PLASMA CELL DISORDERS:NEW STRATEGIES FOR

SCREENING , DIAGNOSIS, PROGNOSIS AND MONITORING

Parameswaran Hari, M.D. Howard S. Robin, M.D.

March 3, 2010

IMPACT OF LABORATORY TESTING

• Diagnostic thinking• Therapeutic choice• Patient outcome• Prevention

BENCE JONES PROTEINThe First Tumor Marker

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.

OSSEOUS MALADIES

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

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.

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

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

SERUM PROTEIN ELECTROPHORESIS

SERUM IMMUNOFIXATIONELECTROPHORESIS

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 T1/2T1/2 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.

IMMUNOELECTROPHORESIS

Urine IEP Urine IFE

TOOLS FOR EVALUATION OF MONOCLONAL GAMMOPATHIES

• SPEP• Immunofixation• UPEP on 24-hour urine• Serum free light chain assay

PLASMA CELLS PRODUCE WHOLE IMMUNOGLOBULIN AND FREE LIGHT CHAINS

Kappa Lambda

Intact Immunoglobulin

Exposed surface

Hidden surface

Kappa

Free Light Chain

Previouslyhidden surface

SERUM FREE LIGHT CHAIN ASSAY

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

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

MRI Heart

septum

ELECTRON MICROSCOPY OF FAT PAD ASPIRATE

TISSUE CONFIRMATION OF AMYLOID:Marrow Biopsy – 10% Plasma Cells

Kappa Light Chains restrictedFat 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

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

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

Condition N Abnormal FLC ratio %

Multiple myeloma (MM)

Symptomatic MM 1706 96

“Non secretory” MM 33 73

Light chain MM 252 100

MGUS 1262 33

AL amyloidosis 467 95

LCDD 28 93

Smoldering MM 345 90

Plasmacytoma 116 47

INCIDENCE OF ABNORMAL sFLC RATIO IN PLASMA CELL GAMMOPATHIES

Mead et al BJH 2004 Aug Drayson et al Blood 2001 Bradwell et al Lancet 2003

SCREENING FOR PLASMA CELL DISORDERS

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

NSPEP+sIFE+uIFE+FLC

SPEP+sIFE +uIFE

SPEP+sIFE+FLC

SPEP+FLC sIFE FLC

N 1877 1851 1821 1828 1770 1632 1395

ALL 98.6 97 97.4 94.3 87 74.3

MM 467 100 98.7 100 100 94.4 96.8

sMM 191 100 100 100 99.5 98.4 81.2

MGUS 524 100 100 97.1 88.7 92.8 42.4

Amyloid 581 98.1 94.2 97.1 96.2 73.8 88.3

Macroglobulinemia 26 100 100 100 100 100 73.1

Plasmacytoma 29 89.7 89.7 89.7 86.2 72.4 55.2

428 Patients with Monoclonal Urinary Protein100 80.8 85.7 93.5 99.5

0

20

40

60

80

100

Ra

te o

f P

os

itiv

e P

ati

en

ts,

%

Urine IFE

Serum PEP

Serum FLC

Serum PEP+IFE

Serum PEP+IFE+FLC

Serum PEP/IFE false negative 28 (6.5%):

Primary Amyloid, n = 19 Plasmacytoma, n = 3 MGUS, n = 3 Multiple Myeloma, n = 2Smoldering MM, n = 1

Serum PEP/IFE or FLC ratio 426 (99.5%) 1 Idiopathic BJ protein MGUS (?contamination)

RELATIVE SENSITIVITY OF ASSAYS

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

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?

Copyright ©2008 American Society of Hematology. Copyright restrictions may apply.

Dispenzieri, A. et al. Blood 2008;111:785-789

RISK OF PROGRESSION TO MYELOMA OR RELATED DISORDER IN 273 PATIENTS WITH SMOLDERING MYELOMA

Kapp

a FL

C

X 100 mg/L

Hb = 13.5 Hb = 13.0 Hb = 9.8

Symptomatic MyelomaFLC Ratio = 28.7

FLC Ratio = 27.8

FOLLOW UP OF PATIENT WITH SMM

24-moTertile (mg/L) N estimate1 (0.6-107) 99 85% 2 (108-746) 102 94%3 (762-71210) 100 73%

Years

TT3: iFLC Baseline Tertiles

Eve

nt

free

su

rviv

al,

%

1 or 2 vs 3, p<0.0011 vs 2, p=0.08

0

20

40

60

80

100

0 1 2 3

SERUM FLC AT DIAGNOSIS IS PROGNOSTIC IN MULTIPLE MYELOMA

Data from Arkansas group using involved FLC and EFS after therapy

TT3: INFERIOR SURVIVAL WITH TOP-TERTILE SFLC LEVEL AT BASELINE

0%

20%

40%

60%

80%

100%

0 12 24 36Months from Enrollment

Tertile 1 (0.06-10.7 mg/dL)Tertile 2 (10.8-74.6 mg/dL)Tertile 3 (76.2-7120 mg/dL)

Deaths / N9 / 99

4 / 10222 / 100

24-MonthEstimate

89% (81,96)94% (88,100)76% (67,86)

p-value Tertiles 1 and 2 vs. 3: <0.001, 1 vs. 2=0.14

Impact of Serum Free Light Chain (SFLC) on Survival

Years

rFLC N 5-yr survival (%)“Low” 46 82±9“High” 47 30±11

Ove

rall

surv

ival

,%

rFLC

P=0.0001

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9

Kyrtsonis MC, et al. Br J Haeml. 2007;137:240-243

SERUM FLC RATIO AT DIAGNOSIS IS PROGNOSTIC IN MM

‘High rFLC’ >3.6 ( )k or < 0.02 ( )lYears

Risk OS,score N mo 0 73 51 1 169 39 2 199 30 3 135 22

ISS & rFLC

Ove

rall

surv

ival

,%

0

20

40

60

80

100

0 2 4 6 8 10 12 14

Snozek CL, et al. Leukemia

>32 (k) or <0.03 (l)

Data using FLC ratio

KEY PROGNOSTIC FACTORS FOR PROGRESSION OF MGUS

Prognostic factor Hazard ratio(95% C.I.)

p value

Abnormal FLC ratio(<0.26 or 1.65)

2.6 <0.001

Serum M protein size 2.4 <0.001

IgA, IgM, or biclonal IgA plus IgM

2.6 <0.001

MGUS: RISK STRATIFICATION MODEL

58%4. High-risk

All 3 factors abnormal

37%3. Hi/Intermediate-risk

Any 2 factors abnormal

21%2. Low/Intermediate-risk

Any 1 factor abnormal

5%1. Low-risk Serum M protein <1.5 gm/dL, IgG subtype,

normal FLC ratio

27%

18%

10%

2%

20 yr. risk of progression after

other causes of death

Absolute risk of progression at 20 yrs.

Risk Group

Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.

Dingli, D. et al. Blood 2006;108:1979-1983

Time to Progression

SOLITARY BONE PLASMACYTOMA

Overall Survival

Normal FLC ratio

Abnormal FLC ratio

Abnormal FLC ratio

Normal FLC ratio

FLC ratio is prognostic in solitary bone plasmacytoma

INVOLVED LC FLC<=60 mg/L FLC>60 mg/L

Hemoglobin(g/dL) N % N % p

<10 6 (10) 10 (26) 0.05

<11.5 11 (19) 20 (51) 0.0009

IgM (g/dL)

>4 3 (5) 13 (33) 0.0004

>7 1 (2) 3 (8) 0.30

B2M (>3 mg/L) 12 (20) 23 (59) 0.0002

WALDENSTROM MACROGLOBULINEMIA:HIGHER DISEASE BURDEN CORRELATES WITH SFLC

In WM patientsMedian 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)

Leleu et al Leuk Lymph 2008

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

SERUM FREE LIGHT CHAIN ASSAY:IMPORTANT PROGNOSTIC MARKER

• Myeloma • Smoldering MM• MGUS• Amyloidosis• Plasmacytoma• Waldenstrom Macroglobulinemia

CASE 3

• 65 yr old man with symptomatic anemia• Urine light chains - >5gms in 24hrs of lambda light chains• SPEP & IFE – IgG lambda 2.2g/dl• Marrow – 62% Plasma Cells• SYMPOMATIC MYELOMA

• Treated with VAD chemotherapy with good response• Creatinine normalised• Complete Remission IFE negative

• Autologous Transplant - 10/2002• Continued in CR till 11/2003

PATIENT WITH MYELOMA NOW IN CR

• Patient presented to clinic with increasing fatigue• CBC:

– Hb 11.3, WBC 5.4, Plts 132– Creatinine 1.4 (baseline)– UPEP – no monoclonal bands– SPEP – no monoclonal protein

• Metastatic Bone Survey – NO new lesions• Serum free light chains: March 10 2006 April 3 2006

– FREE KAPPA CHAINS 12.7 mg/L 1.5

– FREE LAMBDA CHAINS 451.0 (H) mg/L 4430

– FREE KAPPA/LAMBDA RATIO 0.03 (L) 0.001

– Rising Free Light Chains – No Clinical Relapse (yet)!

No evidence of relapse by M spike or clinical criteria

CT Scan of Abdomen and PelvisCT SCAN OF CHEST/ ABDOMEN/PELVIS

Soft tissue plasmacytomas with light chain escape

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

Protein Half LifeIgG 20–25 daysIgA 6 daysIgM 6–8 daysFree Kappa 2–4 hoursFree Lambda 3–6 hours

Rapid clinical confirmation of effective therapy

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.

ROC OF FLC REDUCTION VS. M SPIKE RESPONSE

Early drop in FLC after 2 cyclesAbsolute FLC value orDelta FLC 40% decline in D FLC has :

77% sensitivity and 68% specificity for ultimate M spike reponse

Why use D FLC ?

SERUM FLC VS. UPEP 24 H FOR FOLLOW UP

Dispenzieri A et al. Blood. 2008;111:4908-4915.

Low correlation between change in M spike by UPEP and FLC changeFOR FOLLOW UP – still need 24 hr UPEP

PR CR sCR Prog

No measurable disease1

AL iFLC ↓ 50%Nl rFLC & CR by IFE & BM

NDiFLC ↑ 50% (>

100 mg/L)

MMdFLC ↓

50%Nl rFLC & CR by IFE & BM

↑ 50% dFLC

Measurable disease1

MMNl rFLC & CR by IFE & BM

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

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

CONCORDANCE WITH IFE – DEPENDS ON WHEN MEASURED

Serial Measurements of FLC in MM can be discordant with immunofixation results.

For e.g – patients undergoing therapy when the FLC normalizes before IFERelapse when FLC becomes abnormal prior to IFE change

Singhal et al. Blood July 2009; 114 , 382648 serial samples from 122 patients in varying stages of therapy.Serum IFE positive in 88% samplesFLC Ratio abnormal in 62% samples

FLC Ratio discordant with FLC in 874 samples

Lower Limit for FLC Ratio

Urine ElectrophoresisNeg --------------------------------------------- Positive

Abnormal FLC ratio

SFLC ESCAPE FROM PLATEAU

• FLC escape precedes clinical relapse

• FLC Ratio abnormal 16 mo prior to UPEP turning positive

• Clinical Relapse vs. Biochemical progression

Time UPEP of positivity

Lambda LC elevation

ON AVERAGE : 6 MOS. EARLIER INDICATION OF RELAPSE THAN SIFE

6-mo Earlier Indication of Relapse

Mosbauer et al. Haematologica 2007:92:275-276

, ( m

g/L)

0

20

40

60

80

100

0 20 40 60 80 100 120

Ove

rall

su

rviv

al (

%)

Months

FLC CR? No. DeathsYes Yes 28 0Yes No 13 1No Yes 14 2No No 32 4

FLC RESPONSE IS BETTER PREDICTOR OF OS THAN IS IFE RESPONSE IN AL

Dispenzieri A, et al. Blood. 2006;107:3378-3383

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

SERUM FLC ASSAY RATIONALE

• Simple test• Predicts outcome• Predicts response to therapy• Correlates with current prognostic markers• Compatible with practice

CLINICIAN’S PERSPECTIVE ON LABORATORY TESTING

• Screening

• Diagnosis

• Prognosis

• Predictive

• Monitoring

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