Anne L Sherwood, PhD

Director of Scientific Affairs

The Binding Site, Inc.

Case Studies

Outline

• Multiple Myeloma Overview

• Three case studies (with audience participation)

Multiple Myeloma

• Cancer of the blood (Haematological malignancy)

• Uncontrolled proliferation of plasma cells derived from a single clone (Plasma cell dyscrasia)

• Accumulation of the myeloma cells in the bone marrow results in:• a. Destruction of bone → pain• b. Abnormal bone marrow function → anemia• c. Secretion of monoclonal protein → intact antibody, free light

chains, or both• d. Suppression of normal antibodies → infection

Palumbo, A, Rajkumar SV. Leukemia 23:449 2009; Kyle , RA, Rajkumar SV. N. Engl. J of Med 351:1860 2004

Multiple Myeloma

• 1% of all cancers in the US

• 2nd most common heme malignancy (#1 NHL)

• 10% of all hematologic malignancies in Caucasians

( Incidence is 4.5 per 100,000 per year)

• 20% of all hematologic malignancies in African Americans

(Incidence is 9.3 per 100,000 per year)

Palumbo, A, Rajkumar SV. Leukemia 23:449 2009

Kyle , RA, Rajkumar SV. N. Engl. J of Med 351:1860 2004

Multiple Myeloma

• The ƒ is constantly increasing due to the aging of the population

• 35% of patients are younger than 65 years of age

• 28% are 65 – 74 years of age

• 37% are > 75 years of age

Palumbo, A, Rajkumar SV. Leukemia 23:449 2009

Kyle , RA, Rajkumar SV. N. Engl. J of Med 351:1860 2004

Risk Factors

• Exposure to pesticides• Agricultural workers

• Vietnam era veterans exposed to Agent Orange

• More common in men

• More common in African Americans

• No apparent association with:• Smoking

• Alcohol consumption

• Familial genetics

Dispenzieri, A. Multiple Myeloma. In: Multiple Myeloma and Related Plasma Cell Disorders. M Gertz and P Greipp, Eds. Springer Press 2004

Signs & Symptoms of Multiple MyelomaNONSPECIFIC

• Bone pain (~58%): lower back, ribs• Fatigue: 32%• Weight loss: 24%• Recurrent infections• Anemia: 73%• Nausea• Hypercalcemia: 13%• Serum creatinine ≥ 2.0 mg/dL: 19%• Hyperviscosity: esp Waldenström’s, IgA• Neurologic: peripheral neuropathy• Asymptomatic or minimal symptoms: 11%

Kyle RA. Mayo Clin Proc. 2003;78:21-33.

Diagnostic Criteria for Symptomatic Multiple Myeloma

C Hypercalcemia

R Renal involvement

A Anemia

B Bone Involvement

(I) Infections CANCER – the CRAB

Vertebral fractures due to myeloma

Plasma cell infiltration in bone

marrow

PET Scans in Multiple Myeloma

Osteolytic Lesions

↑ macrophage

inflammatory

protein 1α

↑ receptor

activator of NF-κB

ligand (RANKL)

↓ osteoprotegerin

Osteoblast

Osteoclast activation

with bone resorption

Kyle RA, Rajkumar SV. N Engl J Med. 2004:351:1860-73.

Types of Myeloma

• Intact immunoglobulin myeloma• Production of intact immunoglobulins (M-protein)

• Commonly called myeloma

• Light chain myeloma• Production of only light chains

• Sometimes called light chain disease

• Nonsecretory myeloma*• M-protein is not present on SPEP, IFE, UPEP, or uIFE

* Now usually referred to as Hyposecretory or Oligosecretory MM

Methods to Detect and/or Quantify Immunoglobulins

• Protein electrophoresis

• Serum (SPEP)

• Urine (UPEP)

• Immunofixation electrophoresis (IFE)

• Capillary zone electrophoresis (CZE)

• Nephelometry and turbidimetry

• Quantitative immunoglobulins: measure levels of IgG, IgA, IgM, IgE, IgD

• Serum free light chain assays: measure levels of kappa, lambda, and their ratio

Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004

Monoclonal

Protein “Spike”

Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd

Edition, 2004

Kyle RA and Rajkumar SV. Cecil Textbook of Medicine,

22nd Edition, 2004

Polyclonal Gammopathy

SPEP With:

IFE – Normal Serum IFE – Elevated Polyclonal IgG

Serum Free Light Chains – Normal Ranges

• Kappa ()

• 95% reference interval: 3.3–19.4 mg/L

• Lambda ()

• 95% reference interval: 5.7–26.3 mg/L

• / ratio

• Normal range: 0.26–1.65

• Renal range: 0.37 – 3.10*

Katzmann J, Clin Chem 48: 1437, 2002

*Hutchison C , BMC Nephrol, 2008

Renal Reference Range

• In renal failure, clearance slows for κ and

• Reference range in normal renal function: 0.26–1.651

• Renal reference range for κ/ ratio: 0.37–3.12,3

• Improved specificity from 93% to 99%

1 Katzmann et al. Clin Chem. 2002;48:1437-1444

2 Hutchison et al. BMC Nephrol. Sept. 2008;9:11

3 Wells et al. Clin Chem 2008 C-91.

Renal Ranges for sFLC

Hutchison et al. BMC Nephrol. Sept. 2008;9:11

0.26–1.65

0.37–3.1

normal

MM

RF

Case Studies

Case #1

• PATIENT HISTORY: A 41 year old businessman from San Diego CA, an avid golfer who loves to engage in playful rough-housing with his 8 year old son, experienced sudden excruciating pain in his mid back during a spirited wrestling match. He iced the injury and took to his bed for a couple of days. Noticing no improvement after exhausting his supply of Vioxx (leftover from a prescription 2 years earlier for a golf injury where he threw his back out), he went to see his primary care physician.

• A Skeletal survey revealed a sizable solitary bone lesion in T11 of his thoracic vertebrae.

• A bone marrow biopsy showed 35% plasma cells in the marrow. Other tests gave the following results:

Calcium: 10.0 mmol (nr = 2.25-2.75)Serum creatinine: 1.2 mg/dL (nr = 0.8 to 1.4 mg/dL)Hemoglobin: 13.0 gm/dL (Female: 12.1 to 15.1 gm/dL; Male: 13.8 to 17.2 gm/dL)

Free Kappa () – 7.2 mg/L (nr = 3.3-19.4 mg/L)Free Lambda () – 607.3 mg/L (nr = 5.7-26.3 mg/L)Ratio (/) – 0.012 (nr = 0.26 – 1.65), (normal renal ratio range = 0.37–3.1)

SPEP

sIFE Example (normal sIFE)

Based on the data presented, what do you think is wrong with this patient?

IIMM- IgG-

(Intact Immunoglobulin multiple myeloma; IgG heavy chain; lambda light chain)

Rationale: Bone lesions, Hypercalcemia, 35% plasma cells in marrow. Classic sharp M spike in gamma region on SPEP. Clear banding in IgG and lanes on sIFE. Results confirmed by Freelite testing.

Conclusion

Case #2

• PATIENT HISTORY: A 68 year old woman from Seattle WA, who enjoys bird watching and trips to the wine country with her husband, and has been fit and generally very healthy all her life, presented with a fractured rib following mild trauma. Over the following months, the pain subsided but she reported feeling breathlessness, vague chest pains and tiredness. Full blood counts, blood biochemistry and chest X-rays all appeared normal at this time. Her symptoms persisted and 9 months after initial presentation, bone scans and X-rays revealed extensive osseous lesions. A bone marrow biopsy showed 72% plasma cells in the marrow. Other tests gave the following results:

• Calcium: 4.05 mmol (nr = 2.25-2.75)

• Serum creatinine: 1.0 mg/dL (nr = 0.8 to 1.4 mg/dL)

• Hemoglobin: 10.7 gm/dL (Female: 12.1 to 15.1 gm/dL; Male: 13.8 to 17.2 gm/dL)

• Free Kappa () – 30 mg/L (nr = 3.3-19.4 mg/L)• Free Lambda () – 6.5 mg/L (nr = 5.7-26.3 mg/L)• Ratio (/) – 4.6 (nr = 0.26 – 1.65), (normal renal ratio range

= 0.37–3.1)

Higher risk of malignant plasma cell disorder is associated with more extreme / ratio

0.05 0.26 1.65 5.0 10.0

Very Low LikelyPossiblePossible InconclusiveLikely

/ ratio :

Risk :

Vermeersch Clin Chem Acta 2009 410: 54-58

0.001 0.26 1.65 1000

Lambda secretor Normal Kappa Secretor

/ ratio

Katzmann J, Clin Chem 2002 48: 1437

SPEP – (No evidence of M protein or unusual banding)

sIFE (Example- normal sIFE )

Based on the data presented, what do you think is wrong with this patient?

Hyposecretory (or Oligosecretory) MM

Rationale:

SPEP and sIFE were both negative. Extensive bone lesions with high calcium, 72% plasma cells in marrow. Freelite analysis showed modest elevation of kappa light chains with / ratio of 4.6 (nr= 0.26-1.65)

Conclusion

Case #3• PATIENT HISTORY: A 72 year old Caucasian male went to see his primary care physician with debilitating bone and joint pain. He had been receiving treatment for several years for a number of chronic conditions, including skin rashes, rheumatoid arthritis and osteoporosis. A comprehensive metabolic panel was run and all test results were found to be normal. There was no evidence of renal impairment, although the patient had been previously diagnosed with congenital multiple renal cysts.

Additional testing was ordered including serum immunofixationelectrophoresis (sIFE) and serum protein electrophoresis (SPEP). The sIFEresults showed no abnormality, but the SPEP demonstrated a broad diffuse area in the gamma region that was initially reported by the lab to be suspicious but 'not clearly monoclonal'. The patient was diagnosed with a monoclonal gammopathy based on these laboratory results. The patient’s physician placed a standing order for SPEP testing to occur every month in order to monitor the patient. The laboratory noticed the frequency of orders as well as the ambiguous finding that lead to the diagnosis of monoclonal gammopathy and recommended serum free light chain testing.

Based on all the findings presented, do you think this patient received a correct diagnosis?

Freelite Results

• Free Kappa () – 250.3 mg/L (nr = 3.3-19.4 mg/L)• Free Lambda () – 134.7 mg/L (nr = 5.7-26.3 mg/L)• Ratio (/) - 1.86 (nr = 0.26 – 1.65), (normal renal ratio range

= 0.37–3.1)

Conclusion

Rationale:Osteoporosis can be a presenting sign of MM or related disorder, especially when found in atypical patients such as men and younger pre-menopausal women. However, the sFLC results indicated that this patient had significantly elevated levels of both kappa and lambda light chains and a marginally abnormal κ/ ratio just above the reference range. In polyclonal gammopathy, intact Igs and sFLCs are both elevated, which may explain the presence of the diffuse broad band in the gamma region of the SPEP gel. Based on this patient’s history, this most likely resulted from chronic inflammation. The unique ability of sFLC testing to differentiate between monoclonal and polyclonal gammopathy helped overturn an initial mis-diagnosis in this case amending the final diagnosis to polyclonal hypergammaglobulinemia caused by inflammation.

Initial diagnosis of monoclonal gammopathy was wrong. Freelite testing confirmed this patient had polyclonal (as opposed to monoclonal) gammopathy.

Questions?