1Cancer Stem Cells, Clonal Heterogeneity and Clonal Tides in Multiple MyelomaLinda M. PilarskiUniversity of Alberta, Canada23 October 2014
2To 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 cloneEach MM clone includes PC, B cells and probably a CD20+ CD34+ cancer stem cell compartment
3Each MM clone has multiple phenotypically defined compartments that harbor the clonotypic IgH “wolves insheep’s clothing”
4MM 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 diseaseLate stage progenitors: Limited self renewal, rapid onset of disease symptoms
5Defining a Cancer Stem Cell 1. Able to generate/regeneratethe malignant cloneExhibits self renewal3. Quiescent and drug resistant4. In MM, may be a complexpopulation comprised ofmultiple differentiation stagesMM remains incurable. This must meanthat current MM therapies fail to killMM stem cells
6MM BM lymphs have chromosomal abnormalities 17 “chromotypes” in BM lymphs from ~45% of MM Pts % of total BM lymphocytes harbor abnormalitiesDebes Marun et al, 2012
7MM stem cell capability appears to reside in both B and plasma cell (PC) compartments“Early stage” chromosomal abnormalities(IgH translocations, del13) appear to arisein B cells and are transmitted to PC as MMclonal expansion and differentiation occursLate abnormalities associated with diseaseprogression (del17p and amp1q21)occur only in PC. This suggests that as diseaseprogresses, PC may acquire some degree ofgenerative capability
8Chromosomally abnormal BM lymphocytes correlate with reduced survival in MMMM 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
9MM cancer stem cells (MM-CSC): Clonotypic CD20+ MM-CSC (B cells) show self renewal,proliferative quiescence, drug resistanceand the ability to regenerate diseaseP1 =100% B cellsP1 and P3= 100% clonotypicControl P6 - B cells P6- Plasma cellsKirshner et al,Blood 2008
10MM Cancer Stem Cells Co-purify with Hematopoietic Progenitor PopulationsCD34-purified cells from MM mobilized blood autograftshave clonotypic transcripts (mean = 31% of CD34+45lo cells)CD34-purified cells from MM autografts includeDNA aneuploid cells (mean= 31% hyperdiploid)CD34-enriched cells from MM autografts engraft humanMM to NOD SCID mice (from 6/7 mobilized bloods)CD34-enriched cells from MM autografts cause MMbone lesions in 76% of xenografted NOD SCID micePilarski and Belch 2002
11Dominant B cell clones prevent expansion of other clones The myeloma clone is a dominant cloneDominant B cell clones prevent expansionof other clonesOther clones arise with loss of dominanceSpeculation and Questions:Transformation events may be frequent in MMDoes MM dominance prevent expansion ofother potentially malignant clones?Do “submissive” clones become progenitorsfor second cancers?
12Myeloma is characterized by “clonal tides” (reviewed by Bahlis, Blood 2012)1. Over time, clonal waves expand and contract2. The assumption is that these represent variantsof the MM clone with acquired genomic changesTidal waves share some common abnormalities3. No direct evidence to confirm that all tidalwaves arise from the same MM “parent”.4. Must be confirmed through IgH VDJ analysis
13Dual 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 abnormalitiesSecond clones persist throughout treatmentDoes treatment stimulate second clones and/orallow submissive clones to escape MM dominance?Do second clones form a pool of progenitors thatcan lead to second cancers?Kriangkum et al. 2013
14Dual clones have different IgH VDJ, geographic locations, frequencies and chromosomalabnormalities (12/46 patients)Patient #6del13amp1q21T(11;14)Clone Clone 2Sternal biopsy Iliac biopsiesCDR3 = 63bp CDR3 = 42 bpVH VH1-1891% of PC % of PCProductive IgH Productive IgH
15Clone 1 and Clone 2 are in different cells (single cell analysis) C1 = 100% of BM PCC2 = 10% of BM B cellsC2 = 25% of PB B cellsC1 = 60% of BM PCC2 = 19% of BM B cellsC2 = 10% of PB B cellsP/NP Biallelic IgH rearrangements
16Expansion of a Second Clone After Treatment, at Relapse Treated for 6 years with thalidomideDiagnosis to year 2 - C2 at low frequency in PB, dormant?Years extensive clonal expansion of C2 in BM
17Frequencies 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 BloodClone 1 (MM) % %median = 30% median = 0.5%Clone % %median = 2.5% median = 1.0%Values = % of mononuclear cells
18IgH Clonal Diversity in MM Pts with Two Clones, Measured by ImmunoSEQ~100,000 to >1 million readsC C C C2ImmSEQ % 25% % %Q-PCR % 24% % %Patient Patient 2Uni-clonal MMMassively Parallel Sequencing by Adaptive Biotechnologies
19The frequency of “second” clones is such that they would likely score as distinct clones in array analysisor whole genome sequencing.Must distinguish between :intra-clonal diversity (related clones)andinter-clonal diversity (unrelated clones).Clonal dynamics in MM involve B lineage expansionsSo……IgH VDJ gene rearrangementsare the ONLY unequivocal definition of relatedness.
20e.g. insertions and deletions identified by CGH Assumptions of commonality/relatedness of tidal clones are based on the presence of shared chromosomal abnormalitiese.g. insertions and deletions identified by CGHIs this sufficient?
21Do chromosomal abnormalities arise prior to IgH VDJ rearrangement? If so, post-IgH rearrangement clonal dynamicsmay reflect unrelated and clonally distincttransforming events. (inter-clonal diversity)Shared abnormalities could be present inmultiple “parent” B cells, leading to independentclonal expansions and discrete clonal tidesClonal tides may include bothintra-clonal and inter-clonal diversity
23Inter- and Intra- clonal Tides: Dynamics of Independent Clones in MM TIMEDominant MM cloneEscape from dominanceClone VDJ1Clone VDJ4Clone VDJ3Clone VDJ2/Intra-clonal tidesIgH VDJ Gene rearrangement
24Clonal 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 diversityand/orUnrelated 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?
25Important to Consider the Potential Impact of Treatment on Clonal DynamicsCompromise dominance of the primary MM cloneand/or compromise the MM niche to allowalternate clonal expansions2. Preferentially deplete some clones but spare othersPreferentially stimulate/inhibit clones or subclonesThe impact of treatment may differ in its effecton families of related clones as compared tothat on collections of unrelated clones
26Conclusions MM cancer stem cells are resting B lymphocytes with clonotypic IgH rearrangementsMM BM B-cells carry the same chromosomalabnormalities as MM plasma cells, and contributeto poor survival3. MM patients can harbor two dominant clonessingle cell analysis & next-gen sequencing4. MM may undergo multiple transformation events -unrelated clones may emerge when clonaldominance wanesInter-clonal diversity may contribute to clonal tidesand possibly to second lymphoid malignancies
27Acknowledgments: Carina Debes Marun Jitra Kriangkum, PhD Andrew R. Belch, MDChristopher Venner, MDThanks to the myeloma patients who so generously donate their BM and bloodAlberta Cancer Foundation and Myeloma Alberta