New Approaches to Staging
and Monitoring
BRIAN DURIE, M.D.
CEDARS SINAI OUTPATIENT CANCER CENTER,
LOS ANGELES
April 2005
Sydney Australia

FOCUS for PRESENTATION
IMPORTANCE OF
PRECISE DIAGNOSIS
PRETREATMENT PROGNOSTIC
FACTOR CLASSIFICATION
NEW UNDERSTANDING OF DISEASE
BIOLOGY FROM IMAGING
PET or CT/PET or MRI
CRITICAL ROLE OF NEW MOLECULAR
PATTERN CLASSIFICATIONS
EXAMPLE OF CIRCULATING NUCLEOTIDES
(RNA/DNA)

HOW PATIENTS PRESENT 2005
BONE ISSUES
30%
ROUTINE PHYSICAL
25%*
ELEVATED urine/serum protein
15%
ANEMIA/FATIGUE
15%
INFECTION
3%
RENAL DYSFUNCTION
2%
MISCELLANEOUS
10%
* IMF: Myeloma Interactive 2003/2004/2005

DIAGNOSTIC WORK UP
Standard
Newer
Complete blood counts
* MRI; PET; CT/PET
Chemistry profile
Sß ; Albumin
* Serum FREELITE
2
CRP; LDH
* Cytogenetics/FISH
SPEP: IFE/IEP
* Molecular testing:
UPEP on 24 hr urine
GEP; SNPs;
Quant. Immunoglobulins
proteomics;
Bone marrow asp/biopsy
RNA/DNA
Skeletal survey
sequencing

NEW CLINICAL CRITERIA **
MGUS
PC < 10%; SPIKE < 3.0G/DL
Smoldering
not MGUS or MM; D/S stage IA
Myeloma (MM*) 1 or more of
Calcium elevation (>10.5 g/dL)
Renal insufficiency (CREAT >2 mg/dL)
Anemia (Hb <10 or 2 g <Normal)
Bone disease (Lytic or Osteopenic)
* MM is same as Durie/Salmon stage IB plus stage II/III A/B
** British J. Haematol. 2003;121:749-757.

STAGING
Prognostic Factor Systems
International Staging System (ISS)
· Serum ß2-microglobulin/Serum Albumin
Anatomic / Functional Staging
Durie & Salmon or variants
· Durie & Salmon Plus Imaging
New Molecular Approaches
RNA/DNA/Proteomics

PROGNOSTIC FACTORS FOR STAGING
International Staging System
M / S. Alb
2
Stage I
Low M < 3.5* plus
2
S. Albumin** > 3.5 G/DL
Stage II 2M < 3.5 but low albumin < 3.5 or
2M : 3.5 < 5.5
Stage III High 2M > 5.5mg/DL
* mg/DL
Based upon 10-15 years follow-up
** Gm/DL

International Staging System
Asia / Europe / North America
Overall Survival by ISS
Asia
100%
Median
Asia
Deaths / N
in Months
ISS Stage I
131 / 255
58 (47,64)
80%
ISS Stage II
159 / 240
38 (31,42)
ISS Stage III
181 / 246
24 (19,30)
60%
40%
20%
North
0%
0
24
48
72
96
120
144
168
192
216
Months from initial chemo treatment
America
Overall Survival by ISS
EuropeOverallSurvivalbyISS
North America
Europe
100%
100%
Median
Deaths / N
in Months
ISS Stage I
539 / 1305 71 (65,75)
80%
Median
ISS Stage II
912 / 1771 52 (49,55)
80%
Deaths / N
in Months
ISS Stage III
894 / 1425 32 (30,36)
ISS Stage I
591 / 747
55 (51,60)
ISS Stage II
1012 / 1146 40 (37,42)
60%
ISS Stage III
943 / 1022 26 (24,29)
60%
40%
40%
20%
20%
0%
0%
0
24
48
72
96
120
144
168
192
216
Months from initial chemo treatment
0
24
48
72
96
120
144
168
192
216
Months from initial chemo treatment

International Staging System
Overall Survival by Extreme Regression Groups
Model 3: Poor Risk Group High B M + High Creatinine or Low Alb or Low PLT
2
Median
Deaths/ N
In Months
Normal Risk
2278 / 3415
45 (43,46)
Sß M 10+ and (Creat 4+ or ALB < 2.5 or PLT < 130)
2
237 / 270
17 (14,21)
Logrank P-value < 0.0001
Months from Initial Chemo Treatment

STAGING WITH FDG PET
Multiple Myeloma FDG PET:
Severe Diffuse (D) and Focal (F) Disease
FL on PET & MRI:
F
F
D
F
D
D
F
D
D
D
FDG PET scan
MRI STIR
of thoracic spine
weighted of
thoracic spine

PET 3-D IMAGING

ANATOMIC/FUNCTIONAL STAGING
Durie / Salmon "PLUS" System
Integration of Imaging
DURIE/SALMON
PLUS
MRI/PET*
upstage
STAGE
Number of lesions
I B
I
0 - 4
II A or B
II
5- 20
III A or B
III
> 20
*Bauer 2002
B: creatinine >2
Durie 2002/2005
Walker 2003/2004
and / or EMD on PET

Significance of PET Imaging
New understanding of myeloma biology
More lesions than we thought
Extramedullary lesions quite common
"CRs" are maybe not
Triggers a need to understand better
At the molecular level
DNA/ RNA/ protein

Prognostic and Predictive Value of
Circulating Nucleic Acids (RNA and DNA)
Increased plasma levels of RNA and DNA
occur with active myeloma (e.g. 1-15 µg/ ml)
Specific non-coding gene sequences
expressed and/ or released into circulation:
repetitive/ ALU DNA
RNA and DNA transported within
microvesicles: "regulatory vesicles" or
exosomes enter circulation
RNA and DNA is active at distant sites via
ribosomes and/ or retro-integration

Sub-Cellular Pathways
Damaged cell secretes
sentinel gene markers
into the periphery to
become Circulating
Nucleic Acids (CNA)
Genes and Proteins Packaged into
Secretory Vesicles

Circulating Nucleic Acids in Multiple Myeloma
· 4 Multiple Myeloma Patients
· Enterovirus "non-coding"
· Fragment of 22q11.2
primers used
· Flanking Region of PPAR
· Entire Alu sequence

Circulating Nucleic Acid Packaging
in Multiple Myeloma
Electron Microscopy from CNA Pellet
Circulating Microvesicle
from Multiple Myeloma
Patient
· 50-200 nm in length
· 1.13-1.16 g/ml density
· Proteolipid complex
· Found in both health and
disease
Sample source: Dr. Brian Durie
EM Prof Luc Montangier

DNA Analysis
Exons v. Repetitive DNA Sequences
Exons
Repetitive DNA
Highly
Organized in
Genome

Multiple Myeloma Contig Analysis
Exclusively
Repeat
Regions
· 344 Total Clones
Analyzed
· Categories Set for Over
and Under Representation
Comparing MM to
Patient 1
Healthy
Patient 2
· 128 Selected Clones Fit
into 9 Homologs

Search for Clones Using
ALU (Repetitive DNA) Primers
Percent of Clone-MM-001
Features of M-001 Clone
Derived from chromosome-
16 non-coding region
Higher level (25%) with
untreated relapse
Level reduced with ongoing
treatment/ response
Repetitive sequence loci
Not found in normals or
other disease states
20

Understanding Myeloma Biology
Why is Myeloma
Multiple?

Release of Regulatory Microvesicles from Cells
Could Seed other sites?

Circulating DNA Sequence Pattern Analysis
Current Findings
Myeloma plasma contains 20-30 "clones" or types of
regulatory microvesicles
Each clone of circulating DNA is a composite of a
regulatory ALU (on/off switch) and flanking sequence
Regulatory microvesicles are released from one site
and could potentially seed to multiple sites
At new sites there is potential for transcription and/or
retro-integration of regulatory sequences with
aberrant gene function, e.g. proliferation, apoptosis,
gene expression producing bone disease, or other
features

Conclusions
Imaging and molecular studies provide
new understanding of myeloma biology
CT/PET and circulating nucleotide
analysis can be the basis for
personalized treatment selection and
monitoring

Document Outline