Extended Use of Serum Free Light Chain as a Biomarker in Lymphoproliferative Disorders A Comprehensive Review

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1 Hematopathology / FLC as a Prognostic Biomarker in B-Cell NHL Extended Use of Serum Free Light Chain as a Biomarker in Lymphoproliferative Disorders A Comprehensive Review Khalil M. Charafeddine, MD, Mark N. Jabbour, MD, Raneem H. Kadi, and Rose T. Daher, PhD Key Words: Non-Hodgkin lymphoma; Chronic lymphocytic leukemia; Serum free light chain; Serum free light chain ratio; Prognosis Abstract Serum free light chain (sflc) assays were shown to improve detection, management, and prognostication in plasma cell disorders. Recently, sflc assays improved detection of M proteins when combined with standard methods of protein electrophoresis/ immunofixation in patients with non-hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL). Incidence of abnormal sflc ratio (sflcr) varied from 0% to 36% and 29.7% to 59% in NHL and CLL, respectively. Increased sflc levels or abnormal sflcr predict shorter overall survival in early-stage CLL. Furthermore, abnormal sflcr correlated with advanced disease stage and poorer outcome. In diffuse large B-cell lymphomas, increased sflc was demonstrated as an independent, adverse prognostic factor for overall/event-free survival. Moreover, abnormal sflcr can be a diagnostic tool in central nervous system lymphomas. Finally, the quantitative FLC assay has the potential to become a new, easily measured biomarker for predicting prognosis and enhanced detection in NHL/CLL. It may be used serially at follow-up evaluations to provide clues to relapse. Normal immunoglobulin secretion is a function of terminally differentiated B cells that proceeds from heavy- and light-chain gene assembly with V(D)J recombination in early bone marrow precursor cells to somatic hypermutation in the germinal centers of secondary lymphatic organs. The result is terminally differentiated B cells that are released into the periphery as plasma cells or long-lived memory B cells with the capacity to produce antibodies with high affinity for the immunizing antigen composed of 2 heavy and 2 light chains. The repertoire of tests used to evaluate and quantify the monoclonal immunoglobulin (Ig) protein (M protein) that indicates the presence of a monotypic population of B cells in the marrow or nodal tissue includes serum and urine protein electrophoresis (SPEP and UPEP) and immunofixation electrophoresis (IFE). Nephelometric measurement of serum Igs can also be used; however, measurement of the M-protein peak is the best method for quantitation of the M spike. 1 Around 3% of multiple myeloma (MM) and most patients with amyloidosis are of nonsecretory types. Therefore, nonsecretory myeloma and light chain diseases are difficult to diagnose and monitor during and after treatment with these aforementioned methods. 2,3 With the emergence of immunoassays specific for Ig free light chains (FLCs) that bind to the hidden area of the light chain in an intact Ig model and do not recognize κ and λ bound to heavy chain, it has become clear that serum FLC (sflc) testing is more sensitive than protein electrophoresis (PEL) and IFE for detecting FLCs. Immunofixation can detect κ and λ FLCs at a minimum concentration of 100 to 150 mg/l, whereas sflc assay has a detection limit of 3 to 4 mg/l. The sflc concentrations have also been shown to correlate with disease activity. 4 These sensitive sflc assays may now allow detection of M proteins produced 890 Am J Clin Pathol 2012;137: Downloaded 890 from

2 Hematopathology / Review Article by other B-cell malignancies, which were only occasionally positive on PEL and IFE. Currently, an abnormal sflc ratio (sflcr) is known to play a role as a prognostic factor in Waldenström macroglobulinemia, MM, and AL amyloidosis. 5-7 Moreover, an abnormal sflcr is considered a risk factor for progression of monoclonal gammopathy of unknown significance (MGUS), solitary plasmacytoma, and smoldering multiple myeloma into MM Although sflc testing has now become a routine test for patients with MGUS, smoldering MM, MM, and AL amyloidosis, 13 its application has been limited in conditions such as B-cell non-hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL). 14 Data presented in this review were selected after an extensive and comprehensive search in the English literature through Medline and PubMed. All relevant references were included. The main search terms used included serum free light chain, free light chain ratio, lymphoproliferative disorder(s), non- Hodgkin lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, central nervous system lymphoma, multiple myeloma, monoclonal gammopathy of unknown significance, prognostic factor, and survival. Different keywords were merged using OR or AND operators. Because of the scarcity of literature related to this topic, the only search limit applied was to articles about humans. The final articles were screened by 2 of us (K.M.C. and R.T.D.). Because all articles and abstracts obtained were included in this review, no selection bias should occur. The aim of this review is to highlight the importance of sflc and sflcr as prognostic factors and indicators of progression in NHL and specifically in CLL diseases similar to its importance in plasma cell dyscrasia. Free Light Chain Assay The sflc assay is a sensitive, latex-enhanced immunonephelometric test that quantitates free κ and λ FLC in serum and urine samples. The test is based on the presence of polyclonal antibodies in the reagent (Freelite, The Binding Site Ltd, Birmingham, UK) that react noncompetitively with Bence Jones proteins individually. The assay showed high sensitivity for κ FLC antibodies reacting with κ-labeled cells up to a dilution of 1:16,000 and no reactivity against polyclonal IgG, monoclonal IgA and IgM κ, and monoclonal λ FLC-coated cells at a dilution less than 1:2. Similar results were obtained with λ antibodies reacting with λ-labeled cells and polyclonal and monoclonal immunoglobulins as well as κ FLC-coated cells. At the same time, the detection limit for these assays is at least 20-fold and 50-fold lower than IFE and PEL, respectively. 4 The studies mentioned in this review used the Freelite serum FLC assay from The Binding Site Ltd (Birmingham, UK) and was adapted to the BNII nephelometer (Dade Behring, Deerfield, IL). In healthy individuals, sflc concentrations depend on the balance between production by plasma cells and renal clearance. They are cleared rapidly with a serum half-life of 2 to 4 hours through the renal glomeruli and then metabolized in the proximal tubules of the nephrons. Normally little protein escapes to the urine, and sflc concentrations have to increase manifold before the absorption mechanisms are overwhelmed. 15 Katzmann et al 2 defined the normal range for κ and λ FLC concentrations and ratio in healthy subjects. Fresh serum samples from 127 healthy subjects (age range, years) and frozen serum samples from 155 donors (age range, years) were collected from a serum bank in Olmsted Country, MN. The 95% reference intervals for κ and λ FLC were 3.3 to 19.4 mg/l and 5.7 to 26.3 mg/l, respectively. The 95% reference interval for the κ/λ ratio was 0.3 to 1.2. However, a 5% false-positive rate is considered a high number in diagnosing monoclonal FLC diseases; therefore, the reference range was expanded to 100% ( ). The sensitivity dropped from 98% to 97%, and the specificity increased from 95% to 100%. The positive and negative predictive values were 100% and 99%, respectively. Any patient with a κ/λ ratio greater than 1.65 or less than 0.26 is considered to have excess κ or λ FLC, respectively. 2 The 100% confidence interval used reduces the likelihood that polyclonal activation of B cells will cause an abnormal ratio and therefore the test must be interpreted in the context of a clinical situation and repeated at a later date. 16 κ and λ sflc concentrations may be abnormal because of a number of conditions including immune suppression, immune stimulation, reduced renal clearance, or monoclonal plasma cell proliferative disorders. Serum samples from patients with either polyclonal hypergammaglobulinemia or renal impairment often have elevated κ and λ FLC because of increased synthesis or reduced renal clearance; however, the sflcr usually remains normal in these situations. 2 However, Hill et al 17 reported falsely elevated sflcr in 14% of patients with polyclonal increase in Igs, with concurrently abnormal glomerular filtration rate, thus suggesting that these conditions can cause false-positive results for sflcr. Nevertheless, a significantly abnormal κ/λ sflcr should only be caused by a plasmaproliferative or lymphoproliferative disorder that secretes excess FLC and disturbs the normal balance between κ and λ secretion. 16 Although the test has major advances, it also has some limitations. First, there can be significant lot-to-lot variation (20% coefficient of variation [CV]) between batches of polyclonal FLC antiserum according to the manufacturer. Moreover, Tate et al 18 showed that CV can change up to 29% and 45% for κ FLC and λ FLC, respectively, on repeated measurements. Their recommendation is to be cautious when following up a patient with serial sflc when different reagent lots are used, because the κ/λ ratio can be doubled in a patient with a stable disease. This issue is mainly present in large multi-institutional Downloaded from Am J Clin Pathol 2012;137:

3 Charafeddine et al / FLC as a Prognostic Biomarker in B-Cell NHL trials, where serious consideration should be given for running samples at a centralized testing facility that performs lot-to-lot comparisons. Second, in the absence of additional offline dilutions, κ FLC, and to a lesser extent, λ FLC results may be lower than the real values. In addition, Tate et al 18 showed that FLC might not dilute in a linear fashion, especially the κ type. False high results, by as much as 10-fold, may occur because of the polymerization of light chains. Finally, changes in amino acid sequence of the light chain cause a change in the FLC epitope configuration, which make them unrecognizable by the FLC reagents. 16 Nakano and Nagata 19 developed an FLC κ and λ enzyme-linked immunosorbent assay (range, μg/l), and showed that there is cross reactivity of FLC κ and λ at 100 mg/l with intact IgGs of 0.11% and 0.12%, respectively. Moreover, the cross-reactivity between κ and λ FLCs is minimal at less than 0.015%. The greater the specificity, the better is one s ability to quantitate κ and λ FLC in the presence of a large excess of serum IgG, IgA, and IgM. This distinction is important, because in healthy individuals and in most patients with myeloma, most of the circulating light chain is bound to heavy chains, thus making less specific reagents a near surrogate for circulating heavy chain measurement. Lymphoproliferative Disorders and sflc Non-Hodgkin lymphoma comprises a large number of subtypes that show distinct biologic, molecular, and cytogenetic characteristics. Moreover, some patients experience clinical remission, some experience disease stabilization for a long period, and others experience rapid medical deterioration. Because of their highly variable clinical courses, identification of molecular and biological prognostic markers becomes mandatory to provide new insights into risk stratification of these patients. Several studies have shown that M protein can be produced in other hematologic malignancies, mainly NHL. Using conventional techniques (PEL/IFE), Alexanian 20 documented the presence of M protein in 7% of 640 patients with diffuse large B-cell lymphoma (DLBCL) and CLL. In a cohort study from the Mayo Clinic which included 430 patients with IgM monoclonal protein, 7% of patients had NHL and 5% had CLL. 21 Lin et al 22 studied 382 patients with lymphoid neoplasm with IgM M protein. Fifty-nine percent of these patients had Waldenström macroglobulinemia, 20% had CLL, 7% had marginal zone lymphoma (MZL), 5% had follicular lymphoma (FL), 3% had mantle cell lymphoma (MCL), 2% had DLBCL, and 4% were miscellaneous. 22 Asatiani et al 23 described 7 (27%) of 26 patients with extranodal MZL having an M-protein spike. Table 1 summarizes the frequency of increased sflcr from various studies in the literature The initial evaluation of the frequency of monoclonal sflc in patients with other B-cell malignancies was published by Martin et al 14 in Frozen serum samples from 226 patients Table 1 Comparison of Different Studies Exploring the Role of Abnormal sflc and sflcr in Different Lymphoproliferative Disorders Abnormal FLC Abnormal sflcr Total No. Reference Disease Study Design of Patients Sample Type No. % No. % Martin et al, Mantle 25 NA NA 9 36 SLL 25 NA NA 6 24 MALT 19 NA NA 3 16 LP 14 NA NA 2 14 BL 17 NA NA 2 12 DLBCL 25 NA NA 2 8 Follicular II 25 NA NA 2 8 Follicular III 25 NA NA 1 4 Follicular I 25 NA NA 0 0 Immunoblastic 8 NA NA 0 0 All NHL Retrospective 208 Frozen serum sample NA NA CLL Retrospective 18 Frozen serum sample NA NA 8 44 and urine sample Ruchlemer et al, CLL Prospective 34 Fresh serum and urine NA NA Vermeersch et al, B-CLL 8 NA NA B-NHL 17 NA NA All Prospective 25 Fresh serum sample NA NA Matschke et al, CLL Retrospective 120 Frozen serum sample NA NA Pratt et al, CLL Retrospective 259 Frozen serum sample NA NA Yegin et al, CLL Prospective 101 Frozen serum sample Landgren et al, NHL Case-Control 40 Fresh serum sample NA NA 8 20 Maurer et al, DLBCL Prospective 295 Fresh serum sample Maurer et al, CLL Prospective 339 Fresh serum sample BL, Burkitt lymphoma; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; FLC, free light chain; FLCr, free light chain ratio; LP, lymphoplasmacytoid lymphoma; MALT, lymphoma of mucosa-associated lymphoid tissue; NA, data not available; NHL, non-hodgkin lymphoma; SLL, small lymphocytic leukemia. 892 Am J Clin Pathol 2012;137: Downloaded 892 from

4 Hematopathology / Review Article (NHL: n = 208, CLL: n = 18) collected at the Mayo Clinic/ Lymphoma SPORE serum bank were tested for M protein with the sflc assay, SPEP, and IFE. Overall, M protein was detected in 24% (54/226) of samples (NHL: n = 46, CLL: n = 8). Of these 54 patients, 34 (63%) were sflc-positive (NHL: n = 27, CLL: n = 7), whereas 35 patients were identified by means of SPEP/IFE (NHL: n = 33, CLL: n = 2). In addition, M protein was detected in 19 patients (NHL: n = 13, CLL: n = 6) with sflc only, and 20 patients (NHL: n = 19, CLL: n = 1) with SPEP/IFE only. Within NHL, the highest incidence of sflc was in patients with MCL (9/25, 36%), followed by small lymphocytic leukemia (SLL) (6/25, 24%), lymphoma of mucosa-associated lymphoid tissue (3/19, 16%), lymphoplasmacytoid lymphoma (2/14, 14%), Burkitt lymphoma (2/17, 12%), DLBCL (2/25, 8%), and FL (3/75, 4%). In conclusion, the sflc assay improved the detection of M proteins when combined with standard SPEP/IFE in a substantial fraction of patients with NHL/CLL. However, the relationship of M protein and sflc to pathogenesis, disease course, response to therapy, prognosis, or survival in these patients with NHL or CLL was not investigated. 14 Chronic Lymphocytic Leukemia Biomarkers and Prognosis CLL is considered the most common type of leukemia in the western world, accounting for approximately 40% of all leukemias. It affects mainly the elderly, with a male-to-female ratio of 2:1 (median age at diagnosis of 72 years). Although clinical staging systems (Rai staging and Binet system) are the mainstay for assessing prognosis in patients with CLL, 32,33 additional classic and biological prognostic factors are used to stratify these patients into good-risk or poor-risk groups, and hence give better, more case-specific disease profiles. With this increased awareness, the focus of research has moved toward new simple prognostic markers with widespread clinical practice. This is mainly important in deciding which patients will receive watch and wait strategy while others will receive treatment. It is well documented that the expression of unmutated Ig heavy chain variable region genes (U-IgVH) and CD38 (a cyclic ADP ribose hydrolase) predicts more aggressive clinical behavior However, the IgVH test requires DNA sequencing, an expensive and time-consuming procedure. Moreover, patients with CLL with U-IgVH have higher levels of zeta-chain associated protein (ZAP-70) (molecular weight 70 kda) than patients with mutated IgVH (M-IgVH). 37,38 It must be noted that both CD38 and ZAP-70 can be analyzed with flow cytometry. Fluorescent in situ hybridization analysis can detect certain chromosomal abnormalities that have prognostic importance. For instance, patients with del(13q) have excellent prognosis (median survival, 133 months), whereas patients with deletions in the short arm of chromosome 17 [del(17p)] involving p53 gene and in the long arm of chromosome 11 [del(11q)] involving ataxia telangiectasia mutated (ATM) gene tend to have a worse prognosis and resistance to therapy (median survival, 79 and 32 months, respectively). 39,40 However, the major drawback limiting routine clinical use of these parameters is the cost and complexity of these technical procedures as well as the underlying necessity for further standardization. Serum Free Light Chain as a New Biomarker Recent data have shown that sflc and sflcr can be a prognostic factor in patients with CLL Table ,31 Pratt et al 27 measured sflc in a larger series of 259 patients with Table 2 Clinical Outcome of Patients With CLL in Different Studies Based on FLC Abnormality Total No. Reference of Patients Study Groups (No. of Patients) TFT OS Pratt et al, NL (159) Med = 117 (0-241) Med = 254 (0-292) AB (110) κ ratio (66) Med = 52 (0-362) Med = 201 (0-362) λ ratio (44) Med = 33 (0-192) Med = 124 (0-223) Matschke et al, NL (41) Med = 109 NA AB (71) κ ratio Med = 78 NA λ ratio Med = 34 NA Maurer et al, NL (176) HR = 1.00 HR = 1.00 Type1: Monoclonal FLC abnormality (57) HR = 5.57 ( ) HR = 5.38 ( ) Type2: Polyclonal FLC abnormality (52) HR = 2.12 ( ) HR = 5.37 ( ) Type3: sflcr-only abnormality (54) HR = 2.75 ( ) HR = 0.38 ( ) Yegin et al, NL sflcr (71) Med = NR (0-130) Med = NR ( ) AB sflcr (30) Med = 32 (0-188) Med = 140 ( ) NL sflc (46) Med = NR (0-146) Med = NR (1-188) AB sflc (55) Med = 32 (0-188) Med = 140 ( ) AB, Abnormal; CI, confidence interval; CLL, chronic lymphocytic leukemia; HR, hazard ratio; Med, median, in months; NA, data not available; NL, normal; NR, not reached; OS, overall survival; sflc, serum free light chain; sflcr, serum free light chain ratio; TFT, time to first treatment. Downloaded from Am J Clin Pathol 2012;137:

5 Charafeddine et al / FLC as a Prognostic Biomarker in B-Cell NHL CLL from 3 UK hospitals (181 untreated/pretreatment and 78 who had received treatment) and reported an abnormal sflcr in 39% (100/259). Abnormal sflcr was a prognostic factor for reduced survival in the whole series (P =.002) as well as for the 181 untreated patients (P =.0001), irrespective of the cause of death. When considering CLL-related deaths (n = 33 patients), abnormal sflcr was a prognostic factor for decreased survival (P =.003) but not for those who died of non-cll causes (n = 34 patients). The time to first treatment (TFT) correlated with an abnormal sflcr when all patients in the study were analyzed (TFT with normal sflcr = 117 months vs TFT with abnormal sflcr = 48 months, P =.001), but not when the untreated patients were analyzed separately (P =.18). This was interpreted by the authors as being insufficient because of low patient numbers. In 8% (8/100) of cases, there was a difference between the type of light chain expressed on the CLL cell surface and the type of abnormal sflc, suggesting an underlying MGUS in these patients or, less likely, false-positive sflc results. Moreover, patients with U-IgVH genes have κ ratio as a poor prognostic factor; in contrast, patients with M-IgVH genes have λ ratio as a poor prognostic factor. Using a Cox regression analysis, Pratt et al 27 identified 4 independent prognostic variables for overall survival, namely, Zap-70, β 2 microglobulin, M-IgVH, and abnormal sflcr. Patients with CLL with an abnormal sflcr were significantly more likely to have U-IgVH, a Zap-70 positivity, a lymphocyte doubling time less than 12 months, and a high β 2 microglobulin. Similarly, in a cross-sectional study involving 84 patients with CLL, Perdigao et al 41 showed a correlation among abnormal sflcr, TFT, IgVH mutational status, and survival (P =.05). In their study of 34 patients with CLL in various stages (median age, 66 years; male-female ratio of 1.9:1; median time from diagnosis, 41.5 months), Ruchlemer et al 24 found an abnormal sflcr in 53% of cases, which mostly correlated with advanced disease stage and increased κ chain. In a separate cohort of 120 patients with CLL (serum samples collected before initiation or 6 months after cessation of treatment), 71 patients (59%) had abnormal sflcr. In addition to improving M-protein detection in CLL, it was shown that abnormal low sflcr (indicating λ FLC involvement) was associated with worse outcome. In addition, no correlation was found between sflc and other prognostic factors (ZAP-70, CD38, cytogenetic markers, and Binet stage), which implies that sflc is an independent prognostic factor in patients with CLL. 26 In their recently published cohort study, Maurer et al 31 classified 339 newly diagnosed patients with CLL into 3 types: type 1 with elevated κ or λ FLC and abnormal sflcr (monoclonal, n = 57 patients), type 2 with elevated κ or λ FLC and normal sflcr (polyclonal, n = 52 patients), and type 3 with normal range κ and λ FLC and abnormal sflcr (monoclonal, n = 54 patients). Subjects were followed up for a median of 47 months (range 1-92 months). Forty-nine percent (163/339) of these patients had sflc abnormality with worse TFT and overall survival than those with normal sflc (Table 2). Their conclusion was that sflc is an important prognostic parameter and is maintained after adjustment for Rai staging, with different types of sflc abnormality affecting prognosis to various degrees (Table 2, hazard ratios [HRs]). 31 In 2010, Yegin et al 28 conducted a retrospective study to assess the prognostic value of sflc levels and sflcr in a cohort of 101 patients with CLL (median age, 62 years; male-female ratio, 68:33) followed up for a median of 29 (range, ) months. sflc levels were found to be high in 55 patients (54.5%), with 30 patients (29.7%) having abnormal sflcr. Median TFT was shorter in patients with high sflc levels (P =.02) but not in low-risk patients with CLL. In addition, median TFT was not statistically different between patients with normal and abnormal sflcr. The median overall survival was shorter in patients with both high sflc levels (P =.01) and abnormal sflcr (P =.05), but this did not remain valid in the multivariate analysis, probably because of the small sample. Furthermore, patients with high sflc levels and abnormal sflcr expressed higher CD38 levels and positivity, thus indicating that these biomarkers are involved in stimulation of B-cell receptor on CLL proliferating cells. Finally, sflc and sflcr did not differ significantly among patients with low, intermediate, and high risk groups. 28 AIDS-Related Lymphoma and sflc Persons infected with human immunodeficiency virus (HIV) have an elevated risk of developing NHL particularly DLBCL, primary central nervous system (CNS) lymphoma, and to a lesser extent, Burkitt lymphoma 42 ; this risk remains increased in the era of effective HIV therapy and is directly associated with CD4 T-lymphocyte counts and the presence of B-cell dysfunction. Moreover, serum Ig level, mainly IgG, is high, reflecting nonspecific polyclonal B-cell activation. 43,44 To evaluate whether acquired immunodeficiency syndrome (AIDS) lymphomas are typically preceded by a precursor state manifested in elevated markers of B-cell activation, Landgren et al 29 evaluated the usefulness of FLC assays in 66 individuals who developed NHL and 225 matched HIVinfected, lymphoma-free controls in a case-control study. The 66 patients with NHL included 27 with DLBCL, 10 with primary CNS lymphoma, 3 with Burkitt lymphoma, and 26 with other/unknown NHL subtypes. They found that increased sflc was a strong predictor of NHL up to 2 to 5 years before diagnosis in a dose-response manner. This strong association between sflc level and development of NHL is mostly noted in patients not receiving highly active antiretroviral therapy (HAART). In addition, Landgren et al 29 speculated that, in addition to CD4 cell counts, elevated sflc levels can help identify patients who might most benefit from starting early HAART therapy. 894 Am J Clin Pathol 2012;137: Downloaded 894 from

6 Hematopathology / Review Article DLBCL and sflc DLBCL is the most common NHL in the United States. The International Prognostic Index (IPI) is currently considered a prognostic score for the aggressive type of DLBCL. However, risk-adaptive treatment approaches related to other biomarkers are needed. Therefore, there is a need for additional prognostic markers to better identify patients who experience a relapse after immunochemotherapy or fail to achieve remission. The 8% frequency of abnormal κ/λ ratio in DLBCL was previously described by Martin et al 14 as one of the lowest compared with other NHL types based on a study of 25 patients (Table 1). A larger study was published recently by Maurer et al 30 including 2 independent cohorts (N0489 and MER with 76 and 219 patients, respectively) of 295 patients with DLBCL. They investigated the association of pretreatment sflc with event-free and overall survival. 30 Elevated sflc concentrations and abnormal κ/λ sflcr were seen in 34% and 12% of the N0489 group and in 31% and 15% of the MER group, respectively. Patients with elevated sflc had inferior overall and event-free survival in both cohorts compared with patients with normal FLC (N0489: event-free survival HR, 3.06; overall survival HR, 3.16; P <.02 for both; MER: event-free survival HR, 2.57; overall survival HR, 3.74; P <.001 for both). All associations remained significant for event-free and overall survival after adjusting for the IPI. Abnormal sflcr, however, was modestly associated with event-free and overall survival in combined groups (event-free survival HR, 1.61; overall survival HR, 1.67, P =.07 for both), with the association only related to a concomitantly elevated sflc regardless of its being monoclonal or polyclonal in nature. Therefore, patients with abnormal sflcr without an elevation of either chain should be considered to be at normal risk. In a multivariate analysis, sflc turned out to have stronger association with event-free survival (HR, 2.26; P <.001) and overall survival (HR, 2.5; P <.001) compared with the 5 IPI components. The association between outcome and sflc was maintained in patients with normal creatinine level, suggesting that renal failure is not an interfering factor. The authors also found an association between elevated sflc and stage, and probably with a more aggressive tumor. 30 In conclusion, this study demonstrates increased sflc to be an independent, adverse prognostic factor for event-free and overall survival rather than for sflcr, and further evaluation of sflc assays as a biomarker in DLBCL is required. Cerebrospinal Fluid FLC in Patients with CNS Lymphoma In their first report, Schroers et al 45 studied the detection of FLC in 21 patients with primary and secondary CNS lymphomas and 14 patients with different neurologic disorders. They showed that, in patients with CNS lymphomas, the FLCr in cerebrospinal fluid (CSF) is higher (range, ) than in control patients (range, ). Concentrations of FLC are lower in CSF than in serum samples in all patients studied. In addition, 52% of lymphoma CSF samples have FLCr above 3.0, indicating the presence of clonally restricted B-cell population. 45 Hildebrandt et al 46 found in their study that all patients (5/5) with lymphomatous meningitis, a CNS complication in patients with NHL, had abnormal CSF κ/λ FLCr, whereas around 67% of patients without definite lymphomatous meningitis had normal CSF κ/λ FLCr. 46 These studies demonstrated that CSF κ/λ FLCr can be used as a diagnostic tool in CNS lymphomas. Further studies are needed to confirm this finding, assess its use after treatment and during disease follow-up, and determine its importance as a prognostic factor in CNS lymphoma. Conclusion In conclusion, quantified sflc levels in B-cell lymphomas, specifically high-grade B-cell lymphoma and SLL/CLL may represent a significant prognostic marker for the detection of bulk and residual disease before and after treatment. 27,28,30 Causes for the increase in sflc levels are multiple and include, among others, old age, elevated creatinine levels with renal dysfunction, 47 and immune disruption or stimulation, 48 with most being related to a polyclonal increase in sflcs. Most important is the tumor-related secretion of light chains secondary to the neoplastic clonal proliferation. In fact, the latter is the most plausible explanation because high-grade lymphomas and SLL/CLL have been shown ex vivo to secrete light chains more than heavy chains, in contradiction to FL, which produces balanced secretions of heavy and light chains. The relative ease of analysis and the quantitative nature of the FLC tests make them ideal for monitoring response to therapy, detecting minimal residual disease, and identifying early relapse after effective treatment has been discontinued. Large cohort studies are needed to further assess the usefulness of sflc and sflcr in stratifying patients at diagnosis and in predicting and/or making a prognosis for clinical outcome in different lymphoproliferative disorders. The finding that patients with CLL with abnormal sflc and sflcr constitutes a separate biological subtype with worse outcome (U-IgVH status, positive Zap-70, lymphocyte doubling time less than 1 year, and high β 2 microglobulin) deserves evaluation in other lymphoproliferative disorders. Furthermore, detecting minimal residual disease in NHL cases is not feasible using flow cytometry on peripheral blood samples in contrast to CLL 14 ; hence, future studies can be helpful in delineating the usefulness of sflc and sflcr as biomarkers in detecting residual disease in these patients. In researching these ideas, longitudinal Downloaded from Am J Clin Pathol 2012;137:

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