Cancer Stem Cells, Clonal Heterogeneity and Clonal Tides in Multiple Myeloma

Linda M. PilarskiUniversity of Alberta, Canada

23 October 2014

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

To Monitor the Myeloma Clone and Response to Treatment:

Each MM clone has multiple phenotypically defined compartments that harbor the clonotypic IgH

“wolves in sheep’s clothing”

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

Defining a Cancer Stem Cell

1. Able to generate/regenerate the malignant clone

2. 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

MM BM lymphs have chromosomal abnormalities

17 “chromotypes” in BM lymphs from ~45% of MM Pts. 2-37% of total BM lymphocytes harbor abnormalities

Debes Marun et al, 2012

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

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

Kirshner et al, Blood 2008

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

Control P6 - B cells P6- Plasma cells

P1 and P3 = 100% clonotypic

P1 = 100% B cells

MM Cancer Stem Cells Co-purify with Hematopoietic Progenitor Populations

Pilarski and Belch 2002

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

The myeloma clone is a dominant clone

1. Dominant B cell clones prevent expansion of other clones2. 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?

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

Dual clones occur frequently in MM

1. 20% of MM patients have a second clone (distinct IgH VDJ)

2. Second clones are frequent (0.01-40% of MNC)3. Second clones harbor genetic abnormalities

4. 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

Dual clones have different IgH VDJ, geographiclocations, frequencies and chromosomal abnormalities (12/46 patients) Patient #6

Clone 1 Clone 2Sternal biopsy Iliac biopsiesCDR3 = 63bp CDR3 = 42 bpVH4-30 VH1-1891% of PC 67% of PCProductive IgH Productive IgH

T(11;14)del13amp1q21

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

Expansion of a Second Clone After Treatment, at Relapse

Diagnosis to year 2 - C2 at low frequency in PB, dormant? Years 2-7 - extensive clonal expansion of C2 in BM

Treated for 6 years with thalidomide

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) 0.1-70% 0.01-41% median = 30% median = 0.5%

Clone 2 0.06-24% 0.1-31% median = 2.5% median = 1.0%

Values = % of mononuclear cells

IgH Clonal Diversity in MM Pts with Two Clones, Measured by ImmunoSEQ

~100,000 to >1 million reads

C1 C2 C1 C2ImmSEQ 74% 25% 84% 9%Q-PCR 37% 24% 30% 3% Patient 1 Patient 2

Uni-clonal MM

Massively Parallel Sequencing by Adaptive Biotechnologies

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 expansionsSo……

IgH VDJ gene rearrangements are the ONLY unequivocal definition of relatedness.

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?

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

IgH VDJ Rearrangement

Hematopoietic Progenitors and/or Pro-B cells

VDJ-1 VDJ-2 VDJ-3 VDJ-4 more ….

Dominant MM clone

Transforming Events &Clonal

Dynamics

Copy Number Aberrations/AneuploidyTIME

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

VD

J Gen

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arra

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Clonal dynamics may involve multiple discrete clones as well as multiple subclones

1. 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

2. 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?

Important to Consider the Potential Impact of Treatment on Clonal Dynamics

1. 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

3. Preferentially stimulate/inhibit clones or subclones

4. The impact of treatment may differ in its effect on families of related clones as compared to that on collections of unrelated clones

Conclusions

1. MM cancer stem cells are resting B lymphocytes with clonotypic IgH rearrangements2. MM BM B-cells carry the same chromosomal abnormalities as MM plasma cells, and contribute to poor survival3. 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 wanes5. Inter-clonal diversity may contribute to clonal tides and possibly to second lymphoid malignancies

Acknowledgments:Carina Debes MarunJitra Kriangkum, PhD

Andrew R. Belch, MDChristopher Venner, MD

Thanks to the myeloma patients who so generously donate their BM and blood

Alberta Cancer Foundation and Myeloma Alberta