MOLECULAR DIAGNOSTICS OF MULTIPLE MYELOMA...MOLECULAR DIAGNOSTICS OF MULTIPLE MYELOMA Christopher H....
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MOLECULAR DIAGNOSTICS MOLECULAR DIAGNOSTICS OF MULTIPLE MYELOMA OF MULTIPLE MYELOMA Christopher H. Cogbill, MD Pathology Services of Kalamazoo, P.C. Bronson Laboratories
MOLECULAR DIAGNOSTICS OF MULTIPLE MYELOMA...MOLECULAR DIAGNOSTICS OF MULTIPLE MYELOMA Christopher H. Cogbill, MD Pathology Services of Kalamazoo, P.C. Bronson Laboratories This afternoon
MOLECULAR DIAGNOSTICS MOLECULAR DIAGNOSTICS OF MULTIPLE MYELOMAOF MULTIPLE MYELOMAChristopher H. Cogbill, MDPathology Services of Kalamazoo, P.C.Bronson Laboratories
Presenter�
Presentation Notes�
This afternoon I will be covering how myeloma is an example of modern molecular medicine giving a tailored approach to oncologic management. This stems from advances in understanding the biology and genetics of myeloma and novel ways of classifying the disease for specific therapy and prognosis.�
Disclosures
Presenter�
Presentation Notes�
I have no financial disclosures to express today, though I do disclose that I have two young kids that may or may not have helped with the preparation of this talk!�
ObjectivesDefine MGUS (monoclonal gammopathy of undetermined significance) and multiple myeloma
Discuss the molecular pathogenesis of the initiation and progression of MGUS and multiple myeloma
Discover diagnostic cytogenetics and molecular techniques for myeloma characterization and risk stratification
Learn diagnostic limitations in plasma cell neoplasmsIntratumoral heterogeneity
In our short time today I hope to cover a few high-yield topics of myeloma and its precursor states to give a better sense of its biological characteristics. We will expand on the discussion of MGUS, its molecular pathogenesis and progression to myeloma as currently understood, current cytogenetic and molecular techniques for the characterization of disease and stratification into unique risk groups, limitations in making the diagnosis and classifying myeloma, and new diagnostics on the horizon that may be coming soon to a patient near you.�
Multiple Myeloma 101
Malignant neoplasm of Plasma cellsTerminally differentiated non‐dividing cell of B‐lymphoid lineage
Synthesize immunoglobulin (antibodies) that are antigen specific
Clonal plasma cells typically secrete one antibody (heavy chain + light chain monoclonal protein)
Incredibly heterogeneous; multiple disease types (low risk to high risk)Genome instability
*Almost all patients with multiple myeloma are cytogenetically abnormal*
All cases thought to progress through precursor disease (MGUS)
Presenter�
Presentation Notes�
Multiple myeloma is ostensibly a malignant neoplasm of plasma cells, which are terminally differentiated, non-dividing cells of B-lymphoid lineage. These are the cells responsible for synthesizing immunoglobulin (antibodies) that are antigen specific in immune responses. A clone of plasma cells typically secretes one antibody type (composed of a heavy chain type and light chain type) giving rise to the M-spike seen when the proteins in serum are subjected to electrophoresis. Proportional to the density of staining toward that protein type, the quantity of such as protein can be estimated. The clinical sequelae of myeloma have been nicely summarized already today and for diagnostic purposes are encompassed by hypercalcemia, renal failure, anemia, and bony lesions. Pathologically, the plasma cell clone proliferates in the bone marrow where conditions are just right. In the normal host without myeloma, plasma cells account for <2% of nucleated cells but in myeloma, neoplastic cells may range from a few % up to nearly 100% of cells and replace most of hematopoiesis. Crucial to understanding myeloma is understanding its heterogeneity. Cases are variable with respect to clinical presentation, morphology (depicted in some of these images), cytogenetics, molecular abnormalities, and proliferative capacity. Like many tumors such as lung cancer, myeloma is really many diseases with a common phenotype. Some are indolent and of low immediate risk while others progress rapidly and leave the bone marrow environment and wreak havoc on the body. What do myeloma cases have in common? For one, almost all myelomas are cytogenetically abnormal reflecting a high degree of genome instability. Second, all cases are thought to progress through some phase of precursor disease (MGUS). �
Disease DefinitionsMGUS – annual risk of progression 1%
Clinical myelomaM‐protein, clonal plasma cells, AND CRAB end‐organ damage
(New) BM plasmacytosis ≥ 60%; (new) abnormal sFLC κ/λ ratio ≥ 100 (kappa) or ≤ 0.01 (lambda); (new) focal BM lesions detected by PET‐CT and/or MRI in asymptomatic individuals
Presenter�
Presentation Notes�
Although the duration of time in each stage varies, myeloma starts from an “early/benign phase”, MGUS, procedes to an “intermediate/indolent phase” (smoldering MM) and achieves a final “advanced stage” (active and ultimately resistant/refractory MM. The criteria for each stage of disease have been discussed previously today but a few points are worth repeating. MGUS, the earliest precursor stage, shows an annual risk of progression to active myeloma of 1%. Smoldering myeloma, generally reflected by a greater plasma cell disease burden, progresses at a higher rate of 10% each year for the first 5 years, 3% annually for the following 5 years, and then 1% per year in the subsequent decade. Dr Zimmerman earlier discussed the clinical and pathologic criteria for Active/clinical myeloma, including some potentially new items to the list in addition to our well-accepted CRAB criteria�
**There are two MGUS types**Lymphoid/lymphoplasmacytic MGUS = IgM MGUS
Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening TrialPrediagnostic serum samples consistently demonstrated MGUS in the years before the malignant diagnosis in 71 patients who developed myeloma in 10 yr f/u
Landgren O, Kyle RA, Pfeiffer RM, et al. Monoclonal gammopathy of undetermined significance
(MGUS) consistently precedes multiple myeloma: a prospective study. Blood. 2009;
113(22):5412‐5417
Presenter�
Presentation Notes�
Before continuing down into the details of myeloma biology, it is important to understand that not all MGUSes are created equally. There are generally two types/diseases of MGUS, the IgM MGUS that is generally associated with lymphomas and the non-IgM MGUS which is associated with plasma cell proliferative disorders like myeloma. In general when MGUS is discussed mostly it is the non-IgM MGUS that is being referred to. Non IgM MGUS has M-protein composed of IgG, A, D or E heavy chains plus a light chain or light chain only MGUS. Some risk factors for progression out of MGUS include a non_IgG M spike, one > 1.5 and an abnormal serum FLC ratio, a relatively new test on the block but one that is readily available. Hypogammaglobulinemia is another risk factor for progression. In these circumstances referral to a hematologist is a necessity. I also list the primary study here that points to MGUS as a precursor state in nearly every case of myeloma. This prospective study collected serum samples from tens of thousands of participants and those samples in71 participants who subsequently developed myeloma consistently demonstrated monoclonal gammopathies.�
Blood 2014,123(3)305-307
Presenter�
Presentation Notes�
It also bears mentioning that MGUS is also a precursor condition for other pathologic conditions not associated with plasma cell tumor growth. These conditions directly related to secretion of M-protein such as MGRS, immunoglobulin deposition disease, and other diseases affecting the kidneys have very morbid consequences but are beyond the scope of discussion today. �
Myeloma Pathogenesis
Presenter�
Presentation Notes�
I’ll be referring to this diagram today several times as a framework for the pathogenesis of myeloma. Although simplistic and becoming a bit outdated now, it is a good architectural backbone on which to start.�
Myeloma Pathogenesis #1Inciting event and cytogenetic abnormalities MGUS
Thought to be product of abnormal plasma cell response to antigenic stimulation
Monoclonal immunoglobulin production
Presenter�
Presentation Notes�
As mentioned, virtually all cases of myeloma pass at some point through the MGUS phase. The initial stimulus generating this asymptomatic stage is usually not known but may be related to inherited genetic variation, radiation exposure, or chronic antigenic stimulus of some type. Based on current standard technologies (eg, FISH), the molecular makeup of myeloma precursor disease states and multiple myeloma are strikingly similar and no defining molecular features unique to multiple myeloma have been identified. we currently lack reliable biologic markers that allow us to predict which multiple myeloma precursor patients will progress, and which will not. standard technologies rather reflect the predominant clonal population and fail to take into account the presence of intratumoral subclonal heterogeneity Fortunately, genetic abnormalities not only provide insights into the biology of disease pathogenesis and evolution, they are also powerful prognostic factors in MM. It is worthwhile to examine the basics of the oncogenesis of this disease before launching into the specifics of diagnosis. Mechanisms responsible for interaction between malignant plasma cells and their microenvironment are as important as the genetic changes involved in the development of the malignant clone because these play an important role in bone destruction, tumor cell growth, survival, and migration; and drug resistance. The MM genome is recognized as being complex at the cytogenetic level In the classical view of the initiation and progression of myeloma, an initiating hit is required to immortalize a myeloma-propagating cells (MPC). Such a cell is then destined to acquire additional genetic hits over time, mediated via translocation, loss of heterozygosity, gene amplification, mutation, or epigenetic changes. The acquisition of additional hits further deregulates the behavior of the MPC leading to the clinically recognized features of MM. �
Myeloma Pathogenesis
Presenter�
Presentation Notes�
It has been proposed that myeloma arises from abnormal naïve B-cells that originate in the lymph nodes and migrate to the bone marrow, which provides a microenvironment conducive to terminal plasma cell differentiation. Disease is generally widely disseminated throughout the axial skeleton, supporting this theory. Generally explains why a random BM bx of posterior iliac crest will identify the clonal population in the majority of patients with even “pre-malignant” plasma cell proliferative disorder like MGUS. Bone marrow microenvironment as important in spread�
TranslocationsOncogene (CCND1, MYC, etc.) juxtaposed to IgH enhancer, resulting in aberrant overexpression
Growth and replication via novel:Transcription factors
Growth factor receptors
Cell cycle mediators
Presenter�
Presentation Notes�
Large-scale cytogenetic abnormalities come in two flavors: structural and numerical abnormalities. Nearly half of MM tumors have driving structural aberrations, which are usually reciprocal translocations. Unlike other B-cell tumors (follicular lymphoma, mantle cell lymphoma) MM exhibits a marked diversity of chromosomal loci involved in IGH translocations�
Translocations – common involved oncogenes
t(11;14) 11q13 –
cyclin D1
t(8;14) 8q24 – c‐myc (usually secondary) or mafA
t(6;14) 6p21 –
cyclin D3
t(4;14) 4p16.3 –
multiple
myeloma set domain & fibroblast growth
receptor 3
t(14;20) 20q11 – mafB
t(14;16) 16q23 – c‐maf
Circos plot
t(12;14) 12p13 –
cyclin D2
CCND2
MAFA
Multiple myeloma is oddAlmost all MM tumors are aneuploid (numerical abnormalities of chromosomes)
Reflects genetic instability
Hyperdiploid group:48‐75 chromosomes usually 49‐56
Generally affects odd‐numbered chromosomes (except 1, 13, 17)
Low incidence of structural chromosome abnormalities
Overexpression of genes on affected chromosomes
Presenter�
Presentation Notes�
IN contrast, the hyperdiploid group of MM tumors has an excessive number of extra chromosomes, associated with recurrent trisomies of the odd chromosomes�
Cytogenetic Abnormalities
High riskIGH‐MMSET(FGFR3); t(4;14)IGH‐MAF; t(14;16)IGH‐MAFB; t(14;20)*IGH‐MAFA; t(8;14)*
Deregulation of the G1/S cell cycle transition point via the overexpression of cyclin D genes, an event shown to be a key early molecular abnormality in myeloma
Presenter�
Presentation Notes�
As a result of studying t(11;14) and t(6;14) translocations which deregulate cyclin D1 and D3, respectively GEP profiling analysis has demonstrated that expression of the cyclin proteins, key regulators of the cell cycle, is increased in almost all MM patients, supporting the hypothesis that there is a potential unifying event in its pathogenesis Key to therapy – cyclin D inhibitors showing promise in vitro�
Myeloma Pathogenesis #2Progression from MGUS to MM
Additional genetic insults
Changes in the bone marrow microenvironment
Presenter�
Presentation Notes�
CMM represents an expansion of altered subclones that are already present at early precursor stages. ; changes in bone marrow microenvironment critical Risk of progression similar regardless of duration of MGUS (i.e. not cumulative risk) 1% / year , every year , risk does NOT decrease even over several decades Random “second hit”, secondary abnormalities for malignant progression Mutations Copy number variations (CNV) Loss of heterozygosity (LOH) Epigenetic modifications (tumor suppressor genes) �
Disruption of the retinoblastoma pathway (checkpoint in cell cycle)
translocations, mutations, loss of, hypermethylation of:
cyclin D kinase inhibitors (CdkI)
CDKN2A
(p16), CDKN2C
Tumor suppressors
RB
inactivation
(chromosome 13
deletion)
TP53 (p53)
deletion or
mutation p21 G1/S dysregulation
Secondary abnormalities –
signaling pathways
MA
F
N‐RAS
K‐RAS
Uncommo
n in MGUS
Presenter�
Presentation Notes�
Dysregulation of osteolytic activity Activation of NF kappa B Pathway normally regulates osteolytic activity Multiple mechanisms; potential target for therapy (e.g proteasome inhibitors (bortezomib / Velcade) Epigenetic dysregulation Histone-modifying enzymes are frequent targets of mutation (consequences unknown) Targets for therapy (histone deacetylase inhibitors, e.g. panobinostat) Global hypomethylation; alteration in microRNA expression Cell proliferation / growth / apoptosis Secondary translocations (c-MYC rearrangement, IGH, etc.) �
Myeloma Pathogenesis #3Extramedullary progression
Peripheral
Solid tissues / body cavities
Presenter�
Presentation Notes�
Bone marrow microenvironment critical, emerging as target of therapy and vital player in progression and extramedullary spread �
multiple myeloma DIAGNOSTICS
Presenter�
Presentation Notes�
We are able to use molecular data in the clinical stetting to stratify patients for clinical risk status and use the information select appropriate treatment Cytogenetics through chromosome analysis, FISH, etc. but the presence of a specific cytogenetic lesion is no longer interpreted in isolation. Clinical and cellular background in which a genetic lesion occurs mediates its prognostic impact�
Fluorescence in situ hybridization (FISH)Hundreds of cells, in interphase (not dividing)
Plasma cell selection:Staining with cytoplasmic immunoglobulin
Cell sorting and plasma cell labeling (purification) with anti‐CD138‐coated magnetic beads
Pros: sensitive, specific
Cons: Labor intensive, very targeted probes
Leukemia
(2009) 23, 2210‐21
Presenter�
Presentation Notes�
Reproducible staging systems available using routine clinical tests e.g. serum albumin and beta 2-microglobulin However, presence of adverse genetic factors significantly subdivides survival among these groups Most data on prognosis useful for risk stratification at initial diagnosis E.g. t(11;14) is unfavorable at relapse �
Presenter�
Presentation Notes�
e.g. MAF translocations at presentation in MM associated with a poor prognosis but when they are present in MGUS they are not associated with adverse outcomes (MAF deregulation alone is not responsible for adverse outcomes but interaction with other genetic events) Standard risk t(11;14) IGH-CCND1 Historically favorable at initial diagnosis But some heterogeneity, may need to look at additional markers Enriched with K-RAS mutations Associated with aggressive plasma cell leukemia phenotype Inferior survival at relapse T(4;14) Adverse prognosis Following conventional chemotherapy or high-dose therapy with stem-cell transplant (HDT) Primarily a result of early relapse Several recent studies: bortezomib overcomes poor prognosis in newly diagnosed and relapse patients Clinical testing of FGFR3 inhibitors well underway 17p deletion Most important molecular cytogenetic factor for prognostication Usually monoallelic Very negative effect on survival in all large series tested More aggressive disease Higher prevalence of extramedullary disease (e.g. plasmacytomas), CNS involvement Hypercalcemia Predicts short duration of response after high-dose therapy and involvement of CNS Uncommon in MGUS 1q gains Chromosome 1 lesions are the most common abnormalities in MM Mostly 1q gains Genes unknown; many proposed involved in proliferation (CKS1B ?), possible targets of therapy Pericentrometric translocations (often in the form of “jumping translocations”) Enriched in high-risk signatures of gene expression profiles �
Poor prognosis is multifactorialNewly diagnosed myeloma pts < 66 yo (IFM – French study)
No proteasome (bortezomib) inhibition
Overall survival according to number of poor prognostic factors
Age > 55
β2‐microglobulin > 5.5 mg/L
(albumin)
t(4;14) (11%)
Del(17p) (5.4%)
1q gains (33%)
JCO June 1, 2012
vol. 30
no. 16
1949-1952
J Clin Oncol
June 1, 2012
vol. 30
no. 16
1949‐1952
Other StudiesPlasma cell DNA content and proliferation
Now flow cytometry‐based at Mayo Clinic
% of cells in S‐phase, DNA index (0.95‐1.05), ploidy, % polytypic plasma cells
Supplants plasma cell labeling index test, now somewhat outdated
No unequivocal genetic or phenotypic markers to differentiate
Presenter�
Presentation Notes�
No unequivocal genetic or phenotypic markers to differentiate Even extensive gene expression profiling studies do not resolve precise differences Still very difficult to predict when/if progression will occur ?17p13/TP53 deletion ?MYC expression/rearrangement ?RAS mutations ?t(4;14) �
Gene Expression Profiling
Presenter�
Presentation Notes�
RNA Gene expression profiling (GEP)*** Promising risk stratification tool, uncovering several genetic signatures Upclassifies some patients as high-risk Derive GE signatures to be used to risk stratify patients; but tests are not specific for a given clinical outcome and high-risk groups defined by this approach also have a range of outcomes. These types o tests can be difficult to interpret in the laboratory and require consistent quality control making their widespread uptake in clinical labs difficult at this time Still need to combine with FISH analysis to obtain all of the necessary information for definition of prognostic groups (see the cytogenetic alterations) Issues with reproducibility; not implemented in clinical milieu�
Genetic Classification Systems
Presenter�
Presentation Notes�
GEP analyses of MGUS have inherent problems. The percentage of plasma cells is low (by definition 10%), so that there is significant contamination with other kinds of cells despite selection of CD138 cells on magnetic beads. In addition, in MGUS patients— unlike in multiple myeloma patients—monoclonal plasma cells are likely to be significantly contaminated with normal plasma cells (due to the relatively low percentage of monoclonal plasma cells in MGUS). Until we have better processing methods and better assays, one has to be cautious when interpreting GEP analyses of plasma cells selected by CD138 expression from MGUS patients. �
Next Generation SequencingWidespread genetic heterogeneity in multiple myeloma. Cancer Cell. Jan 13, 2014; 25(1): 91–101
Presenter�
Presentation Notes�
High-resolution comprehensive genomics tools DNA Array comparative genomic hybridization (aCGH) Single nucleotide polymorphism (SNP) arrays Next-generation sequencing (NGS)*** �
The Future of Molecular Diagnostics
Morgan GJ and MF Kaiser. Hematology Am Soc Hematol Educ Program 2012;2012:342‐9
Clonal heterogeneity (genomic chaos)Multiple subclones at time of diagnosis
Certain clones dominate the malignancy at any one point in disease course
Hazard to targeted therapyMutations often present in subclones (e.g. BRAF)
Model for treatment resistance; lack of curability
Blood.
2012 Aug 2;120(5):927‐8.Morgan GJ, Walker BA, Davies FE. The genetic architecture
of multiple myeloma. Nat Rev Cancer. 2012;12(5):335‐348.
Presenter�
Presentation Notes�
The transition of MGUS to plasma cell leukemia has been traditionally represented as a linear pathway. However, it is more likely that the pathway to myeloma is through branching pathways typical of those that are associated with the evolution of species. The key molecular events leading to disease evolution last in subsequent generations but multiple distinct subclones may develop. In the setting of advantageous abnormalities or selective pressure from therapy, certain subclones may replace those once predominant. Genomic evolution: 1) stable genomes (no difference between diagnosis and relapse); 2) linear evolution in which the relapse clone apparently derives from the major subclone at diagnosis but continues to diversify through additionally acquired lesions; and 3) branching (nonlinear) models in which the relapse clone clearly derives from a minor subclone that is barely present at diagnosis �
Clinical Examples
Mayo FISH panel‐13/13q‐, RB1/LAMP1
t(11;14), CCND1/IGH
t(14;var), IGH
17p‐, TP53/Cen17
+3/+7, Cen3/Cen7
+9/+15, Cen9/Cen15
1q gain, TP73/CKS1B
t(8;var), MYC
Reflex if IGH rearrangement not with CCND1:
t(4;14)(p16.3;q32) FGFR3/IGH
t(14;16)(q32;q23) IGH/MAF
t(14;20)(q32;q12) IGH/MAFB
t(6;14)(p21;q32) CCND3/IGH
Presenter�
Presentation Notes�
13q deletion: thought crucial early event for clonal expansion or progression event (e.g. with t(11;14)) unknown oncogenesis but may lead to progression due to haploinsufficiency of RB1 closely associated with nonhyperdiploid cases and high-risk genetic features (e.g. found in 90% of t(4;14) cases) probably not independently significant unless picked up by conventional cytogenetics (indicating proliferating clone) �
Presenter�
Presentation Notes�
Consensus opinion that takes into account genetically determined risk status and various treatment strategies currently available; in conjunction with host factors, disease stage, and other prognostic factors On and off clinical trials Extramedullary disease/relapse is major limitatino �
Future DirectionRole of molecular/cytogenetic testing after treatment
Role of minimal residual disease in optimizing management and prognosis
Effect of proteasome inhibitors, IMiDs, and total therapy (TT) on prognosis –will conventional prognostic indicators still stand?
Comprehensive genomic tools for risk stratification and targeted therapy
Intratumoral heterogeneityTechnologies generally reflect the predominant clonal population
Take‐awayMyeloma is extremely heterogeneous but always progresses through a benign precursor stage (MGUS, smoldering disease)
MGUS is of two types: IgM and non‐IgM
MGUS progresses at an annual rate of 1% to myeloma requiring treatment
Genomic instability is present from the earliest stages
FISH is the mainstay of current molecular diagnostics
Next generation sequencing is likely to play a very significant role in the near‐term future
Risk stratification is multifactorial; clinical, laboratory, genetic data
Clonal heterogeneity is a major limitation to diagnosis and effective therapy
