El curso clínico del MM es muy heterogéneo, lo que lleva a pensar que,

Transcripción

1 Multiple myeloma

2 El curso clínico del MM es muy heterogéneo, lo que lleva a pensar que, bajo el término de mieloma se engloben enfermedades diferentes desde el punto de vista genético, y cuyo comportamiento está marcado por una gran heterogeneidad genética. Los avances en el conocimiento de las bases genéticas del MM, la aproximación genómica están suponiendo: Propuestas de clasificación molecular del MM Descripción de nuevos marcadores con impacto en el pronóstico Elección de nuevas terapias Desarrollo de nuevas terapias Numerosos ensayos clínicos en marchap

3 MM arise from an asymptomatic premalignant proliferation of monoclonal PCs derived from a post germinal center B cells. Multistep genetic and microenvironement changes lead to the transformation into a malignant neoplasm.

4 Which is the genomic portrait of myeloma?

5 An initiating hit is required to immortalize a myeloma-propagating cell. That cell is destined to acquire aditional genetic hits over time Genetic abnomalities alter the expression of adhesion molecules, as well as responses to gowth stimuli in the microenvoronement. These interactions increase tumor growth

6 Primary early cytogenetic abnormalities in MM Cytogenetic abnormality Feature Frequency Translocation ** IgH translocations (14q32) 3 Cyclin D translocation t(11;14) 1,2 ( t(6;14) 1,2 MMSET translocation t(4;14) # MAF translocation t(14;16) 1-3,a t(14;20) 1,a Other translocations t(8;14) 2 t(14;18) 2 Upregulation of cyclin D1 Overexpression of IRF4/MUM1 Upregulation of FGFR3 and MMSET Dysregulation of c-maf Dysregulation of c-maf Overexpression of c-myc Overexpression of BCL2 40% 15-20% 2% 15% 5-10% 2% 1% Rare Deletion del(13q) 1,2,a del(17p) 1,3,a Loss of Rb1 Loss of p53 50% 10% Hyperdiploid 1,3 Favourable prognosis 45% Hypodiploidy 1,a Unfavourable prognosis 40% High risk mutation/abnormality. **genes placed under the control of the strong enhancers of the heavy chain (IGH) loci leading to their deregulation #2 subgroups. Low b2 microglogulin and hemoglobin >OS after tandem trasplant and high-dose therapy

7 Primary cytogenetic abnormalities in MM (two major groups) MM non-hyperdiploid (50%) (<48 ó >75 chromosomes) Translocations of the immunoglobulin gene (IGH 14q32), deletions, monosomies (hypodiploid) MM hyperdiploid (50%) (48 a 75 chromosomes) Trisomies of odd chromosomes: 3, 5, 7, 9, 11, 15, 19 y 21,

8 MM NHD: eventos genéticos primarios, translocaciones IGH recurrentes (a diferencia de linfomas en los que el partner de la translocación es específico de tipo histológico, en MM muy heterogéneo, hasta 30 partners: EVENTO MOLECULAR COMUN (desrregulación de ciclinas)

9 Over time there is accumulation of secondary changes

10 Eventos cromosómicos secundarios: relacionados con progresión Inestabilidad cariotípica Inactivación p53. del(17p) Ganancias 1q (35%). No se ve en GMSI As. con proliferación, y sobreexpresión de CKS1B, Asociada con t(4;14) y del13. Pérdidas 1p. Asociada con resistencia Tx IgH secundarias. Mediadas por mecanismos distintos a las células B Gen MYC (c- (15%) >> N- > L-). Alta agresividad Tx IgL (κ>>λ)

11 More recently: genetic abnormalities: chromosome patients in IFM trial Major negative prognostic factors for OS and PFS Confirms importance of 1p deletion from previous studies 2 1 1p/1q (CDKN2C/CKS1B) Total Therapy 2 IFM and trials Hebraud B, ASH 2012: 933

12 Análisis citogenético convencional: cariotipos complejos Sólo el 30 % de los pacientes presentan cariotipos alterados. La mayoría de los pacientes, cariotipo normal Bajo nivel de infiltración de CP en las muestras Bajo índice mitótico de las CP Presencia de translocaciones crípticas En los casos con cariotipos alterados, muy complejos

13 Strong association among adverse lesions Boyd et al, Leukemia 2011 Adverse FISH abnormalities t(4;14) t(14;16) t(14;20) +1q/1p 17p-

14 Boyd et al, Leukemia 2011 OS of each of the adverse FISH lesions when they occur in isolation compared with samples lacking any adverse lesions OS graded by number of adverse lesions 42 meses 61 meses 9 meses 24 meses Prognostic model in myeloma based on co-segregating adverse FISH lesions

15 The survival of patients with high-risk MM has remained poor despite aggressive therapy, However, some studies show that first generationnew regimens improve but not overcome outcome of high-risk cytogenetics Better than control Worse than control Conventional chemotherapy Bortezomib regimens IMiDs regimens Bergsagel L, Blood 2013

16 Eventos genéticos secundarios Mutaciones N-RAS, K-RAS, FGFR3, P53 Proliferación mediada por Rb1. Inactivación de CDKN2A y C Desrregulación epigenética: - alteración de expresión de mrnas (p53) - modificaciones de metilación (en t(4;14)>) Mutaciones de pathway NFKb: Activación NFκB en un 40% de los pacientes. TRAF3 (Regulador negativo NFκB)

17 Over time there is accumulation of secondary changes

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19 Definición de riesgo citogenético (high risk cytogenetics)

20 Risk Stratification: qué factores determinan enfermedad alto riesgo? Host characteristics Age Co-morbidities: e.g renal failure; spinal cord compression Tumor characteristics ISS stage Estramedullary disease High risk Cytogenetics Plasma cell leukemia Lactate dehydrogenase level Proliferative indices (high proliferation rate/labelling index)

21 Cytogenetic analysis is one of the most important prognosis factors in MM: and is mandatory at diagnosis to define high risk cytogenetic patients High-Risk (25%) Standard-Risk, neutral (75%) * All others including: (FISH) FISH*** del 17p t(4;14) t(14;16) 1q gain/1p deletion ANY ABNORMALITY DETECTED BY CONVENTIONAL KARYOTYPING - Hyperdiploid - t(11;14) - t(6;14) - 5q amplification - FISH del13q without t(4;14) and/or del17p ***Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorde (Ross et al, 2012)

22 Prognosis is influenced by association with other abnormalities & number of involved cells Negative Positive or Neutral - t (4;14) worst if associated -Hyperdiploidy - Del 17p (p53).number of cells* - 1q gains - 12p deletions - 5q amplification - t (11;14) - 13q by cytogenetics *It seems that higher proportion of PC berinf 17p deletions is associated with shorter survival (the cut-off is controversial) Perez Simon, Blood 1999; Fonseca Blood 2003; Chang Bllod 2005; Gutierrez Leukemia 2007; Avet- Loisseau JCO 2010 & Blood 2011; Boyd Leukemia 2011, Kumar Blood 2012

23 Cytogenetic analysis in MM in the laboratory Methodologies for the detection of chromosomal abnormalities Karyotype (G-banded) and FISH Genomic approaches have not been implemented in most clinical laboratories and are not standardized tools for risk stratification

24 Tipo de muestras MM (% CP) Cariotipo (mo heparina) 72h sin estimular FISH (mo heparina) MOLECULAR (mo edta) Reservar CD138 criopreservadas Qué panel de sondas FISH utilizar?

25 Normal BM Acute leukemia Myeloma Because of Low infiltration of tumoral cells in the bone marrow

26 and low mitotic index, only 30% of MM patiens have abnormal karyotypes BM Heparin 70% Normal karyotype Karyotype 72h culture El informe genético: citogenética convencional

27 FISH Fonseca, Blood 2003 Molecular Cytogenetics FISH (based on fluorescence) Does not require metaphases, high resolution, but: Only explores specific target regions

28 The plasma cells need to be selected enabling an unambiguous identification FISH analysis is performed in purified PC Immunomagnetic separation Flow cytometry, FACSAria II cell sorter Cell sorting results in a pure PC population which enables further analyses to be performed

29 Cytogenetic analysis in MM patients (High risk cytogenetics) Procedure: multi step MM sample karyotype (total BM) 72h culture FISH (purified PC) 2-3 steps with specific probes (100 PC per probe should be scored) FISH MM 1 (step 1) Translocations IGH P53 1p/1q FISH MM 2 (step 2) t(4;14) t(14,16) FISH MM 3 (extended) Hyperdiploidy Extended panel (guidelines)

30 Presence of trisomies in patients with t(4;14), t(14;16), t(14;20), or P53 deletion in MM ameliorates the usual adverse impact associated with these abnormalities n=370 n=370 n=114 n=6 6 n=4 8 High-risk FISH: t(4;14), t(14;16), t(14;20) and P53 deletion Trisomies: 3, 7, 9, 11, 13, 15 and 17 Kumar S, Blood 2012

31 FISH MM step 3 Extended panel: Hyperdyploidy t(11;14) t(14;20) 13q 12 Not mandatory HYPERDIPLOIDY D5S23 / D5S721 CEP 9 CEP 15 VS38/CD138*alexa594

32 Finally the Cytogenetic Report ISCN 2013 An International System for Human Cytogenetic Nomenclature (2013)

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34 In summary All patients with MM have a cytogenetic study in order to identify the chromosomal abnormalities with prognostic value. The cytogenetic study (karyotype and FISH in purified PC using the consensus panel) should be performed at the time of diagnosis. The cytogenetic study also is carried out at relapse in those cases initially classified as genetic standar-risk Cytogenetics is not a tool for disease follow up. Genomic approaches: GEP, or mutation analysis (NGS) have not been implemented in most clinical laboratories at diagnosis and are not standardized tools for predicting outcome (nowadays, flow citometry is the standar)

35 Risk Stratification: the future Host characteristics Age Co-morbidities: e.g renal failure; spinal cord compression Tumor characteristics ISS stage Estramedullary disease Cytogenetics Plasma cell leukemia Lactate dehydrogenase level Proliferative indices (high proliferation rate/labelling index) GEP signatures Arrays SNPs Mutations (NGS)

36 Genetic markers with prognostic significance

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42 Next generation sequencing (NGS) in MM Hairy-cell leukemia BRAF mutations ~ 100% Waldenstrom's Macroglobulinemia MYD88 mutations ~ 90% Chronic lymphocytic leukemia SF3B1 mutations ~ 15% Multiple myeloma?

43 Initial genome sequencing and analysis of MM Even for a dominant ly actin gene, such as NRAS or KRAS, they identify variation in the size of clone carrying the mutation, pointing to intraclonal heterogeneity and to potential difficulties in the use of targeted strategies 38 multiple myeloma patients: an average of 35 nonsynonimus mutatios per case. (8 in leukaemias; 540 in epithelial tumors). Few recurrent mutations. Confirm known mutations ERK pathway: KRAS in 27%, NRAS in 24%, BRAF 4%)* A few novel genes (FAM46C in 13%, DIS3 in 11%) deregulation of pathways is pathogenically important rather than the regulation of a specific gene (NFKb, ERK, PI3K) Chapman et al, Nature 2011

44 Initial genome sequencing and analysis of MM Chapman et al, Nature 2011

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46 Keats, Blood 2012 Magrangeas, Leukemia 2012 Three distinct patterns of genomic evolution Stable genomes: no differences between diagnosis and relapse clones. Low-risk cytogenetics Linear evolution: the relapse clone apparently derives from the major subclone at diagnosis, but continues to diversify through additionally acquired lesions. High-risk cytogenetics Branching (nonlinear) model: the relapse clone clearly derives from a minor subclone, barely present at diagnosis. These changes are best explained by the existence of clonal heterogeneity at diagnosis. High-risk cytogenetics

47 Next generation sequencing (NGS) in MM 203 multiple myeloma patients 11 genes mutados (5 previos) Lohr et al, Cancer Cell 2014

48 Genome sequencing and analysis of MM 1. MM tumors are highly heterogenous 1. Point mutations in the most significanttly mutated genes were found to be clonal in some patients and subclonal in others (NRAS, KRAS, BRAF initiators or potentiators) 1. Important clinical implications. For example BRAF V600E with BRAF inhibition. Treating patients harboring subclonal BRAF inhibitors may stimulate the growth of BRAF-wild type tumor cells. 2. It is important to enumerate the extent of clonal heterogeneity in patients for targeted therapy. Efective targeted therapy will require either drug combinations targeting distintc subclones or using targeted therapies only in patients for whom the drug target is entirely clonal. Lohr et al, Cancer Cell 2014

49 Intraclonal architectural heterogeneity at diagnosis and at different stages of disease progression over time Clonal competition with alternating dominance in multiple myeloma Keats, Blood 2012; Walker, Blood 2012; Egan, Blood 2012

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51 Comentarios -La heterogeneidad clonal debe ser además evaluada en el contexto de ensayos clínicos con combinaciones de fármacos o con Terapias secuenciales con distintos mecanismos de acción para evaluar las mejores opciones terapéuticas en consolidación y mantenimiento tras la selección clonal secundaria a los tratamientos de inducción. (alterar la secuencia de los tratamientos) -La emergencia de subclones en MM bajo presión selectiva e muy importante en el contexto de tratamientos de mantenimiento o tratamiento de estados asintomáticos de la enfermedad -La heterogeneidad intraclonal y la evolucion clonal en el MM subyacen a la resistencia al tratamiento (además del mecanismo de mutación de la diana terapeútica).

52 Estrategias de estratificación, NGS

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54 GEP15 Cereblon t(4;14) del(1p) GEP80 Gracias PSDM4 1q amp NGS & clonality GEP70