Serum free light chain (FLC) levels are abnormal in a significant percentage of lymphoid malignancies. Here we discuss the utility of serum FLC measurement and the serum FLC ratio in chronic lymphocytic leukemia (CLL) management, reviewing its potential as a simple, cost-effective, prognostic biomarker of disease outcome and overall survival that is accessible to widespread clinical practice.
by Dr Alison M. Levoguer, Dr Alex Legg and Dr Richard G. Hughes

CLL has a heterogeneous clinical course
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B lymphocytes in blood, bone marrow and lymphoid tissues. It is the most common form of leukemia in Western populations, has a male to female ratio of 2:1 (median diagnosis age = 72 years) and an incidence rate of 4.1 cases per 100,000 people per year, which has remained stable over the last three decades. The median survival of patients with CLL is around 10 years, however, individual prognosis is highly variable: some patients live for decades with no need for treatment whilst others have a rapidly aggressive clinical form [1]. Deciding when and how to treat patients as they become symptomatic compared to a conservative “watch and wait” strategy, which best maintains a good quality of life for these individuals, has long been the prime issue of CLL management. A clear consensus on best first line treatments remains elusive and, to date, improved survival from early therapy has not been convincingly demonstrated nor is there an effective definition of the truly “high risk” patient. Furthermore, the World Health Organization classification considers both CLL and small lymphocytic lymphoma (SLL) as one single disease entity. CLL exhibits by definition a lymphocytosis in the blood of at least 5 x 109 B lymphocytes /L (5000/µL), the threshold required by CLL guidelines, whilst SLL has lymph node or tissue involvement.

In spite of therapeutic advances CLL remains incurable and its etiology poorly characterized. Clarity regarding the role of “nature versus nurture” in CLL is needed; families exist where multiple CLL cases have been diagnosed. This strongly suggests involvement of a genetic component [2]; both this and the role of environmental factors are areas of active study. Given such a diverse clinical background there is a pressing need for good clinical staging of disease and traditionally this has followed one of two systems – either Rai staging based on progressive accumulation of malignant cells and its sequelae (lymphocytosis, lymphadenopathy, hepatosplenomegaly, anemia and thrombocytopenia) or the Binet system which looks specifically at five sites of involvement (cervical, axillary and inguinal lymph nodes, spleen and liver).

The need for biomarkers to establish CLL prognosis
A variety of biomarkers for genetic features and treatment outcome have been described in CLL which, taken together, allow a “disease profile” to be built up for each clinical case. These include i) clinical stage ii) leukemic cell morphology iii) lymphocyte doubling time iv) infiltration pattern in bone marrow trephine biopsies v) p53 function  vi) cytogenetic abnormalities and vii) serum factors such as beta – 2 microglobulin (β-2M) [3]. The most important and consistent abnormalities are cytogenetic (detectable by fluorescence in situ hybridisation (FISH) and comprising deletions of 13q-, 11q- , 17p- and 6q- and trisomy 12). Mutational status of the immunoglobulin heavy chain variable gene locus (IGVH) gives rise to two distinct clinical behaviors of disease – IGVH mutated and IGVH non – mutated forms. Cases with somatic hypermutation in IGVH have a more indolent clinical course and approximately twice the survival time of unmutated cases. Determining such IGVH mutations still requires dedicated equipment for DNA sequencing; it is time-consuming and expensive, and as such limited to a research tool. Because of this, many attempts have been made to identify different surrogate markers two of which are described below.

CD38 (cyclic ADP ribose hydrolase) is a glycoprotein found on the surface of CD4+, CD8+, B and NK cells. It is encoded on chromosome 4 and is a marker of cell activation. Loss of CD38 function is associated with impaired immune responses [4]. ZAP-70, a member of the protein – tyrosine kinase family and normally expressed on T and NK cells has a critical role in the initiation of T-cell signaling. Recent data suggest that membrane ZAP-70 expression offers potential as an accurate proxy for IGVH gene mutational status [5]. CD38 and ZAP-70 expression can both be obtained, relatively simply, by flow cytometry, thus placing IGVH assessment within the reach of any lab set up to perform this technique. Generally ZAP-70 positive CLL patients are likely to be at a more advanced clinical stage and, together with CD38+ cases, are more likely to have unmutated IGVH [6]. To ensure optimal clinical decisions regarding treatment are taken, all of the aforementioned prognostic markers will require assessment on an individual patient basis [5].
The role of serum free light chain (FLC) in B-cell malignancy management
Since 2001 an immunoassay that allows the measurement of free serum kappa (κ) and lambda (λ) immunoglobulin light chains in serum to a level of 2–4 mg/L has been available. This is a significantly greater sensitivity than that of the older more conventional methods such as immunofixation (IFE) which detects FLCs at a minimum concentration of 100–150 mg/L [7]. Use of the serum FLC assay permits detection of monoclonal protein in some patients where this would have been previously undetectable. An abnormal FLC ratio has been shown to have prognostic value in the progression of monoclonal gammopathy of undetermined significance (MGUS) [8], smoldering myeloma [9], solitary plasmacytoma of bone [10] as well as multiple myeloma at diagnosis [11].

Serum free light chain ratio as a biomarker for CLL
An assessment of the κ/λ FLC ratio can give prognostic information on CLL patient outcome, [12] and for a subset of patients where the tumor is producing significant quantities of free light chain the rapid half life of κ and λ free light chains means that quantitative FLC assays may give a prompt indication of CLL treatment response. This is a logical extension of the already well documented clinical value of the serum FLC assay in light chain only multiple myeloma (LCMM), AL amyloidosis, non-secretory (NSMM) and oligo-secretory multiple myeloma [13]. The first evaluation of serum FLC κ/λ ratio in non-Hodgkin’s lymphoma and chronic lymphocytic leukemia (CLL) in 2007 at the Mayo Clinic found an abnormal serum FLC ratio at unspecified time points in 8 out of 18 (44%) CLL patients and 9 out of 25 (36%) mantle cell lymphoma patients [14].

Subsequently in a much larger three UK center retrospective patient cohort, Guy Pratt and colleagues [15] measured serum FLC for 181 untreated/pre-treatment CLL patients alongside an additional 78 patients further into their disease course who had already received treatment. An abnormal serum FLC ratio was found in 39% (100/259) of the overall CLL cohort [Figure 1] and was prognostic for decreased survival when measured at initial diagnosis for both the entire cohort of 259 patients and the 181 untreated CLL patient cohort [Figure 2]. For the whole group of 259 cases, presence of an abnormal serum FLC ratio correlated with a significantly shorter time to first treatment (P=0.001) on average 69 months earlier than those patients with a normal κ/λ ratio [Figure 3]. 

In summary CLL patients with abnormal sFLC ratios were more likely to have:
(i) Unmutated IGVH status
(ii) Zap-70 positivity
(iii) Lymphocyte doubling time < 12 months
(iv) Higher β-2M
than CLL patients with a normal ratio [15]. The latter two (iii-iv) suggest that abnormal serum FLC ratios in CLL indicate tumor mass and/or proliferation. This has been further characterized recently by the observation that serum FLC >50mg/mL with an abnormal κ/λ ratio can independently identify a group of CLL patients with progressive disease and a poorer outlook [16].

In a separate cohort of 120 CLL patients’ sera (collected before treatment initiation or six months following cessation of therapy), 71 patients (59%) had an abnormal FLC ratio, which was associated with a worse outcome, particularly with a low abnormal FLC ratio [17].

Independently, Yegin and colleagues in Turkey [18] have performed a retrospective study of 101 CLL patients at Gazi University Hospital and reported abnormal/high serum FLC in 55 CLL patients (54.5%), with 30 CLL patients (29.7%) having an abnormal serum FLC ratio – these individuals had shorter median overall survival, an effect detected even in early stage patients. Furthermore, CLL patients with high serum FLC levels and an abnormal serum FLC ratio had significantly higher CD38 expression levels suggesting that elevated serum FLC, the FLC ratio and CD38 expression are associated biomarkers for stimulation of the B-cell receptor on CLL proliferating cells.

Conclusions
Despite the heterogeneous clinical course of CLL the universal factor that characterizes the disease course is abnormal B-cell proliferation with ZAP-70 and CD38 expression giving additional information regarding the level of cellular abnormality. The utility of serum free light chain measurements - indicating polyclonal elevation early in the evolution of the disease or an abnormal sFLC ratio - requires further analysis in larger cohort studies. Such studies will more fully dissect out what this means in terms of patient stratification at initial diagnosis and how it can be helpful in long term clinical management by prompt indication of worse outcome and shorter survival time. It may be that an abnormal sFLC ratio is a biomarker for a biologically different subtype of CLL.
Clarifying the role of serum FLC as a prognostic marker for CLL and its various subtypes may be pertinent in patient management since this is a simple, quick serum test. In the next generation of studies if the serum FLC assay and the κ/λ ratio are utilized on patients treated and followed in a uniform manner the true usefulness of this biomarker as a predictive and/or prognostic indicator for tumor response and overall disease survival may be determined.

References
1. Gribben JG. How I treat CLL up front. Blood 2010; 115(2): 187-197.
2. Montserrat E, Moreno C. Chronic lymphocytic leukaemia: a short overview. Ann Oncol 2008; 19 Suppl 7: vii320-325
3. Mainou-Fowler T, Dignum HM, Proctor SJ, Summerfield GP. The prognostic value of CD38 expression and its quantification in B cell chronic lymphocytic leukemia (B-CLL). Abstract. Leuk Lymphoma Mar 2004; 45(3):455-62.
4. Deaglio S, Mehta K, Malavasi F. Human CD38: a (r)evolutionary story of enzymes and receptors. Leukemia Research 2001; 25: 1-12.
5. Hassanein NM, Perkinson KR, Alcancia F, Goodman BK, Weinberg JB, Lagoo AS. A Single Tube, Four-Color Flow Cytometry Assay for Evaluation of ZAP-70 and CD38 Expression in Chronic Lymphocytic Leukemia. Am J Clin Pathol 2010; 133 (5):708-17
6. Rassenti LZ et al. Relative value of ZAP-70, CD38, and immunoglobulin mutation status in predicting aggressive disease in chronic lymphocytic leukemia.Blood 2008; 112(5): 1923-1930.
7. Bradwell AR et al. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001; 47(4): 673-680.
8. Rajkumar SV et al. Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance.Blood 2005; 106(3): 812-817.
9. Dispenzieri A et al. Immunoglobulin free light chain ratio is an independent risk factor for progression of smoldering (asymptomatic) multiple myeloma. Blood 2008; 111(2): 785-789.
10. Dingli D et al. Immunoglobulin free light chains and solitary plasmacytoma of bone. Blood 2006; 108(6): 1979-1983.
11. Kyrtsonis MC. Prognostic value of serum free light chain ratio at diagnosis in multiple myeloma. Br J Haematol 2007;137(3): 240-243.
12. Pratt G. The evolving use of serum free light chain assays in haematology. Br J Haematol 2008; 141(4): 413-422.
13. Katzmann JA et al. Diagnostic performance of quantitative kappa and lambda free light chain assays in clinical practice. Clin Chem 2005; 51(5): 878-881.
14. Martin W et al. Serum-free light chain-a new biomarker for patients with B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia.Transl Res 2007; 149(4): 231-235.
15. Pratt G et al. Abnormal serum free light chain ratios are associated with poor survival and may reflect biological subgroups in patients with chronic lymphocytic leukaemia. Br J Haematol 2009; 144(2): 217-222.
16. Pratt G, Harding S, Fegan C, Pepper CJ, Oscier D, Gardiner A, Holder R, Bradwell A, Mead G. Serum FLC levels at presentation have independent prognostic significance in CLL and levels above 50mg/L identify patients with progressive disease. Blood (ASH Annual Meeting Abstracts), Nov 2009; 114: 2355.
17. Matschke, J, Eisele L, Sellmann L, Duehrsen U, Duerig J, Nückel H. Abnormal free light chain ratios in chronic lymphocytic leukemia: a new prognostic factor? Blood (ASH Annual Meeting Abstracts), Nov 2009; 114: 1237.
18. Yegin ZA et al. Free light chain: A novel predictor of adverse outcome in chronic lymphocytic leukemia. Eur J Haematol 2010; 84(5): 406-411

The authors
Dr Alison M. Levoguer & Dr Alex Legg
Scientific Affairs
and
Dr Richard G. Hughes
Senior Research Scientist
Binding Site
Birmingham
UK