Presentation webinar March 3, 2010

1. PLASMA CELL DISORDERS:NEW STRATEGIES FOR SCREENING , DIAGNOSIS, PROGNOSIS AND MONITORING ParameswaranHari, M.D. Howard S. Robin, M.D. March 3, 2010

2. IMPACT OF LABORATORY TESTING Diagnostic thinking Therapeutic choice Patient outcome Prevention

3. BENCE JONES PROTEINThe First Tumor Marker OSSEOUS MALADIES Multiple Myeloma Bence-Jones, Henry (1813-1873), British nephrologist and clinical pathologist. 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. 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.

4. MONOCLONAL GAMMOPATHIES . Disorders Signs and symptoms Bone pain or fractures Elevated serum and/or urine proteins Anemia and fatigue Renal dysfunction 1026 patients Mayo 1992

5. OBJECTIVES 1. Develop new strategies to screen for and diagnose monoclonal plasma cell gammopathies. 2. Evalulate new effective strategies for monitoring relapse and response to therapy of patients with plasma cell disorders. 3. Acquire knowledge regarding the prognostic value of current tests for monoclonal gammopathies.

6. CASE 1 Previously healthy 75 yr old man Physically active – runs 3 miles /day Exertional dyspnea while running Gave up running after a month Chest discomfort & increasing SOB Cardiopulmonary Tests: Stress test – No inducible ischemia Catheterization – 40% LAD stenosis CT chest – No Pulmonary embolism Echocardiogram – Left Ventricular Hypertrophy

7. DYSPNEA OF UNKNOWN ORIGIN Clinical picture of increasing right heart failure Elevated NT Pro BNP of 1500 Repeat echocardiogram Diastolic dysfunction and worsening septal hypertrophy Differential diagnosis Restrictive cardiomyopathy Cardiac amyloidosis Referral to hematology clinic Serum Protein Electrophoresis (SPEP) Immunofixation (IFE) Urine Protein Electrophoresis (UPEP) – 24 hr urine All Negative for Monoclonal Spike

8. SERUM PROTEIN ELECTROPHORESIS

9. SERUM IMMUNOFIXATIONELECTROPHORESIS

10. PHYSIOLOGY OF LIGHT CHAINS ≤ 1 g/d 500mg/d from normal B and plasma cells k:l production 2:1 Lambda is dimeric and has longer T1/2 T1/2 varies from hrs to 1-2 d (renal function) Filtered freely by glomeruli then reabsorbed and metabolised by prox. tubules Tubules can absorb upto 10-30 gms per day.

11. IMMUNOELECTROPHORESIS Urine IEP Urine IFE

12. TOOLS FOR EVALUATION OF MONOCLONAL GAMMOPATHIES SPEP Immunofixation UPEP on 24-hour urine Serum free light chain assay

13. PLASMA CELLS PRODUCE WHOLE IMMUNOGLOBULIN AND FREE LIGHT CHAINS Kappa Lambda

14. Free Light Chain Kappa Exposed surface Hidden surface Previously hidden surface SERUM FREE LIGHT CHAIN ASSAY Intact Immunoglobulin

15. SERUM FREE LIGHT CHAINS FLC reference range: k 3.3 – 19.4 mg/L l 5.7 – 26.3 mg/L k/l ratio 0.26 - 1.65

16. SCREENING FOR CARDIAC AMYLOIDOSIS Serum Free Light Chains Free kappa – 530 mg/L Free lambda – 16 Increased k:l ratio (33.13) Urine – 950mg/24h of albumin Cardiac MRI scan with Gadolinum

17. MRI Heart septum

18. ELECTRON MICROSCOPY OF FAT PAD ASPIRATE TISSUE CONFIRMATION OF AMYLOID: Marrow Biopsy – 10% Plasma Cells Kappa Light Chains restricted Fat Pad Aspirate - Positive AL AMYLOIDOSIS: SUSPECT Presence of Amyloid Related Syndrome SCREEN Monoclonal plasma cell disorder PROVE Positive Tissue Biopsy (Fat pad, Marrow, Organ) AL by typing – IHC, sequencing etc

19. SCREENING & DIAGNOSIS OF PLASMA CELL DISORDERS Plasma Cell Disorders are a spectrum of illnesses: Myeloma (MM) AL Amyloidosis Light Chain Deposition MGUS (Monoclonal Gammopathy of Unknown Significance) Smoldering MM Plasmacytoma Plasma Cell Leukemia

20. IDEAL SCREENING PANEL FOR MONOCLONAL GAMMOPATHY Serum Protein Electrophoresis ( SPEP) SPEP + Serum Immunofixation (SIFE) SPEP + SIFE + sFLC SPEP + sFLC SPEP + SIFE + sFLC+ UPEP Most comprehensive

21. INCIDENCE OF ABNORMAL sFLC RATIO IN PLASMA CELL GAMMOPATHIES Mead et al BJH 2004 Aug Draysonet al Blood 2001 Bradwellet al Lancet 2003

22. SCREENING FOR PLASMA CELL DISORDERS Screening panels for detection of monoclonal gammopathies. Katzmann JA et al Clin Chem. 2009 Aug;55(8):1517-22.

24. SERUM FREE LIGHT CHAIN ASSAY Serum assay Eliminates need to collect urine specimen More sensitive than SPEP or SIFE Indications: Diagnosis of Plasma Cell Disorders Prognosis & Risk Stratification Response Assessment on therapy Disease Monitoring on follow up

25. CASE 2 A 74 year old woman presented with elevated total protein August 2003 Routine Labs revealed high total protein 9.8 g/dL Normal : CBC, Ca++, Bone X ray screen, Creatinine SPEP & SIFE – IgG Kappa 3.2 g/dl Marrow 23% Plasma cells kappa light chain clone Diagnosis – Smoldering MM (SMM) Risk of Progression to MM?

26. RISK OF PROGRESSION TO MYELOMA OR RELATED DISORDER IN 273 PATIENTS WITH SMOLDERING MYELOMA Dispenzieri, A. et al. Blood 2008;111:785-789 Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.

27. FOLLOW UP OF PATIENT WITH SMM X 100 mg/L Symptomatic Myeloma FLC Ratio = 28.7 Kappa FLC FLC Ratio = 27.8 Hb = 13.5 Hb = 13.0 Hb = 9.8

28. TT3: iFLC Baseline Tertiles 100 80 1 or 2 vs 3, p<0.001 1 vs 2, p=0.08 60 Event free survival, % 40 24-moTertile (mg/L) N estimate 1 (0.6-107) 99 85% 2 (108-746) 102 94% 3 (762-71210) 100 73% 20 0 0 1 2 3 Years SERUM FLC AT DIAGNOSIS IS PROGNOSTIC IN MULTIPLE MYELOMA Data from Arkansas group using involved FLC and EFS after therapy

29. TT3: INFERIOR SURVIVAL WITH TOP-TERTILE SFLC LEVEL AT BASELINE 100% 80% 60% 24-Month 40% Deaths / N Estimate Tertile 1 (0.06-10.7 mg/dL) 9 / 99 89% (81,96) Tertile 2 (10.8-74.6 mg/dL) 4 / 102 94% (88,100) 20% Tertile 3 (76.2-7120 mg/dL) 22 / 100 76% (67,86) p-value Tertiles 1 and 2 vs. 3: <0.001, 1 vs. 2=0.14 0% 0 12 24 36 Months from Enrollment Impact of Serum Free Light Chain (SFLC) on Survival

30. ISS & rFLC 100 Risk OS,score N mo 0 73 51 1 169 39 2 199 30 3 135 22 80 0 1 2 3 4 5 6 7 8 9 60 Overall survival,% 40 20 0 0 2 4 6 8 10 12 14 Years >32 (k) or <0.03 (l) Snozek CL, et al. Leukemia SERUM FLC RATIO AT DIAGNOSIS IS PROGNOSTIC IN MM Data using FLC ratio rFLC 100 80 60 Overall survival,% P=0.0001 40 rFLC N 5-yr survival (%) “Low” 46 82±9 “High” 47 30±11 20 0 Years ‘High rFLC’ >3.6 (k) or < 0.02 (l) Kyrtsonis MC, et al. Br J Haeml. 2007;137:240-243

31. KEY PROGNOSTIC FACTORS FOR PROGRESSION OF MGUS

32. 20 yr. risk of progression after other causes of death 2% 10% 18% 27% MGUS: RISK STRATIFICATION MODEL Absolute risk of progression at 20 yrs. Risk Group 5% 1. Low-risk Serum M protein <1.5 gm/dL, IgG subtype, normal FLC ratio 2. Low/Intermediate-risk Any 1 factor abnormal 21% 3. Hi/Intermediate-risk Any 2 factors abnormal 37% 4. High-risk All 3 factors abnormal 58%

33. SOLITARY BONE PLASMACYTOMA FLC ratio is prognostic in solitary bone plasmacytoma Time to Progression Overall Survival Normal FLC ratio Abnormal FLC ratio Normal FLC ratio Abnormal FLC ratio Dingli, D. et al. Blood 2006;108:1979-1983 Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.

34. WALDENSTROM MACROGLOBULINEMIA: HIGHER DISEASE BURDEN CORRELATES WITH SFLC In WM patients Median sFLC > 60 mg/L was associated with lower hemoglobin and higher B2M sFLC Lower involved FLC 20 mg/L vs. 36 mg/L (p=0.0003) in IgM-MGUS than WM patients Abnormal ratio in 23% vs. 76% (p<0.001) Leleuet al LeukLymph 2008

35. PROGNOSTIC USES IN OTHER SITUATIONS Recovery of renal function in MM Where monitoring for Light Chain elevation is crucial After renal transplant for Light chain deposition disease in remission Sensitive marker for NHL risk in HIV infection 2 -5 years prior to actual Landgren et al J Clin Oncol. 2010 Feb 10;28(5):773-9

36. SERUM FREE LIGHT CHAIN ASSAY:IMPORTANT PROGNOSTIC MARKER Myeloma Smoldering MM MGUS Amyloidosis Plasmacytoma WaldenstromMacroglobulinemia

39. CT Scan of Abdomen and Pelvis CT SCAN OF CHEST/ ABDOMEN/PELVIS Soft tissue plasmacytomas with light chain escape

40. TREATMENT OF LIGHT CHAIN SECRETING PLASMACYTOMAS Patient received D-PACE (Dexamethasone,Cisplatin, Adriamycin,Cytoxan, Etoposide) chemotherapy 2 cycles After cycle 1 ----------------- After cycle 2 FREE KAPPA CHAINS <0.1 (L) mg/L too low FREE LAMBDA CHAINS 741.0 (H) mg/L 2.7mg/L FREE KAPPA/LAMBDA RATIO 0.01 (L) --- CT Scan – complete resolution of masses FLC – resolution of involved LC elevation ProteinHalf Life IgG 20–25 days IgA 6 days IgM 6–8 days Free Kappa 2–4 hours Free Lambda 3–6 hours Rapid clinical confirmation of effective therapy

41. FREE LIGHT CHAIN AS A MARKER OF RESPONSE IN MM Stored samples from a mature ECOG clinical trial (E9486) data Assess serum FLC at baseline and after 2 months of alkylator based therapy in 399 patients FLC response after 2 months of therapy Superior to early M-protein measurement to predict overall response, but did not predict for survival endpoints Dispenzieri A et al.Blood. 2008;111:4908-4915.

42. ROC OF FLC REDUCTION VS. M SPIKE RESPONSE Early drop in FLC after 2 cycles Absolute FLC value or Delta FLC 40% decline in D FLC has : 77% sensitivity and 68% specificity for ultimate M spike reponse Why use D FLC ?

43. SERUM FLC VS. UPEP 24 H FOR FOLLOW UP Low correlation between change in M spike by UPEP and FLC change FOR FOLLOW UP – still need 24 hr UPEP Dispenzieri A et al.Blood. 2008;111:4908-4915.

44. 1 Measurable includes serum M-protein ≥ 10 g/L or a urine M-protein ≥ 200 mg/day for myeloma patients (100 mg/day for AL patients). Gertz MA et al. Am J Hematol. 2005;79:319-328. Durie BG et al. Leukemia. 2006;20:1467-1473. RESPONSE CRITERIA IN MM AND AL INCORPORATE FLC

45. CAVEATS Stringent CR – not well validated for minimal residual disease and long term outcomes SFLC Ratio abnormal despite good remission Oligoclonal reconstitution de Larrea et al Blood. 2009 Dec 3;114(24):4954-6 Temporal discordance when measured as rate of drop or rise of FLC is not concurrent with M spike Can lead to erroneous conclusions

46. CONCORDANCE WITH IFE – DEPENDS ON WHEN MEASURED Singhal et al. Blood July 2009; 114 , 38 2648 serial samples from 122 patients in varying stages of therapy. Serum IFE positive in 88% samples FLC Ratio abnormal in 62% samples FLC Ratio discordant with FLC in 874 samples Serial Measurements of FLC in MM can be discordant with immunofixation results. For e.g – patients undergoing therapy when the FLC normalizes before IFE Relapse when FLC becomes abnormal prior to IFE change

47. Abnormal FLC ratio Lower Limit for FLC Ratio Urine Electrophoresis Neg --------------------------------------------- Positive SFLC ESCAPE FROM PLATEAU Lambda LC elevation FLC escape precedes clinical relapse FLC Ratio abnormal 16 mo prior to UPEP turning positive Clinical Relapse vs. Biochemical progression Time UPEP of positivity

48. ON AVERAGE : 6 MOS. EARLIER INDICATION OF RELAPSE THAN SIFE , ( mg/L) 6-mo Earlier Indication of Relapse Mosbauer et al. Haematologica 2007:92:275-276

49. Overall survival (%) FLC CR? No. Deaths Yes Yes 28 0 Yes No 13 1 No Yes 14 2 No No 32 4 Months CP1312670-3 FLC RESPONSE IS BETTER PREDICTOR OF OS THAN IS IFE RESPONSE IN AL Dispenzieri A, et al. Blood. 2006;107:3378-3383

50. SERUM SFLC ASSAY SCREENING for PLASMA CELL DISORDERS DIAGNOSIS of PLASMA CELL DISORDERS PROGNOSTIC VALUE Monoclonal gammopathy of undermined significance Smoldering myeloma Symptomatic myeloma Plasmacytoma AL amyloidosis HEMATOLOGIC RESPONSE AL amyloidosis “Non-secretory” myeloma Stringent complete response in multiple myeloma ( not fully validated) Light chain deposition disease EARLY RESPONSE TO THERAPY RARE SITUATIONS Free Light Chain escape HIV and NHL Waldenstrom

51. SERUM FLC ASSAY RATIONALE Simple test Predicts outcome Predicts response to therapy Correlates with current prognostic markers Compatible with practice

52. CLINICIAN’S PERSPECTIVE ON LABORATORY TESTING Screening Diagnosis Prognosis Predictive Monitoring

53. GOALS OF PLASMA CELL DYSCRASIA TESTING Improve predictive value of screening Identify candidates for biopsy Identify candidates to treat Determine optimal therapy Predict response to therapy