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

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

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 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

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

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

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?

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, geographic locations, frequencies and chromosomal abnormalities (12/46 patients) Patient #6 del13 amp1q21 T(11;14) Clone 1 Clone 2 Sternal biopsy Iliac biopsies CDR3 = 63bp CDR3 = 42 bp VH4-30 VH1-18 91% of PC 67% of PC Productive IgH Productive IgH

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 Treated for 6 years with thalidomide Diagnosis to year 2 - C2 at low frequency in PB, dormant? Years 2-7 - extensive clonal expansion of C2 in BM

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 C2 ImmSEQ 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 expansions So…… IgH VDJ gene rearrangements are the ONLY unequivocal definition of relatedness.

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?

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

Hematopoietic Progenitors and/or Pro-B cells 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/Aneuploidy TIME

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

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?

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

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

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