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The prognostic utility and the association of serum light chains (free and total) and absolute lymphocyte count in patients with newly diagnosed diffuse large B-cell lymphoma

Leukemia Research

Highlights

 

  • Elevated sFLC were more frequent than abnormal sTLC in patients with DLBCL.
  • Patients with elevated sFLC more frequently showed adverse clinical features.
  • Both elevated sFLC andκ/λratio were strongly associated with clinical outcomes.
  • Not abnormal sTLC, but elevated sFLC was found to be associated with low ALC.
  • sFLC and low ALC remain independent prognostic factors after adjusting for IPI.

Abstract

In this study, serum free and total light chains (sFLC/sTLC) were measured in 108 serum samples of therapy-naïve patients with DLBCL. Clinicopathologic data and survival outcomes were analyzed according to the results of sFLC/sTLC measurements. Moreover, the association of sFLC/sTLC with absolute monocyte count (AMC) and absolute lymphocyte count (ALC) was evaluated. Elevated sFLC and abnormalκ/λratio was present in 42.6% (51/108) and 4.6% (5/108) of patients, respectively. sTLC was successfully measured in 107 serum samples, abnormal sTLC and abnormalκ/λratio was found in 28.0% (30/107) and 26.2% (28/107) of patients, respectively. Patients with elevated sFLC more frequently displayed adverse clinical characteristics, including age (P = 0.001), B symptoms (P = 0.022), low ALC (P = 0.024) and hyperglobulinemia (P = 0.012). Patients with elevated sFLC had an inferior overall survival (OS) (P = 0.012) and tended to have shorter progression-free survival (PFS) (P = 0.061) compared to patients with normal sFLC. Abnormal sTLC or abnormal sTLC ratio showed no significant association with clinical outcomes, with exception of abnormal concurrentκandλ. Only association of sFLC and ALC with survival remained significant after adjusting for the International Prognostic Index (IPI). The measurement of sFLC and ALC at diagnosis might be useful for the prognostic stratification of patients and sTLC measurement was of little prognostic utility in DLBCL.

Keywords: Diffuse large B cell lymphoma, Free light chains, Total light chains, Absolute monocyte count, Absolute lymphocyte count, Prognostication.

1. Introduction

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma (NHL). The treatment of DLBCL has changed since 2002 when rituximab was added to CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy (R-CHOP)[1] and [2]. The move to an immunochemotherapy approach has meaningfully improved the progression-free survival (PFS) and overall survival (OS) rates in DLBCL[3] and [4]. The International Prognostic Index (IPI), which includes parameters such as age, stage, number of extranodal sites, performance status and serum lactate dehydrogenase (LDH) levels, is well-known scoring system for risk stratification of DLBCL patients at diagnosis. However, the prognostic discriminating power of IPI alone diminished in the rituximab or immunochemotherapy era[5] and [6]. Gene-expression profiling, immunohistochemistry-based detection of prognostic biomarkers and early interim analysis with positron emission tomography (PET) had been explored as predictors that may identify high-risk patients[7], [8], [9], [10], and [11]. Despite the active research in these areas, the identification of prognostic factors that are inexpensive, widely available and easily interpretable in the care of patients with DLBCL is still in the development stage at this time.

The serum free light chains (sFLC) are produced by monoclonal and/or polyclonal B-cell populations, including kappa (κ) and lambda (λ) light chains that are not attached to heavy chain [12] . sFLC abnormalities can occur in different ways[13] and [14]: monoclonal elevated FLC (elevatedκand/orλwith abnormal FLC ratio), polyclonal elevated FLC (elevatedκand/orλwith normal FLC ratio), and ratio-only FLC abnormality (normal rangeκandλwith abnormal FLC ratio). Currently, sFLC testing has become a routine test for patients with monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM), and AL amyloidosis [15] . Several studies also showed that sFLC abnormalities were associated with poor outcome in patients with chronic lymphocytic leukemia (CLL) [16] , mantle cell lymphoma (MCL) [17] , Hodgkin lymphoma (HL) [18] and DLBCL [4] . However, the data on sFLC are limited in DLBCL, and the discrepancies of sFLC abnormalities among different studies are remarkable. Therefore, further efforts are needed to determine the definite role of sFLC as a novel prognostic marker, before it is brought into routine clinical practice in DLBCL. In addition, very few quantitative data exist on serum total light chains (free and bound, sTLC) in DLBCL, especially in Chinese population, and the clinical significance of measuring sFLC and sTLC in the same population is still unknown.

Recently, there is now an increasing interest in the role of monocytes and lymphocytes in the pathogenesis of lymphoproliferative disorders. It has showed that high absolute monocyte count (AMC) or low absolute lymphocyte count (ALC) at diagnosis have an adverse impact on survival in HL[19] and [20]and NHL[21], [22], [23], [24], [25], [26], [27], and [28]. In advanced HL, lymphopenia has been a well-established prognostic marker, and is included in the international prognostic score [29] . However, whether AMC and ALC could be included in the new prognostic model for DLBCL should be further evaluated. In addition, the relationship of the possible promising prognostic parameters, such as sFLC, sTLC, AMC, and ALC, should be explored.

In this study, we investigated the possibility of using sFLC and sTLC levels as tumor markers for Chinese patients with newly diagnosed DLBCL. We also evaluated the prognostic value of sFLC/TLC abnormalities and the relationship of sFLC, sTLC and AML, ALC in these patients.

2. Materials and methods

2.1. Study population

From September 2007 to May 2011, 108 patients with DLBCL treated at the Cancer Hospital and Institute of Chinese Academy of Medical Sciences and Peking Union Medical College (the vast majority of these patients received R-CHOP or other R-CHOP-like regimens) were included in this study. All patients were diagnosed as DLBCL with histologic features and an immunophenotypic evaluation. Serum samples were collected at the time of inclusion before any chemotherapy. Medical records of all patients were reviewed to collect demographic, clinical and pathologic information. Informed consent was obtained from all patients and this study was approved by the institutional review board.

2.2. Serum FLC measurements

The Freelite assay (The Binding Site Ltd., Birmingham, United Kingdom) was used to measure serumκandλFLC concentrations on stored research serum samples. Results were analyzed based on previously established normal ranges. Elevated sFLC was defined as aκconcentration higher than 1.94 mg/dL or aλconcentration higher than 2.63 mg/dL, and abnormalκ/λsFLC ratio was defined as aκ/λsFLC ratio outside of (0.26, 1.65). Elevated sFLC levels with normal and abnormalκ/λratio were considered polyclonal and monoclonal, respectively. Patients with normal FLC levels but an abnormalκ/λratio were considered to have ratio-only sFLC abnormality.

2.3. Serum TLC measurements

SerumκandλsTLC concentrations were tested by usingκandλlight chain kits (Beckman Coulter, America). Normal sTLC concentrations andκ/λsTLC ratio were defined according to the manufacture's recommendations as follows:κ: 598–1329 mg/dL;λ: 280–665 mg/dL;κ/λratio: 1.47–2.95.

2.4. Statistical analyses

The data were summarized as appropriate and the statistical analyses were performed by SPSS version 17.0 software (SPSS, Chicago, IL). Treatment response was evaluated based on the international response criteria for lymphoma [30] . PFS was defined as the time from diagnosis to disease progression, retreatment, or death due to any cause. OS was defined as the time from diagnosis to death due to any cause. Patients without an event or death were censored at time of last known follow-up. Chi-square test and Wilcoxon rank-sum tests were used to examine the association of sFLC/TLC abnormalities with clinical, prognostic, and demographic variables. Cox proportional hazards regression models and Kaplan–Meier curves were used to assess the association of sFLC/TLC abnormalities with clinical outcomes. A two sidedPvalue of 0.05 was considered statistically significant.

3. Results

3.1. Patient characteristics

The clinicopathologic characteristics of the 108 patients are listed in Table 1 . The median age of these patients at diagnosis was 52 years (range: 16–82 years), and 46.3% of them were males. B symptoms presented in 31 patients (28.7%). Forty-eight patients (44.4%) had Ann Arbor stage III or IV disease, 31 patients (28.7%) had two or more extranodal sites, and only seven patients (6.5%) had a performance status of ≥2. Serum lactate dehydrogenase levels were higher than normal in 46 patients (42.6%). The IPI was calculated as low risk (L-IPI) in 56 patients (51.8%), low-intermediate risk (LI-IPI) in 31 patients (28.7%), high-intermediate risk (HI-IPI) in 11 patients (10.2%) and H-IPI in 10 patients (9.3%). The median of AMC and ALC were 0.39 × 109/L (range 0.06–1.67) and 1.61 × 109/L (range 0.30–15.0), respectively. When the cutoff value of AMC and ALC was set at 0.63 × 109/L and 1.0 × 109/L, respectively, as previous studies reported [31] , 24 patients (22.2%) had monocytosis and 20 patients (18.5%) had lymphopenia. In addition, seven patients (6.5%) had hyperglobulinemia (globulin levels were higher than the upper limit of normal) and no patient had elevated creatinine levels in this study population ( Table 1 ).

Table 1 Association of clinicopathologic characteristics with sFLC/TLC abnormalities.

Variables Total patients N = 108 (%) sFLC (n = 108) sTLC (n = 107)
    Elevated FLC N = 51 (%) Normal FLC N = 57 (%) P-value Abnormal TLC N = 30 (%) Normal TLC N = 77 (%) P Abnormal ratio N = 28 (%) Normal ratio N = 79 (%) P-value
Age at diagnosis, yrs
 Median 52 60 41 <0.001 65.5 46 <0.001 53 52 0.702
 Range 16–82 17–82 16–70   21–82 16–81   17–77 16–82  
  ≥60 36 (33.3) 25 (49.0) 11 (19.3) 0.001 15 (50.0) 20 (26.0) 0.017 8 (28.6) 27 (34.2) 0.587
Male 50 (46.3) 21 (41.2) 29 (50.9) 0.313 15 (50.0) 35 (45.5) 0.672 16 (57.1) 34 (43.0) 0.199
B symptoms 31 (28.7) 20 (39.2) 11 (19.3) 0.022 11 (36.7) 19 (24.7) 0.215 7 (25.0) 23 (29.1) 0.677
Ann Arbor stage: III–IV 48 (44.4) 27 (52.9) 21 (36.8) 0.093 17 (56.7) 30 (39.0) 0.097 11 (39.3) 36 (45.6) 0.565
LDH > ULN 46 (42.6) 23 (45.1) 23 (40.4) 0.618 11 (36.7) 35 (45.5) 0.409 11 (39.3) 35 (44.3) 0.645
Performance status≥2 7 (6.5) 4 (7.8) 3 (5.3) 0.879 3 (10.0) 4 (5.2) 0.398 0 (0) 7 (8.9) 0.186
No. of extranodal sites ≥2 31 (28.7) 16 (31.4) 15 (26.3) 0.562 11 (36.7) 20 (26.0) 0.273 4 (14.3) 27 (34.2) 0.046
IPI       <0.001     0.464     0.056
 0–1 56 (51.8) 22 (43.1) 34 (59.6)   14 (46.7) 42 (54.5)   19 (67.8) 37 (46.8)  
 2 31 (28.7) 15 (29.4) 16 (28.1)   7 (23.3) 23 (29.9)   7 (25.0) 23 (29.1)  
 3 11 (10.2) 7 (13.7) 4 (7.0)   3 (10) 8 (10.4)   1 (3.6) 10 (12.7)  
 4–5 10 (9.3) 7 (13.7) 3 (5.3)   6 (20) 4 (5.2)   1 (3.6) 9 (11.4)  
AMC ≥ 0.63 × 109/L 24 (22.2) 12 (23.5) 12 (21.1) 0.757 5 (16.7) 19 (24.7) 0.372 6 (21.4) 18 (22.8) 0.882
ALC ≤ 1.0 × 109/L 20 (18.5) 14 (27.5) 6 (10.5) 0.024 9 (30.0) 11 (14.3) 0.061 7 (25.0) 13 (16.5) 0.319
Globulin > ULN 7 (6.5) 7 (13.7) 0 (0.0) 0.012 6 (20.0) 1 (1.3) 0.002 1 (3.6) 6 (7.6) 0.620
Creatinine > ULN 0 (0.0) 0 (0.0) 0 (0.0) / 0 (0.0) 0 (0.0) / 0 (0.0) 0 (0.0) /

Abbreviations: sFLC, serum free light chain; sTLC, serum total light chain; LDH, lactate dehydrogenase; ULN, upper limit of normal; No., number; IPI, International Prognostic Index; AMC, absolute monocyte count; ALC, absolute lymphocyte count.

3.2. sFLC abnormalities of patients with DLBCL

In this study cohort, 46 patients (42.6%) had elevated sFLC-κ(medianκconcentration 2.56 mg/dl) and 30 patients (27.8%) had elevated sFLC-λ(median concentration 3.13 mg/dl). Therefore, 51 patients (47.2%) had elevatedκorλ(termed as elevated sFLC), and 25 patients (23.2%) had elevated bothκandλ. Among these patients, five patients (4.6%) harbored abnormalκ/λsFLC ratio with elevatedκorλ, no patient had ratio-only sFLC abnormality; five and 46 patients had monoclonal and polyclonal elevated sFLC, respectively ( Table 2 ).

Table 2 Results of serum free/total light chains measurements in newly diagnosed DLBCL.

sFLC/TLC measurement n (%) Median measurement (range), mg/dl
sFLC (n = 108)
 Elevated κ 46 42.6 2.56 (1.97–6.82)
 Elevated λ 30 27.8 3.13 (2.65–5.94)
 Elevated ratio 5 4.6 1.83 (1.67–4.51)
 Elevated κ and λ 25 23.2 /
 Elevated κ or λ 51 47.2 /
 Elevated κ or λ with abnormal ratio (monoclonal elevated FLC) 5 4.6 /
 Elevated κ or λ with normal ratio (polyclonal elevated FLC) 46 42.6 /
sTLC (n = 107)
 Reduced κ 4 3.7 521.50 (405.00–560.00)
 Elevated κ 9 8.3 1750.00 (1350.00–2300.00)
 Reduced λ 2 1.9 237.50 (224.00–251.00)
 Elevated λ 24 22.4 763.50 (677.00–4070.00)
 Reduced ratio 27 25.2 1.31 (0.19–1.46)
 Elevated ratio 1 0.9 6.52 (6.52–6.52)
 Abnormal κ and λ 9 8.3 /
 Abnormal κ or λ 30 28.0 /
 Abnormal κ or λ with abnormal ratio 11 10.3 /
Abnormal sFLC and sTLC 22 20.6 /

Abbreviations: sFLC, serum free light chain; sTLC, serum total light chain;κ, Kappa;λ, Lambda.

3.3. sTLC abnormalities of patients with DLBCL

TLC was successfully measured in 107 serum samples. The detail results are showed in Table 2 . Compared with the normal reference range, 13 patients (12.1%) had abnormal sTLC-κ, including four had reduced and nine had evaluatedκlight chain. Twenty-six patients (24.3%) had abnormal sTLC-λ, including two had reduced and 24 had evaluatedλlight chain. Therefore, 30 patients (28.0%) had abnormalκorλ(termed as abnormal sTLC), and nine patients (8.3%) had abnormal bothκandλ. Besides, 28 patients (26.2%) had abnormalκ/λsTLC ratio, including 27 had reduced and one had evaluatedκ/λratio. Of these 28 patients with abnormalκ/λratio, 11 had abnormalκorλ, 17 had neither abnormalκnorλ. In addition, of the 30 patients with abnormalκorλsTLC, 22 patients (73.3%) also accompanied with evaluated sFLC ( Table 2 ).

3.4. Relationship between sFLC/TLC abnormalities and clinicopathologic variables

As indicated in Table 1 , elevated sFLC was found to be more frequently in elders (P = 0.001), patients with B symptoms (P = 0.022), higher risk scores (P < 0.001) and hyperglobulinemia (P = 0.012). Patients with elevated sFLC and normal sFLC did not differ significantly with respect to gender, Ann Arbor stage, serum LDH levels, involvement of more than two extranodal sites. However, an advanced Ann Arbor stage tended to be more frequent among patients with elevated sFLC rather than among those with normal sFLC (P = 0.093). In addition, elevated sFLC was associated with ALC (P = 0.024), but did not correlate with AMC (P = 0.757).

Abnormal sTLC was also found to be more frequently in elders (P = 0.017) and patients with high globulin levels (P = 0.002). There were no significant correlation between abnormal sTLC and other clinicopathologic features. However, an advanced Ann Arbor stage and low ALC tended to be more frequent among patients with abnormal sTLC rather than among those with normal sTLC (P = 0.097 andP = 0.061, respectively). In addition, patients with abnormal sTLCκ/λratio tended to have less than two extranodal sites involvement and lower risk scores than patients with normal sTLCκ/λratio (P = 0.046 andP = 0.056, respectively). However, no other association was observed between abnormal sTLCκ/λratio and clinicopathologic variables.

3.5. Survival analysis

The median follow-up, calculated by using the reverse Kaplan–Meier method, was 32.4 months. Of the total group of 108 patients evaluated, 21 patients were dead and 87 patients were alive. On univariate analysis, tumor stage (P < 0.001), performance status (P < 0.001) and low ALC (P = 0.001) were significantly associated with PFS, but only elevated sFLC (P = 0.017) and low ALC (P = 0.001) had significant association with OS. Patients with elevated sFLC had an inferior OS (P = 0.017) and tented to have poorer PFS (P = 0.066) compared to patients with normal sFLC. Patients with low ALC also had worse PFS (P = 0.001) and OS (P = 0.001) compared to patients with high ALC ( Table 3 ). As components of the IPI are important and commonly used prognostic factors in DLBCL, we included five components of the IPI in a multivariate analysis with both sFLC and ALC as dichotomized variables. As summarized in Table 3 , after adjusted for IPI, advanced stage (P = 0.004) and low ALC (P = 0.013) was significantly associated with PFS, but only elevated sFLC (P = 0.027) and low ALC (P = 0.004) exhibited significant association with OS. PFS and OS Kaplan–Meier survival curves for sFLC and ALC are displayed inFig 1 and Fig 2, respectively. Besides, we found that the five patients with abnormalκ/λsFLC ratio showed significantly associated with inferior PFS and OS, and patients with elevated bothκandλlight chain had poor PFS and OS ( Fig. 1 ). Furthermore, we novelty found that polyclonal elevated sFLC still retained its prognostic value even the five patients with altered sFLC ratio were excluded from the survival analysis (data not shown). These results demonstrated that elevated sFLC and low ALC were independent prognostic factors for OS.

Table 3 PFS and OS Cox proportional hazards models.

Model PFS OS
  HR 95% CI P-value HR 95% CI P-value
Univariate
 Age 1.303 0.723–2.759 0.489 1.657 0.697–3.936 0.253
 Sex 1.532 0.308–3.245 0.266 1.273 0.535–3.031 0.585
 Stage 6.343 2.578–15.610 <0.001 1.657 0.697–3.936 0.253
 LDH 1.936 0.931–4.028 0.077 2.073 0.873–4.923 0.098
 Extranodal sites 1.993 0.951–4.175 0.068 2.180 0.903–5.263 0.083
 Performance status 0.171 0.065–0.453 <0.001 0.292 0.086–0.999 0.050
 Elevated sFLC 2.021 0.954–4.282 0.066 3.163 1.224–8.178 0.017
 Abnormal sTLC 1.713 0.802–3.660 0.165 1.693 0.692–4.144 0.249
 Low ALC (≤1.0 × 109/L) 3.522 1.659–7.478 0.001 4.126 1.734–9.819 0.001
Multivariable
 Elevated sFLC 1.886 0.841–4.230 0.124 3.081 1.136–8.355 0.027
 Age ≥60 years 0.922 0.390–2.179 0.854 1.200 0.461–3.121 0.709
 Performance status ≥2 0.214 0.068–0.675 0.009 0.374 0.900–1.560 0.177
 Two or more extranodal sites 1.081 0.436–2.677 0.867 1.576 0.549–4.526 0.398
 Stage III/IV 4.124 1.588–10.712 0.004 2.077 0.746–5.782 0.162
 LDH > ULN 1.361 0.615–3.012 0.447 1.706 0.664–4.383 0.267
 Low ALC (≤1.0 × 109/L) 2.824 1.24–6.432 0.013 4.007 1.568–10.240 0.004

Abbreviations: sFLC, serum free light chain; sTLC, serum total light chain; LDH, lactate dehydrogenase; ULN, upper limit of normal; PFS, progression free survival; OS, overall survival.

gr1

Fig. 1 Progression-free survival (A, C, E) and overall survival (B, D, F) Kaplan–Meier survival curves by serum free light chain (sFLC) in the DLBCL population.

gr2

Fig. 2 Progression-free survival (A, C) and overall survival (B, D) Kaplan–Meier survival curves by absolute lymphocyte count (ALC) in the DLBCL population (A, B) and in the patients with elevated serum free light chain (sFLC) (C, D).

We also compared survival curves according to sTLC levels. Though patients with abnormal sTLC tended to have inferior PFS and OS than patients with normal sTLC, the difference was not significant. The same result was observed between patients with abnormal and normalκ/λsTLC ratio. However, we found that patients with abnormal concurrentκandλhad significantly shorter PFS and OS (P < 0.001 andP = 0.002, respectively) than others in our group of patients ( Fig. 3 ).

gr3

Fig. 3 Progression-free survival (A, C, E) and overall survival (B, D, F) Kaplan–Meier survival curves by serum total light chain (sTLC) in the DLBCL population.

4. Discussion

We report here the first study showing the prognostic value and the association of sFLC/TLC with peripheral blood cell count in a cohort of Chinese DLBCL patients. The results demonstrated that elevated sFLC were more frequent than abnormal sTLC in patients with DLBCL. Both elevated sFLC andκ/λratio were strongly associated with clinical outcome. However, abnormal sTLC was not found to be a significant prognostic indicator, with the exception of an abnormality of concomitantκandλ. In addition, not abnormal sTLC, but elevated sFLC was found to be associated with low ALC. The present study demonstrated that only sFLC and low ALC remained statistically significant prognostic factors of OS after adjusting for IPI.

In the present study, 47.2% patients had an elevated sFLCκorλconcentrations. This frequency was much higher than that found in previous studies[4] and [32]. It is known that several plausible causes can lead to elevated sFLC in patients with DLBCL, host effects, such as renal dysfunction, advanced age, immune disruption or stimulation [33] . In addition, tumor could also drive the excess of the residual light chain that detected in serum [4] . In this study, all patients had normal renal function, elevated sFLC measurements associated with unfavorable outcome are correlated with the presence of adverse clinical features at diagnosis, including age, stage, B symptoms, low ALC and high globulin levels. Although various factors could contribute to the difference of sFLC measurements in different study cohorts, patients’ immune status but not renal dysfunction was one of the causes led to elevated sFLC in our study cohort. However, we might not draw a definite conclusion that the level of sFLC exhibited a distinguishing feature in Chinese DLBCL patients. Further studies are needed to explore whether sFLC levels in DLBCL had geographical or racial differences.

To date, the data on the prognostic value of sFLC are limited in DLBCL. Maurer et al. [4] firstly reported that elevated sFLC was strongly associated with inferior PFS and OS, and independent of IPI. More notably, when sFLC was included in a multivariable model with the IPI components, sFLC was the largest single predictor of outcome. Jardin et al. [32] recently also reported that elevated sFLC was strongly associated with PFS and OS in DLBCL patients on univariate analysis, but after adjusted for IPI, it is only significantly associated with OS. Herein, we found that elevated sFLC was still strongly associated with OS after adjusting for IPI. However, we did not observed significant association of sFLC with PFS. In addition, it was noteworthy that a cohort of patients harbored elevated bothκandλsFLC had inferior PFS and OS. Though these results exhibited some differences, it clearly demonstrated that elevated sFLC was associated with unfavorable outcomes in patients with DLBCL.

Alongside the absolute levels of the individualκandλsFLC, theκ/λsFLC ratio is used to assess monoclonality and monitor disease. However, the proportion percentage ofκ/λsFLC ratio varied widely. In the present study, the proportion of patients with abnormalκ/λFLC ratio only accounted to 4.6%, which is much lower than Maurer et al. (14.0%) and Jardin et al. (9.3%) reported[4] and [32]. Previous studies showed that the presence of an abnormalκ/λsFLC ratio at diagnosis was a poor prognostic factor for patients with MCL, MM and DLBCL[4], [17], [32], and [34]. However, in DLBCL, Maurer et al. [4] showed that only an abnormalκ/λsFLC ratio with corresponding elevatedκorλwas associated with an inferior outcome. Patients with abnormalκ/λsFLC ratio without an elevation of either chain should be considered normal for risk. In our study, all these five patients with abnormal sFLC ratio corresponded with either elevatedκorλ, and had inferior outcome. Unfortunately, there was no patient with ratio-only sFLC abnormality, we could not further comprehensively analysis the prognostic value of abnormal sFLC ratio in our study cohort. Due to the limitation of small sample size, our data support the need for large-scale and prospective biomarker studies to validate the role of abnormalκ/λsFLC ratio in DLBCL.

Besides the detection of sFLC, it is now possible to detect the entire immunoglobulins in serum, not only according to the heavy chain isotype, and more precisely according to the combined heavy and light chain (HLC) isotypes. It has reported that the HLC ratio is a factor for predicting PFS in MM [35] , and was associated with unfavorable outcomes in patients with DLBCL treated by R-CHOP [32] . In the clinical practice, measurement data of sTLC (free and bound light chains) were used for the diagnosis and treatment of various diseases, including liver and kidney disease and blood protein diseases. The isotype of light and heavy chains, the concentrations of polyclonal IgG, IgA and IgM should be analyzed, if abnormal immunoglobulins were identified in patients’ serum. All these analysis could help to estimate prognosis, and to identify whether patients are vulnerable to disease infection due to the deficiency of polyclonal immunoglobulins. In the current study, we measured sTLC and sFLC in the same cohort of patients. We found that the prevalence of sTLC was lower than that of sFLC. Majority of patients (73.3%) with abnormal sTLC accompanied with elevated sFLC. However, less than half of patients (44.0%) with elevated sFLC accompanied with abnormal sTLC. Moreover, the clinical features of patients with abnormal sTLC were not same to those of patients with elevated sFLC. Patients with abnormal sTLC or abnormal sTLC ratio showed no significant difference in clinical outcomes, compared to patients with normal sTLC or sTLC ratio. Only patients with abnormal bothκandλshowed significantly unfavorable clinical outcomes. These results suggested that sFLC and sTLC measurements reflected, at least partially, different biological processes related or unrelated to the tumor but also different sensitivities of the respective assays. Therefore, sTLC measurement was of less prognostic utility, compared with sFLC measurement in newly diagnosed DLBCL patients.

Several studies showed that both high AMC and low ALC had negative prognostic value in NHL[21], [22], [23], [24], [25], [26], [27], and [28], when used the best cut-off level of ≥0.63 × 109/L and ≤1.0 × 109/L, respectively[5] and [31]. However, our results did not coincide exactly with previous studies. We observed that only ALC at diagnosis associated with sFLC levels and had prognostic significance even after adjusting of IPI components. AMC did not show any significant association with survival. The variances of sample size, clinicopathologic characters and the distribution of risk stratification of study cohorts may contribute to the different results. To our knowledge, we report for the first time the association of ALC and sFLC levels in patients with newly diagnosed DLBCL. Previous study [36] showed that low ALC may be associated with a preexisting immunosuppressed condition and may be a consequence of lympholytic cytokines produced by the lymphoma cells, or a combination of both or other factors. Lymphopenia is not only a parameter correlated to survival but also a biological mechanism stimulating tumor progression in NHL. Therefore, both elevated sFLC and low ALC may be influenced by patients’ immune status and tumors. However, the actual mechanisms of the association between elevated sFLC and lymphopenia in DLBCL remain unclear and are probably multifactorial. It will be an interesting and important question to answer in future studies. In addition, the true prognostic role of AMC in DLBCL warrants confirmatory testing in an independent series of patients with DLBCL.

Previous studies[37] and [38]showed that sFLC assay was more sensitive than immunofixation electrophoresis (IFE) or serum protein electrophoresis (SPE) methods detecting for FLCs. However, we failed to provide comparison in term of sensibility/specificity of sFLC assay with IFE or SPE methods in the same series of patients. Although our study has some obvious limitations, including the fact that the characteristics of underlying disease or patients’ related conditions were excluded from consideration, it is retrospective and confirmatory with small sample size, patients distributed unevenly with short follow-up time. Our results still could demonstrate some insights into establishing new prognostic model to identify patients at different risk for death from DLBCL.

In summary, we have provided evidence that sFLC correlate with ALC and disease outcome in DLBCL. In contrast, sTLC measurement was of little prognostic utility. Furthermore, sFLC and ALC remained as significant parameters for outcome prediction in a multivariate analysis that included the five IPI components. Clearly, sFLC measurement and ALC provided additional information to standard laboratory tests, which may be of considerable importance in DLBCL patient management.

Conflict of interest statement

The authors declare that no conflict of interest exists.

Acknowledgment

This study was supported in part by grant from National Science and Technology Major Project of China (2012ZX09303012).

Authors contributions:YKS, XHH, PL, JLY, SYZ, YQ, SY, LG, XHH designed this study; XHH, NNZ, JFW, JRY, YF and DL performed the acquisition, analysis and interpretation of the data; NNZ wrote the draft; XHH, NNZ, JF revised the manuscript. We all authors participated in revising and approved the submission of the manuscript.

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Footnotes

Department of Medical Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing Key Laboratory of Clinical Study on Anticancer Molecular Targeted Drugs, Beijing 100021, China

lowast Corresponding author at: Department of Medical Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing Key Laboratory of Clinical Study on Anticancer Molecular Targeted Drugs, 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021, China. Tel.: +86 10 87788293; fax: +86 10 67705068.

1 These two authors contributed equally to this work.