Predicting Response to Thalidomide-
Dexamethasone Combination in
Multiple Myeloma by Gene Expression
Profiling of Tumor cells at Diagnosis
Shaji Kumar, P. Leif Bergsagel, Philip R Greipp, Jessica Haug, Michael Kline,
Wee Joo Chng, Emily Blood, John A Lust, Morie A Gertz, Rafael Fonseca, and
S. Vincent Rajkumar. Hematology, Mayo Clinic, Rochester, MN; Hematology
and Oncology, Mayo Clinic, Scottsdale, AZ and Eastern Cooperative Oncology
Group (ECOG) Statistical Center, Dana-Farber Cancer Institute, Boston, MA.
Background
· Thalidomide + Dexamethasone is an effective combination
in MM
· Over two-thirds of patients respond to this combination
· Ability to identify patients unlikely to respond can help
individualize therapy and avoid unnecessary toxicity
· Gene expression profiling of tumor cells can potentially
correlate expression of individual genes with likelihood of
response
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Methods
· Patient samples from the ECOG E1A00 clinical trial were
studied
Thalidomide +
Dexamethasone
· Newly Diagnosed MM
Dexamethasone
· Additionally, patient samples from Mayo Clinic Phase II
study of Thalidomide +Dexamethasone
· Bone marrow samples from 50 patients were available
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Methods
· 30 patients received Thalidomide + Dexamethasone
· 20 patients received Dexamethasone alone
· Laboratory data from diagnosis and during therapy
was reviewed
· Response: A decrease in serum M protein by 50% or a
decrease in 50% of urine M protein in the absence of a
serum M spike
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Methods
· Bone marrow mononuclear cells were separated from
aspirates by Ficoll or ACK lysis
· Tumor cells were isolated by CD138 selection
· Purity confirmed by cytospin slide
· Total RNA isolated using Qiagen RNeasy, cDNA
generated
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Methods
· The cDNA was hybridized onto an Affymetrix U133A
chip and scanned using a Genechip 300 scanner and
GeneChip 5.0 software (Affymetrix)
· The CEL files from the analysis were analyzed using
Genespring 7.2® software (Agilent Technologies);
preprocessed using GCRMA followed by per gene
normalization to median.
· The output was further analyzed using Ingenuity®
Pathway Analysis program.
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Objectives
1. Can we identify genes that can reliably predict an
individual patient's likelihood of responding to the
combination of thalidomide and dexamethasone?
Class Prediction Analysis
2. Can comparison of gene expression profiles from
non-responders and responders provide insights
into mechanisms of drug activity and resistance
Ingenuity Pathway Analysis
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Predictors of response
· Class Prediction analysis on 30 patients receiving Thal
+ Dex combination
· Support Vector Machines; Golub (signal to noise ratio)
method
· Using the set of 30 chips as training set, we arrived at
set of 25 genes that correctly classified non-
responders from responders
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Non-responders
Responders
Genes
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Non-responders
Responders
Non- responders
Patient Number
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Chr Loc
Gene Name
Protein
4q12
SEC3L1
Exocyst complex component 1
3q26.1
SMC4L1
Structural maintenance of chromosomes (SMC) family member
12q12-
TUBA6
Tubulin alpha 6
q14
14q22
CNIH
Cornichon homolog
7q11
WBSCR5
Linker for activation of T cells family, member 2
16p12-13
NFATC2IP
Nuclear factor of activated T-cells
Xp22
CD99
MIC2, T cell activation
1p31
LEPR
Leptin receptor gene-related protein
1p32
USP1
Member of the ubiquitin-specific processing (UBP) family of proteases
1p35
FLJ11730
Sarcoma antigen NY-SAR-91
10q11
CSTF2T
Cleavage stimulation factor
10q23
FAM35A
Family with sequence similarity 35, member A
11p15
ILK
Integrin-linked kinase, apoptosis suppressor
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Correlation with Primary IgH
translocations
· None of the differentially predictor genes were
associated with the five common translocations in MM
(11q13 (cyclin D1), 6p21 (cyclin D3), 4p16 (FGFR3-
MMSET), 16q23 (c-maf), or 20q11 (maf-B)).
· However all 5 non-responders had one of the primary
IgH translocations (11q13 -1 pt, 6p21 -1 pt, 4p16 -2 pts,
20q11 -1pt)
· In comparison, 7 of the 25 responders had one of the
translocations.
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Response and Primary IgH
Translocations
· 7 of 12 patients (56%) with a primary IgH
translocation responded to Thal Dex
versus
· 18 of 18 patients (100%) with no primary IgH
translocations identified responded to Thal Dex
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Pathway analysis
· Ingenuity Pathway Analysis Program
· 521 genes which were two fold or more differentially
expressed between responders vs non responders
· Functionally grouped networks were identified to
which 289 specific genes were grouped "focus genes"
· Canonical pathways into which the genes could be
grouped were examined
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Canonical pathways
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Conclusion
· We have identified a set of 15 genes that can
potentially predict for response to thalidomide and
dexamethasone
· Genes and networks involved in immune response
and cytokine signaling were up regulated in non-
responders
· Although patients with and without primary IgH
translocations respond well to thalidomide
dexamethasone, those without the translocations had
much higher response rates.
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Future Direction
· Validation will be performed on a larger group
· Custom arrays with specific genes, could allow
personalization of drug therapy for this disease.
· Implications for future classification and
treatment approaches to myeloma.
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