Proportions of bone marrow lymphocyte subsets at diagnosis may predict survival in patients with newly diagnosed diffuse large B‐cell lymphoma independently of the international prognostic index
Authors: Luke D. Zurbriggen, Jacob D. Gundrum, Seema M. Policepatil, Sai Ravi K. Pingali, Gordon G. Zeng, Ronald S. Go
Journal: American Journal of Hematology (2011)

There is substantial evidence to support that the immune system plays a role in the outcome of lymphoma. For a long time, it is known that lymphoma can regress spontaneously, [1] and immunotherapy such as interferon and vaccine have some activity against the disease [2, 3]. More recently, several studies in diffuse large B-cell lymphoma (DLBCL) have shown the potential value of the assessment of peripheral blood absolute lymphocyte count (PBALC) as well as nonmalignant lymphocyte subsets in the blood and the lymph node obtained at diagnosis in predicting clinical outcome [4-12]. Finally, molecular profiling of DLBCL show that genes associated with lymph node signature (host response) are predictive of overall survival (OS) independent of the commonly used International Prognostic Index (IPI) [13]. These studies of the effect of immune system on DLBCL have focused primarily on the blood and lymph node but not the bone marrow. In our retrospective study, we showed that that a higher proportion of total T-cells in the bone marrow as measured by flow cytometry was predictive of a better OS and disease-specific survival (DSS) independent of IPI in patients with newly diagnosed DLBCL treated with anthracycline-based chemotherapy. In contrast to previous studies, we did not find PBALC to have predictive importance. As bone marrow aspiration and biopsy is generally performed as part of DLBCL staging, enumeration of bone marrow lymphocyte subsets by flow cytometry may provide additional prognostic information in patients with newly diagnosed DLBCL and deserves further investigation. Since the bone marrow is a major component of the host immune system and bone marrow biopsy is routinely a part of lymphoma staging work-up, we studied the prognostic significance of bone marrow lymphocyte subsets at diagnosis as determined by flow cytometry in patients with primary nodal DLBCL. Out of 168 DLBCL patients who were seen at our institution, 86 patients met the study criteria and were included. Reasons for exclusion for the other 82 patients were bone marrow biopsy not performed at diagnosis or it was suboptimal (32), suboptimal or no flow cytometry performed on the aspirate (22), palliative care only or received nonanthracycline-based chemotherapy (9), primary central nervous system lymphoma (8), primary cutaneous lymphoma (4), concomitant low grade lymphoma (3), Richter's transformation (3), and infection with human immunodeficiency virus (1). The reasons for not performing a bone marrow biopsy at the time of initial staging included stage IV disease as a result of other organ involvement, presence of major comorbidities, aggressive disease presentation resulting in death before staging could be completed, and treatment with palliative intent. The clinical features of and treatment received by all patients with primary nodal non-HIV-related DLBCL, including those who did not meet study criteria, are summarized in Table I. For those who met the study criteria, the median age at diagnosis was 65 years and most (55.8%) were males. They were predominantly low or intermediate risk by IPI (88.4%). All patients received either cyclophosphamide/doxorubicin/vincristine/prednisone/rituximab, CHOP + R, (72.1%) or CHOP/CHOP-like (27.9%) chemotherapy. Only 17.4% had bone marrow involvement by DLBCL. After a median follow-up of 45.5 months (range, 1.2–116.9), 32 patients (37.2%) have died, mostly (19) of lymphoma or treatment complications. Univariate analysis showed that lower IPI scores, and higher proportions of bone marrow total lymphocytes, B-cells, T-cells (total and subsets), and NK-cells, but not ALC in the blood, were associated with better OS and DSS (Table II). Treatment with CHOP + R was associated with a better DSS but not OS. When evaluated in a multivariate model (Table III), higher proportion of total T-cells and lower IPI scores remained significant independent predictors of better OS and DSS. OS according to proportions of CD3+ cells is shown in Fig. 1. Overall survival according to proportion of bone marrow CD3+ cells. CHOP, cyclophosphamide/doxorubicin/vincristine/prednisone; R, rituximab. It is now known that survival in patients with DLBCL is determined by both clinical and biologic factors. For the clinical factors, IPI score is the summation and is routinely used in practice [14]. Biologic factors pertain to features of the DLBCL of which the host response to the tumor is a key component [13]. Our study investigated the prognostic significance of the host immune system, using bone marrow lymphocyte proportion as a surrogate, in patients with DLBCL. We found that higher proportion of total lymphocytes in the bone marrow conferred better survival. This was also true of the various lymphocyte subsets. The most significant lymphocyte subset was total T-cells, as it was prognostically independent of the IPI score. However, ALC was not found to be significant. Taken together, this study shows that the status of host immunity in the bone marrow may play a role in the outcome of DLBCL. Our findings are supported by earlier studies that showed proportions of certain lymphocyte subsets in the tumor tissue or the blood may be important in the prognosis of patients with DLBCL. Ansell et al. studied the proportions of T-cells subsets and NK-cells in the lymph nodes by flow cytometry [8]. Increased number of CD4+ cells, but not total T- or NK-cells, predicted a better 5-year failure-free survival and OS. The focus of this study was the importance of tumor microenvironment rather than the systemic host immunity. In our study, the total T-cells in the bone marrow was stronger predictor of survival than T-cell subsets. Plonquet et al. showed that the lymphocyte subsets in the peripheral blood at diagnosis were quantitatively abnormal in the majority of patients. Patients with higher peripheral blood NK-cell count had a better response (complete and partial) to chemotherapy and longer event-free survival. However, the total T-cells and T-cell subsets were not significantly associated with clinical outcome. They did not provide data on the prognostic significance of ALC [11]. Finally, Muris et al. demonstrated that higher proportions of activated cytotoxic T-cells in lymph node biopsy, as determined by CD3 and granzyme B expression, were associated with failure to reach a complete remission as well as shorter progression-free survival and OS independent of the IPI score [10]. The discrepancies seen among these studies and ours with regards to the most clinically significant lymphocyte subset may be due to the potential differences in the natural lymphocyte composition of the various host immune compartments (blood, lymph node, and bone marrow) and the method used to enumerate the lymphocytes (immunohistochemistry or flow cytometry). While several studies have shown that high peripheral blood ALC before chemotherapy may also be predictive of a better outcome, this was not confirmed in our study [4-7]. This suggests that the bone marrow may be a better surrogate of host immunity compared to blood or that analysis of blood lymphocyte subsets may be more informative than total ALC. None of the published retrospective studies on ALC and DLBCL investigated lymphocyte subsets. This was because flow cytometric analyses of the peripheral blood were not routinely performed. Future studies comparing ALC and peripheral blood lymphocyte subsets may clarify this issue. Our study has several limitations. First, we excluded 42.3% of our patients with primary nodal non-HIV DLBCL. The predominant reason was the lack of a bone marrow biopsy performed at diagnosis. The latter was most frequently due to patients electing to choose palliative treatment because of serious comorbidities or personal reasons. As we pointed out earlier, the ineligible group were older and had a higher risk IPI score (Table I). Therefore, there was a selection bias towards a healthier group of DLBCL patients. However, the data are more reflective of patients who undergo CHOP ± R chemotherapy, since we excluded those who did not have anthracycline-based chemotherapy. Second, only 2/3 of the patients in our study were treated with CHOP + R. Nevertheless, based on our analyses, CD3% was prognostically more important than type of treatment received with respect to OS (Table II) and retained its prognostic significance even after controlling for treatment type with respect to DSS (Table III). Third, our sample size was relatively small. Therefore, our study is considered exploratory in nature. The results are hypothesis-generating rather than conclusive and will need to be validated by other groups. Finally, we did not apply statistical correction for multiple hypothesis testing. Although we appreciate the idea and understand the potential of concluding something significant when it fact it is insignificant when testing multiple variables, we believe that a tighter control over this error should be reserved for future definitive or controlled studies. However, if we apply a very conservative Bonferonni adjustment of 20 tests (one per univariate factor and each of OS or DS analysis), we end up with a significance level of 0.0025. At this level, for OS analysis, the total lymphocyte %, CD19%, CD16%, and IPI scores of 2–3 all become insignificant. If we keep this threshold for the multivariate modeling (and still force in IPI), CD3 remains significant. Therefore, our main conclusion that CD3% may add prognostic information over and above IPI does not change. More and more retrospective data are emerging on the predictive and prognostic significance of blood or tissue lymphocyte subsets in patients with DLBCL even in the era of rituximab-based therapy. Because of its potential to improve upon the IPI, analysis of lymphocyte subsets as a surrogate of immune function should be evaluated in prospective studies. The use of flow cytometry for quantification is particularly attractive due to its ease of use, amenability to standardization, and more routine incorporation into DLBCL clinical practice of diagnosis and staging. Patient population. This study was approved by the Gundersen Lutheran Institutional Review Board. DLBCL patients diagnosed and treated at our institution from 2000–2009 were identified through our cancer registry. We included in the analyses all patients who had primary nodal DLBCL and had flow cytometric analyses of their bone marrow aspirates at the time of diagnosis. Patients with human immunodeficiency virus infection, Richter transformation, concurrent diagnosis of low grade lymphoma, and DLBCL arising from the central nervous system and skin were excluded. None of the patients who were included in the study were treated with steroids before bone marrow biopsy was performed. We obtained the PBALC from blood counts performed before any steroids were given. For those patients with marrow involvement by DLBCL, we did not subtract the malignant lymphocytes in the calculation of lymphocyte subsets. Therefore, the proportion of CD19+ cells, represent both the clonal and nonclonal populations of B-cells in the marrow as a proportion of the total nucleated cells. Response criteria and measurement of survival. We assessed whether complete response was achieved or not for each patient using the guidelines from the non-Hodgkin lymphoma international workshop [15]. Both OS and DSS were measured from the time of initial diagnosis. Statistical analyses. Patient characteristics were summarized by using descriptive statistics. When comparing the eligible versus ineligible patients, we used chi-square tests for categorical variables and Wilcoxon rank-sum test for age. We examined the significance of the following variables in predicting OS and DSS: IPI score (0–1 vs. 4–5 and 2–3 vs. 4–5), chemotherapy received (CHOP + R vs. CHOP or CHOP-like), PBALC, and proportions of total lymphocyte and lymphocyte subsets [B-cells (CD19+), total T-cells (CD3+), helper-T cells (CD3+/CD4+), cytotoxic-T cells (CD3+/CD8+), NK-cells (CD16+/CD56+), and ratio of CD4+ to CD8+ cells] in the bone marrow. We used the method of Contal and O'Quigley to determine cut-off points for lymphocyte subsets. Implementation of the method was via a SAS macro created by Jay Mandrekar et al. available through the GNU General Public License. Univariate survival analyses were performed according to the method of Kaplan and Meier. We also studied the prognostic effect of the various clinical variables using univariate and multivariate Cox proportional hazards models, where we assessed hazard ratios and corresponding P values to compare survival outcomes. Significant univariate variables form the basis of the multivariate modeling where a stepwise elimination method was used after forcing IPI into the models. P values <0.05 were considered statistically significant. We would like to thank Austin Zeng for collecting the flow cytometry lymphocyte subset data. Luke D. Zurbriggen*, Jacob D. Gundrum*, Seema M. Policepatil , Sai Ravi K. Pingali , Gordon G. Zeng , Ronald S. Go§ ¶, * Department of Medical Research, Gundersen Lutheran Medical Foundation, La Crosse, WI, Department of Medical Education, Gundersen Lutheran Medical Foundation, La Crosse, WI, Department of Pathology, Gundersen Lutheran Health System, La Crosse, WI, § Section of Hematology, Department of Internal Medicine, Gundersen Lutheran Health System, La Crosse, WI, ¶ Center for Cancer and Blood Disorder, Gundersen Lutheran Health System, La Crosse, WI.