Serum Free Light Chains


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  • Protein electrophoresis strip showing (1) normal plasma, (2) polyclonal hypergammaglobulinaemia, (3) serum M protein, and (4) urine M protein (Bence Jones proteinuria) and albuminuria.
  • Serum Free Light Chains

    1. 1. PLASMA CELL DISORDERS:NEW STRATEGIES FOR SCREENING , DIAGNOSIS, PROGNOSIS AND MONITORING<br />ParameswaranHari, M.D. <br />Howard S. Robin, M.D.<br />March 3, 2010<br />
    2. 2. IMPACT OF LABORATORY TESTING<br />Diagnostic thinking<br />Therapeutic choice<br />Patient outcome<br />Prevention<br />
    3. 3. BENCE JONES PROTEINThe First Tumor Marker<br />OSSEOUS MALADIES<br />Multiple Myeloma<br />Bence-Jones, Henry (1813-1873), British nephrologist and clinical pathologist. <br />In 1848 Bence-Jones published an article announcing his discovery of a special protein in the urine of patients suffering from softening of the bones. <br />The first physician to note the occurrence of xanthine in urine, Bence-Jones was also a recognized authority on diseases of the stomach and kidneys.<br />
    4. 4. MONOCLONAL GAMMOPATHIES<br />.<br />Disorders<br />Signs and symptoms<br />Bone pain or fractures<br />Elevated serum and/or urine proteins<br />Anemia and fatigue<br />Renal dysfunction<br />1026 patients Mayo 1992<br />
    5. 5. OBJECTIVES<br />1. Develop new strategies to screen for and diagnose monoclonal plasma cell gammopathies.<br />2. Evalulate new effective strategies for monitoring relapse and response to therapy of patients with plasma cell disorders.<br />3. Acquire knowledge regarding the prognostic value of current tests for monoclonal gammopathies. <br />
    6. 6. CASE 1<br />Previously healthy 75 yr old man<br />Physically active – runs 3 miles /day<br />Exertional dyspnea while running<br />Gave up running after a month<br />Chest discomfort & increasing SOB<br />Cardiopulmonary Tests:<br />Stress test – No inducible ischemia<br />Catheterization – 40% LAD stenosis<br />CT chest – No Pulmonary embolism<br />Echocardiogram – Left Ventricular Hypertrophy<br />
    7. 7. DYSPNEA OF UNKNOWN ORIGIN<br />Clinical picture of increasing right heart failure<br />Elevated NT Pro BNP of 1500<br />Repeat echocardiogram<br />Diastolic dysfunction and worsening septal hypertrophy<br />Differential diagnosis <br />Restrictive cardiomyopathy<br />Cardiac amyloidosis<br />Referral to hematology clinic<br />Serum Protein Electrophoresis (SPEP)<br />Immunofixation (IFE)<br />Urine Protein Electrophoresis (UPEP) – 24 hr urine <br />All Negative for Monoclonal Spike<br />
    10. 10. PHYSIOLOGY OF LIGHT CHAINS <br />≤ 1 g/d<br />500mg/d from normal B and plasma cells<br />k:l production 2:1<br />Lambda is dimeric and has longer T1/2<br />T1/2 varies from hrs to 1-2 d (renal function)<br />Filtered freely by glomeruli then reabsorbed and metabolised by prox. tubules <br />Tubules can absorb upto 10-30 gms per day.<br />
    11. 11. IMMUNOELECTROPHORESIS<br />Urine IEP<br />Urine IFE<br />
    12. 12. TOOLS FOR EVALUATION OF MONOCLONAL GAMMOPATHIES<br />SPEP<br />Immunofixation<br />UPEP on 24-hour urine<br />Serum free light chain assay<br />
    14. 14. Free Light Chain<br />Kappa<br />Exposed surface<br />Hidden surface<br />Previously<br />hidden <br />surface<br />SERUM FREE LIGHT CHAIN ASSAY<br />Intact Immunoglobulin<br />
    15. 15. SERUM FREE LIGHT CHAINS<br />FLC reference range:<br />k 3.3 – 19.4 mg/L <br />l 5.7 – 26.3 mg/L <br />k/l ratio 0.26 - 1.65<br />
    16. 16. SCREENING FOR CARDIAC AMYLOIDOSIS<br />Serum Free Light Chains<br />Free kappa – 530 mg/L<br />Free lambda – 16<br />Increased k:l ratio (33.13)<br />Urine – 950mg/24h of albumin <br />Cardiac MRI scan with Gadolinum<br />
    17. 17. MRI Heart<br />septum<br />
    18. 18. ELECTRON MICROSCOPY OF FAT PAD ASPIRATE<br />TISSUE CONFIRMATION OF AMYLOID:<br />Marrow Biopsy – 10% Plasma Cells <br /> Kappa Light Chains restricted<br />Fat Pad Aspirate - Positive<br />AL AMYLOIDOSIS:<br />SUSPECT<br />Presence of Amyloid Related Syndrome <br />SCREEN<br />Monoclonal plasma cell disorder <br />PROVE<br />Positive Tissue Biopsy (Fat pad, Marrow, Organ) <br />AL by typing – IHC, sequencing etc <br />
    19. 19. SCREENING & DIAGNOSIS OF PLASMA CELL DISORDERS<br />Plasma Cell Disorders are a spectrum of illnesses:<br />Myeloma (MM) <br />AL Amyloidosis<br />Light Chain Deposition<br />MGUS (Monoclonal Gammopathy of Unknown Significance)<br />Smoldering MM<br />Plasmacytoma<br />Plasma Cell Leukemia<br />
    20. 20. IDEAL SCREENING PANEL FOR MONOCLONAL GAMMOPATHY<br />Serum Protein Electrophoresis ( SPEP) <br />SPEP + Serum Immunofixation (SIFE)<br />SPEP + SIFE + sFLC<br />SPEP + sFLC<br />SPEP + SIFE + sFLC+ UPEP <br />Most comprehensive<br />
    21. 21. INCIDENCE OF ABNORMAL sFLC RATIO IN PLASMA CELL GAMMOPATHIES<br />Mead et al BJH 2004 Aug Draysonet al Blood 2001 Bradwellet al Lancet 2003<br />
    22. 22. SCREENING FOR PLASMA CELL DISORDERS<br />Screening panels for detection of monoclonal gammopathies.<br />Katzmann JA et al Clin Chem. 2009 Aug;55(8):1517-22.<br />
    23. 23. 100<br />80.8<br />85.7<br />93.5<br />99.5<br />100<br />80<br />60<br />Rate of Positive Patients, %<br />40<br />20<br />0<br />Urine IFE<br />Serum PEP<br />Serum FLC<br />Serum PEP+IFE<br />Serum PEP+IFE+FLC<br />RELATIVE SENSITIVITY OF ASSAYS <br />428 Patients with Monoclonal Urinary Protein<br />Serum PEP/IFE false negative 28 (6.5%):<br />Primary Amyloid, n = 19<br /> Plasmacytoma, n = 3<br /> MGUS, n = 3<br /> Multiple Myeloma, n = 2<br /><ul><li>SmolderingMM, n = 1</li></ul>Serum PEP/IFE or FLC ratio 426 (99.5%)<br /> 1 Idiopathic BJ protein<br /> MGUS (?contamination)<br />
    24. 24. SERUM FREE LIGHT CHAIN ASSAY<br />Serum assay<br />Eliminates need to collect urine specimen<br />More sensitive than SPEP or SIFE<br />Indications:<br />Diagnosis of Plasma Cell Disorders<br />Prognosis & Risk Stratification<br />Response Assessment on therapy<br />Disease Monitoring on follow up<br />
    25. 25. CASE 2<br />A 74 year old woman presented with elevated total protein August 2003<br />Routine Labs revealed high total protein 9.8 g/dL<br />Normal : CBC, Ca++, Bone X ray screen, Creatinine<br />SPEP & SIFE – IgG Kappa 3.2 g/dl<br />Marrow 23% Plasma cells kappa light chain clone<br />Diagnosis – Smoldering MM (SMM)<br />Risk of Progression to MM?<br />
    26. 26. RISK OF PROGRESSION TO MYELOMA OR RELATED DISORDER IN <br />273 PATIENTS WITH SMOLDERING MYELOMA<br />Dispenzieri, A. et al. Blood 2008;111:785-789<br />Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.<br />
    27. 27. FOLLOW UP OF PATIENT WITH SMM<br />X 100 mg/L<br />Symptomatic <br />Myeloma<br />FLC Ratio = 28.7<br />Kappa FLC<br />FLC Ratio = 27.8<br />Hb = 13.5<br />Hb = 13.0<br />Hb = 9.8<br />
    28. 28. TT3: iFLC Baseline Tertiles<br />100<br />80<br />1 or 2 vs 3, p<0.001<br />1 vs 2, p=0.08<br />60<br />Event free survival, %<br />40<br />24-moTertile (mg/L) N estimate<br />1 (0.6-107) 99 85% <br />2 (108-746) 102 94%<br />3 (762-71210) 100 73%<br />20<br />0<br />0<br />1<br />2<br />3<br />Years<br />SERUM FLC AT DIAGNOSIS IS PROGNOSTIC IN MULTIPLE MYELOMA<br />Data from Arkansas group using involved FLC and EFS after therapy <br />
    29. 29. TT3: INFERIOR SURVIVAL WITH TOP-TERTILE SFLC LEVEL AT BASELINE<br />100%<br />80%<br />60%<br />24-Month<br />40%<br />Deaths / N<br />Estimate<br />Tertile 1 (0.06-10.7 mg/dL)<br />9 / 99<br />89% (81,96)<br />Tertile 2 (10.8-74.6 mg/dL)<br />4 / 102<br />94% (88,100)<br />20%<br />Tertile 3 (76.2-7120 mg/dL)<br />22 / 100<br />76% (67,86)<br />p-value Tertiles 1 and 2 vs. 3: <0.001, 1 vs. 2=0.14<br />0%<br />0<br />12<br />24<br />36<br />Months from Enrollment<br />Impact of Serum Free Light Chain (SFLC) on Survival<br />
    30. 30. ISS & rFLC<br />100<br />Risk OS,score N mo<br /> 0 73 51<br /> 1 169 39<br /> 2 199 30<br /> 3 135 22<br />80<br />0<br />1<br />2<br />3<br />4<br />5<br />6<br />7<br />8<br />9<br />60<br />Overall survival,%<br />40<br />20<br />0<br />0<br />2<br />4<br />6<br />8<br />10<br />12<br />14<br />Years<br />>32 (k) or <0.03 (l) <br />Snozek CL, et al. Leukemia<br />SERUM FLC RATIO AT DIAGNOSIS IS PROGNOSTIC IN MM<br />Data using FLC ratio<br />rFLC<br />100<br />80<br />60<br />Overall survival,%<br />P=0.0001<br />40<br />rFLC N 5-yr survival (%)<br />“Low” 46 82±9 <br />“High” 47 30±11 <br />20<br />0<br />Years<br />‘High rFLC’ >3.6 (k) or < 0.02 (l)<br />Kyrtsonis MC, et al. Br J Haeml. 2007;137:240-243<br />
    32. 32. 20 yr. risk of progression after other causes of death<br />2%<br />10%<br />18%<br />27%<br />MGUS: RISK STRATIFICATION MODEL<br />Absolute risk of progression at 20 yrs.<br />Risk Group<br />5%<br />1. Low-risk<br />Serum M protein <1.5 gm/dL, IgG subtype, <br />normal FLC ratio <br />2. Low/Intermediate-risk <br />Any 1 factor abnormal<br />21%<br />3. Hi/Intermediate-risk<br />Any 2 factors abnormal<br />37%<br />4. High-risk<br />All 3 factors abnormal<br />58%<br />
    33. 33. SOLITARY BONE PLASMACYTOMA<br />FLC ratio is prognostic in solitary bone plasmacytoma<br />Time to Progression<br />Overall Survival<br />Normal FLC ratio<br />Abnormal FLC ratio<br />Normal FLC ratio<br />Abnormal FLC ratio<br />Dingli, D. et al. Blood 2006;108:1979-1983<br />Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.<br />
    34. 34. WALDENSTROM MACROGLOBULINEMIA:<br />HIGHER DISEASE BURDEN CORRELATES WITH SFLC<br />In WM patients<br />Median sFLC > 60 mg/L was associated with lower hemoglobin and higher B2M<br />sFLC<br />Lower involved FLC 20 mg/L vs. 36 mg/L (p=0.0003) in IgM-MGUS than WM patients <br />Abnormal ratio in 23% vs. 76% (p<0.001) <br />Leleuet al LeukLymph 2008<br />
    35. 35. PROGNOSTIC USES IN OTHER SITUATIONS<br />Recovery of renal function in MM<br />Where monitoring for Light Chain elevation is crucial<br />After renal transplant for Light chain deposition disease in remission <br />Sensitive marker for NHL risk in HIV infection 2 -5 years prior to actual<br />Landgren et al J Clin Oncol. 2010 Feb 10;28(5):773-9<br />
    36. 36. SERUM FREE LIGHT CHAIN ASSAY:IMPORTANT PROGNOSTIC MARKER<br />Myeloma <br />Smoldering MM<br />MGUS<br />Amyloidosis<br />Plasmacytoma<br />WaldenstromMacroglobulinemia<br />
    37. 37. CASE 3<br />65 yr old man with symptomatic anemia<br />Urine light chains - >5gms in 24hrs of lambda light chains<br />SPEP & IFE – IgG lambda 2.2g/dl<br />Marrow – 62% Plasma Cells<br />SYMPOMATIC MYELOMA<br />Treated with VAD chemotherapy with good response<br />Creatinine normalised<br />Complete Remission IFE negative<br /><ul><li>Autologous Transplant - 10/2002
    38. 38. Continued in CR till 11/2003</li></li></ul><li>PATIENT WITH MYELOMA NOW IN CR<br />Patient presented to clinic with increasing fatigue<br />CBC:<br />Hb 11.3, WBC 5.4, Plts 132<br />Creatinine 1.4 (baseline)<br />UPEP – no monoclonal bands<br />SPEP – no monoclonal protein<br />Metastatic Bone Survey – NO new lesions<br />Serum free light chains: March 10 2006 April 3 2006<br />FREE KAPPA CHAINS 12.7 mg/L 1.5<br />FREE LAMBDA CHAINS 451.0 (H) mg/L 4430<br />FREE KAPPA/LAMBDA RATIO 0.03 (L) 0.001<br />Rising Free Light Chains – No Clinical Relapse (yet)!<br />No evidence of relapse by M spike <br />or clinical criteria<br />
    39. 39. CT Scan of Abdomen and Pelvis<br />CT SCAN OF CHEST/ ABDOMEN/PELVIS<br />Soft tissue plasmacytomas with light chain escape<br />
    40. 40. TREATMENT OF LIGHT CHAIN SECRETING PLASMACYTOMAS<br />Patient received D-PACE (Dexamethasone,Cisplatin, Adriamycin,Cytoxan, Etoposide) chemotherapy<br />2 cycles <br />After cycle 1 ----------------- After cycle 2 <br />FREE KAPPA CHAINS <0.1 (L) mg/L too low<br />FREE LAMBDA CHAINS 741.0 (H) mg/L 2.7mg/L<br />FREE KAPPA/LAMBDA RATIO 0.01 (L) ---<br />CT Scan – complete resolution of masses<br />FLC – resolution of involved LC elevation<br />ProteinHalf Life<br />IgG 20–25 days<br />IgA 6 days<br />IgM 6–8 days<br />Free Kappa 2–4 hours<br />Free Lambda 3–6 hours<br />Rapid clinical confirmation of effective therapy<br />
    41. 41. FREE LIGHT CHAIN AS A MARKER OF RESPONSE IN MM<br />Stored samples from a mature ECOG clinical trial (E9486) data<br />Assess serum FLC at baseline and after 2 months of alkylator based therapy in 399 patients <br />FLC response after 2 months of therapy <br />Superior to early M-protein measurement to predict overall response, but did not predict for survival endpoints<br />Dispenzieri A et al.Blood. 2008;111:4908-4915.<br />
    42. 42. ROC OF FLC REDUCTION VS. M SPIKE RESPONSE<br />Early drop in FLC after 2 cycles<br />Absolute FLC value or<br />Delta FLC <br />40% decline in D FLC has : <br /> 77% sensitivity and 68% specificity for ultimate M spike reponse<br />Why use D FLC ?<br />
    43. 43. SERUM FLC VS. UPEP 24 H FOR FOLLOW UP<br />Low correlation between change in M spike by UPEP and FLC change<br />FOR FOLLOW UP – still need 24 hr UPEP <br />Dispenzieri A et al.Blood. 2008;111:4908-4915.<br />
    44. 44. 1 Measurable includes serum M-protein ≥ 10 g/L or a urine M-protein ≥ 200 mg/day<br /> for myeloma patients (100 mg/day for AL patients). <br />Gertz MA et al. Am J Hematol. 2005;79:319-328.<br />Durie BG et al. Leukemia. 2006;20:1467-1473.<br />RESPONSE CRITERIA IN MM AND AL INCORPORATE FLC<br />
    45. 45. CAVEATS<br />Stringent CR – not well validated for minimal residual disease and long term outcomes<br />SFLC Ratio abnormal despite good remission<br />Oligoclonal reconstitution<br />de Larrea et al Blood. 2009 Dec 3;114(24):4954-6<br />Temporal discordance when measured as rate of drop or rise of FLC is not concurrent with M spike<br />Can lead to erroneous conclusions<br />
    46. 46. CONCORDANCE WITH IFE – DEPENDS ON WHEN MEASURED<br />Singhal et al. Blood July 2009; 114 , 38<br />2648 serial samples from 122 patients in varying stages of therapy.<br />Serum IFE positive in 88% samples<br />FLC Ratio abnormal in 62% samples<br />FLC Ratio discordant with FLC in 874 samples<br />Serial Measurements of FLC in MM can be discordant with immunofixation results.<br />For e.g – patients undergoing therapy when the FLC normalizes before IFE<br />Relapse when FLC becomes abnormal prior to IFE change<br />
    47. 47. Abnormal FLC ratio <br />Lower Limit for FLC Ratio<br />Urine Electrophoresis<br />Neg --------------------------------------------- Positive<br />SFLC ESCAPE FROM PLATEAU<br />Lambda LC elevation<br />FLC escape precedes clinical relapse<br />FLC Ratio abnormal 16 mo prior to UPEP turning positive<br />Clinical Relapse vs. Biochemical progression<br />Time UPEP of positivity<br />
    48. 48. ON AVERAGE : 6 MOS. EARLIER INDICATION OF RELAPSE THAN SIFE<br />, ( mg/L)<br />6-mo Earlier Indication of Relapse<br />Mosbauer et al. Haematologica 2007:92:275-276<br />
    49. 49. Overall survival (%)<br />FLC CR? No. Deaths<br /> Yes Yes 28 0<br /> Yes No 13 1<br /> No Yes 14 2<br /> No No 32 4<br />Months<br />CP1312670-3<br />FLC RESPONSE IS BETTER PREDICTOR OF OS THAN IS IFE RESPONSE IN AL<br />Dispenzieri A, et al. Blood. 2006;107:3378-3383<br />
    50. 50. SERUM SFLC ASSAY<br />SCREENING for PLASMA CELL DISORDERS<br />DIAGNOSIS of PLASMA CELL DISORDERS<br /> <br />PROGNOSTIC VALUE<br />Monoclonal gammopathy of undermined significance<br />Smoldering myeloma<br />Symptomatic myeloma<br />Plasmacytoma<br />AL amyloidosis<br /> <br />HEMATOLOGIC RESPONSE<br />AL amyloidosis<br />“Non-secretory” myeloma<br />Stringent complete response in multiple myeloma ( not fully validated)<br />Light chain deposition disease<br />EARLY RESPONSE TO THERAPY<br />RARE SITUATIONS <br />Free Light Chain escape <br />HIV and NHL<br />Waldenstrom<br />
    51. 51. SERUM FLC ASSAY RATIONALE<br />Simple test<br />Predicts outcome<br />Predicts response to therapy<br />Correlates with current prognostic markers<br />Compatible with practice<br />
    52. 52. CLINICIAN’S PERSPECTIVE ON LABORATORY TESTING<br />Screening<br />Diagnosis<br />Prognosis<br />Predictive<br />Monitoring<br />
    53. 53. GOALS OF PLASMA CELL DYSCRASIA TESTING <br />Improve predictive value of screening<br />Identify candidates for biopsy<br />Identify candidates to treat<br />Determine optimal therapy<br />Predict response to therapy<br />