1. Cancer Stem Cells, Clonal Heterogeneity and
Clonal Tides in Multiple Myeloma
Linda M. Pilarski
University of Alberta, Canada
23 October 2014

2. To Monitor the Myeloma Clone and Response to Treatment:
Essential to change the focus from screening only the plasma cell (PC) compartment of MM to looking at the entirety of a MM clone
Each MM clone includes PC, B cells and probably a CD20+ CD34+ cancer stem cell compartment

3. Each MM clone has multiple phenotypically defined compartments that harbor the clonotypic IgH
“wolves in
sheep’s clothing”

4. MM Cancer Stem Cell Function is Multi-Faceted and Influenced by Parameters of Maturation and Time
Early stage progenitors: Extensive self renewal, prolonged time to detectable disease
Late stage progenitors: Limited self renewal, rapid onset of disease symptoms

5. Defining a Cancer Stem Cell
1. Able to generate/regenerate
the malignant clone
Exhibits self renewal
3. Quiescent and drug resistant
4. In MM, may be a complex
population comprised of
multiple differentiation stages
MM remains incurable. This must mean
that current MM therapies fail to kill
MM stem cells

6. MM BM lymphs have chromosomal abnormalities
17 “chromotypes” in BM lymphs from ~45% of MM Pts % of total BM lymphocytes harbor abnormalities
Debes Marun et al, 2012

7. MM stem cell capability appears to reside in
both B and plasma cell (PC) compartments
“Early stage” chromosomal abnormalities
(IgH translocations, del13) appear to arise
in B cells and are transmitted to PC as MM
clonal expansion and differentiation occurs
Late abnormalities associated with disease
progression (del17p and amp1q21)
occur only in PC. This suggests that as disease
progresses, PC may acquire some degree of
generative capability

8. Chromosomally abnormal BM lymphocytes
correlate with reduced survival in MM
MM patients with abnormalities in both PC+ lymphs have significantly worse survival compared to those with abnormalities only in PC (p=0.016)
Debes Marun et al, 2012

9. MM cancer stem cells (MM-CSC): Clonotypic
CD20+ MM-CSC (B cells) show self renewal,
proliferative quiescence, drug resistance
and the ability to regenerate disease
P1 =
100% B cells
P1 and P3
= 100% clonotypic
Control P6 - B cells P6- Plasma cells
Kirshner et al,
Blood 2008

10. MM Cancer Stem Cells Co-purify
with Hematopoietic Progenitor Populations
CD34-purified cells from MM mobilized blood autografts
have clonotypic transcripts (mean = 31% of CD34+45lo cells)
CD34-purified cells from MM autografts include
DNA aneuploid cells (mean= 31% hyperdiploid)
CD34-enriched cells from MM autografts engraft human
MM to NOD SCID mice (from 6/7 mobilized bloods)
CD34-enriched cells from MM autografts cause MM
bone lesions in 76% of xenografted NOD SCID mice
Pilarski and Belch 2002

11. Dominant B cell clones prevent expansion of other clones
The myeloma clone is a dominant clone
Dominant B cell clones prevent expansion
of other clones
Other clones arise with loss of dominance
Speculation and Questions:
Transformation events may be frequent in MM
Does MM dominance prevent expansion of
other potentially malignant clones?
Do “submissive” clones become progenitors
for second cancers?

12. Myeloma is characterized by “clonal tides”
(reviewed by Bahlis, Blood 2012)
1. Over time, clonal waves expand and contract
2. The assumption is that these represent variants
of the MM clone with acquired genomic changes
Tidal waves share some common abnormalities
3. No direct evidence to confirm that all tidal
waves arise from the same MM “parent”.
4. Must be confirmed through IgH VDJ analysis

13. Dual clones occur frequently in MM
% of MM patients have a second clone
(distinct IgH VDJ)
Second clones are frequent ( % of MNC)
Second clones harbor genetic abnormalities
Second clones persist throughout treatment
Does treatment stimulate second clones and/or
allow submissive clones to escape MM dominance?
Do second clones form a pool of progenitors that
can lead to second cancers?
Kriangkum et al. 2013

14. Dual clones have different IgH VDJ, geographic
locations, frequencies and chromosomal
abnormalities (12/46 patients)
Patient #6
del13
amp1q21
T(11;14)
Clone Clone 2
Sternal biopsy Iliac biopsies
CDR3 = 63bp CDR3 = 42 bp
VH VH1-18
91% of PC % of PC
Productive IgH Productive IgH

15. Clone 1 and Clone 2 are in different cells (single cell analysis)
C1 = 100% of BM PC
C2 = 10% of BM B cells
C2 = 25% of PB B cells
C1 = 60% of BM PC
C2 = 19% of BM B cells
C2 = 10% of PB B cells
P/NP Biallelic IgH rearrangements

16. Expansion of a Second Clone After Treatment, at Relapse
Treated for 6 years with thalidomide
Diagnosis to year 2 - C2 at low frequency in PB, dormant?
Years extensive clonal expansion of C2 in BM

17. Frequencies of MM clone and Second Clones
in Genomic DNA of MNC for 12 MM patients
(single cell PCR and/or real time quantitative PCR)
BM Blood
Clone 1 (MM) % %
median = 30% median = 0.5%
Clone % %
median = 2.5% median = 1.0%
Values = % of mononuclear cells

18. IgH Clonal Diversity in MM Pts with Two Clones,
Measured by ImmunoSEQ
~100,000 to >1 million reads
C C C C2
ImmSEQ % 25% % %
Q-PCR % 24% % %
Patient Patient 2
Uni-clonal MM
Massively Parallel Sequencing by Adaptive Biotechnologies

19. The frequency of “second” clones is such that they
would likely score as distinct clones in array analysis
or whole genome sequencing.
Must distinguish between :
intra-clonal diversity (related clones)
and
inter-clonal diversity (unrelated clones).
Clonal dynamics in MM involve B lineage expansions
So……
IgH VDJ gene rearrangements
are the ONLY unequivocal definition of relatedness.

20. e.g. insertions and deletions identified by CGH
Assumptions of commonality/relatedness of tidal clones are based on the presence of shared chromosomal abnormalities
e.g. insertions and deletions identified by CGH
Is this sufficient?

21. Do chromosomal abnormalities arise prior to IgH VDJ rearrangement?
If so, post-IgH rearrangement clonal dynamics
may reflect unrelated and clonally distinct
transforming events. (inter-clonal diversity)
Shared abnormalities could be present in
multiple “parent” B cells, leading to independent
clonal expansions and discrete clonal tides
Clonal tides may include both
intra-clonal and inter-clonal diversity

22. Hematopoietic Progenitors and/or Pro-B cells
IgH VDJ Rearrangement
Hematopoietic Progenitors and/or Pro-B cells
VDJ VDJ VDJ VDJ more ….
Dominant
MM clone
Transforming
Events &
Clonal
Dynamics
Copy Number Aberrations/Aneuploidy
TIME

23. Inter- and Intra- clonal Tides: Dynamics of Independent Clones in MM
TIME
Dominant MM clone
Escape from dominance
Clone VDJ1
Clone VDJ4
Clone VDJ3
Clone VDJ2/Intra-clonal tides
IgH VDJ Gene rearrangement

24. Clonal dynamics may involve multiple discrete clones as well as multiple subclones
Related clones arise from the same B cell
- may acquire additional genetic aberrations
- clones will share the same IgH VDJ
- intra-clonal diversity
and/or
Unrelated clones derive from different B cells
- may arise from a shared pre/pro B expansion
- clones have different IgH VDH rearrangements
- clones may have the same genetic abnormalities
- inter-clonal diversity
- clinical significance ? second malignancies?

25. Important to Consider the Potential Impact
of Treatment on Clonal Dynamics
Compromise dominance of the primary MM clone
and/or compromise the MM niche to allow
alternate clonal expansions
2. Preferentially deplete some clones but spare others
Preferentially stimulate/inhibit clones or subclones
The impact of treatment may differ in its effect
on families of related clones as compared to
that on collections of unrelated clones

26. Conclusions MM cancer stem cells are resting B lymphocytes
with clonotypic IgH rearrangements
MM BM B-cells carry the same chromosomal
abnormalities as MM plasma cells, and contribute
to poor survival
3. MM patients can harbor two dominant clones
single cell analysis & next-gen sequencing
4. MM may undergo multiple transformation events -
unrelated clones may emerge when clonal
dominance wanes
Inter-clonal diversity may contribute to clonal tides
and possibly to second lymphoid malignancies

27. Acknowledgments: Carina Debes Marun Jitra Kriangkum, PhD
Andrew R. Belch, MD
Christopher Venner, MD
Thanks to the myeloma patients who so generously donate their BM and blood
Alberta Cancer Foundation and Myeloma Alberta