ 0.019), CD19 ⬍ 85/μL (p ⫽ 0.043), CD3/CD19 ratio ⱖ 13 (p ⫽ 0.036) and CD4/CD19 ratio ⱖ 10 (p ⫽ 0.002). At multivariate analysis, advanced stage (p ⫽ 0.017), positive
Figure 1. Receiver operating characteristic curve (ROC) and area under the curve (AUC) for natural killer cells (a), CD19 cells (b), CD3/CD19 ratio (c), CD4/CD10 ratio (d).
CD4/CD19 cell ratio in Hodgkin lymphoma 1599 Table II. Response rate. Patient characteristics Advanced vs. early stage Int-PET positive vs. negative Bone marrow involvement vs. not WBC ⱖ 15 vs. ⬍ 15 ⫻ 103/μL NK cells ⬍ 200 vs. ⱖ 200/μL CD19 ⬍ 85 vs. ⱖ 85/μL CD3/CD19 ⱖ 13 vs. ⬍ 13 CD4/CD19 ⱖ 10 vs. ⬍ 10
Discussion Response rate (%)
p-Value
65 vs. 83 50 vs. 93 33 vs. 81 59 vs. 83 70 vs. 85 71 vs. 86 52 vs. 88 54 vs. 80
0.031 ⬍ 0.001 0.043 0.021 0.049 0.039 0.007 0.003
Int-PET, interim positron emission tomography; WBC, white blood cell count; NK, natural killer.
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int-PET (p ⬍ 0.001) and CD4/CD19 ⱖ 10 (p ⫽ 0.008) were independent prognostic factors of PFS (Table III).
CD4/CD19 ratio and int-PET stratified groups Patients were stratified into four groups: group 1 included patients with a negative int-PET and CD4/CD19 ratio ⬍ 10; group 2 included positive int-PET and CD4/CD19 ratio ⬍ 10; group 3 included negative int-PET and CD4/CD19 ratio ⱖ 10; and group 4 included positive int-PET and CD4/CD19 ratio ⱖ 10. The 30-month PFS rates (Figure 3) for each group were: 76% (group 1); 10% (group 2); 69% (group 3); and 50% (group 4), p ⬍ 0.0001.
cHL is curable by chemotherapy, combined modality therapy (chemotherapy plus radiotherapy) or radiotherapy alone. The ABVD regimen is considered standard care, producing a high rate of durable complete responses [21–23]. However, even after this treatment, 5–10% of early and 20–40% of advanced stage patients either progress during treatment or relapse following an initial response [10,12,23]. Pretreatment identification of patients with a high likelihood of failing to benefit from standard treatment is of paramount importance. Several investigators have attempted to identify such patients by formulating prognostic models based on clinical and routine laboratory parameters [13,14]. None of the described models, however, has proven capable of selecting a sizeable group of patients with a sufficiently low PFS to necessitate their inclusion in protocols using first-line intensified treatment. In addition, the clinical variables included in these models do not provide insight into the biology of cHL or guidance for its rational treatment [13,14]. Some markers based on the presumed pathogenesis of cHL have been reported to have a prognostic value for patients with cHL, but there is no consensus on biologic markers that add prognostic value to the clinical parameters [24–32].
Figure 2. Progression-free survival based on CD19 cells (a), natural killer cells (b), CD3/CD19 ratio (c), CD4/CD19 ratio (d).
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F. Gaudio et al. Table III. Univariate and multivariate analysis for progression-free survival. Univariate analysis
Multivariate analysis
Patient characteristics
HR
95% CI
p-Value
HR
95% CI
p-Value
Advanced stage Bone marrow involvement IPS ⱖ 3 Int-PET positive NK cells ⬍ 200/μL CD19 ⬍ 85/μL CD3/CD19 ratio ⱖ 13 CD4/CD19 ratio ⱖ 10
3.3 2.5 1.8 3.5 1.5 2.2 2.8 3.8
1.5–6.3 1.9–5.8 0.6–2.3 0.4–2.6 0.5–4.2 1.7–5.4 1.4–5.9 1.8–7.5
0.003 0.001 0.032 ⬍ 0.001 0.019 0.043 0.003 0.002
1.5 — — 3.2 — — — 2.3
1.4–5 — — 1.3–5.1 — — — 1.5–4.8
0.017 — — ⬍ 0.001 — — — 0.008
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IPS, International Prognostic Score; Int-PET, interim positron emission tomography; NK, natural killer.
In patients with advanced-stage HL, the early response after two courses of ABVD chemotherapy, when evaluated with int-PET scan, shows important prognostic significance [33–34]. It is considered to be similar to a test of chemosensitivity, and overrides all conventional prognostic factors, including IPS score. Advanced-stage patients after two courses of ABVD are projected to achieve a 2-year PFS of ⬎ 90%, while a 2-year PFS of ⬍ 10% is expected in int-PET⫹ patients. cHL is characterized by a minority of tumor cells surrounded by a large amount of non-neoplastic cells [1,2]. A better knowledge of the biology of the disease, particularly the interaction between HRS cells and the surrounding cells, is essential in order to improve its diagnosis and treatment. Although the composition of the background infiltrate is heterogeneous, most cells are CD4 ⫹ lymphocytes. Recent studies of HL have shown that infiltrating lymphocytes in cHL lymph nodes are predominantly CD4⫹, CD25 ⫹ regulatory T cells, which have suppressive functions and induce a profoundly immunosuppressive environment [35–37]. In this retrospective study of 118 newly diagnosed cases of cHL, we have shown that there is important prognostic information to be gained from evaluating peripheral blood
B, T and NK cell count at diagnosis and, in particular, the CD3/CD19 and CD4/CD19 ratios. Using the χ2 test, we compared the response rate in various groups of patients defined by known prognostic factors, the peripheral blood B, T and NK cell count, CD3/CD19 and CD4/CD19 ratios. We found that in univariate analysis, advanced stage, positive int-PET, bone marrow involvement, WBC ⬎ 15 ⫻ 103/L, NK cells ⬍ 200/μL, CD19 ⬍ 85/μL, CD3/ CD19 ratio ⱖ 13 and CD4/CD19 ratio ⱖ 10 negatively influenced the response to treatment (Table III). At multivariate analysis, int-PET positivity was the strongest prognostic factor for PFS (p ⬍ 0.001), followed by CD4/CD19 ⱖ 10 (p ⫽ 0.008) and advanced stage (p ⫽ 0.017). We confirm earlier findings of a high positive predictive value of fluorodeoxyglucose (FDG)-PET for HL [32,17]. We categorized the patients into four groups based on positive versus negative int-PET and CD4/CD19 ratio ⬍ 10 versus CD4/CD19 ratio ⱖ 10. Patients in the group with negative int-PET and CD4/CD19 ratio ⬍ 10 experienced superior PFS in comparison with any other group. Early interim FDG-PET, CD4/CD19 ratio and advanced stage identify a group with a marker of high tumor burden, low host immunity and/or immunosuppressive microenvironment, where a vast majority are destined to have short-term progression or relapse. This could be important in the selection of patients for early treatment intensification. To our knowledge, the present study is the first to show the important prognostic value of the peripheral blood B, T and NK cell count at diagnosis. This retrospective study provides evidence that new biological markers taking into account the surrounding microenvironment could be predictive of response to treatment and survival in patients with cHL. These results warrant further studies in a larger group of patients with cHL. Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.
References Figure 3. Superior progression-free survival observed in patients in group 1 compared with other groups. Group 1 ⫽ negative interim positron emission tomography (PET) scan and CD4/CD19 cell ratio ⬍ 10; group 2 ⫽ positive interim PET scan and CD4/CD19 cell ratio ⬍ 10; group 3 ⫽ negative interim PET scan and CD4/CD19 cell ratio ⱖ 10; and group 4 ⫽ positive interim PET scan and CD4/CD19 cell ratio ⱖ 10.
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