MOLECULAR GENETICS OF B CELL LYMPHOMAS: AN UPDATE Michel Trudel, MD, FRCPC Shaikh Khalifa Medical Center

B CELL LYMPHOMAS: MOLECULAR PATHWAYS Pasqualucci & Dalla-Favera, 2002

MICROARRAY EXPRESSION PROFILING: PLATFORMS

MICROARRAY EXPRESSION PROFILING: MOLECULAR INTERACTIONS

MICROARRAY EXPRESSION PROFILING IDENTIFICATION OF:  genes involved in pathogenesis / progression, localization / spread, etc  novel genes / disease categories (“class discovery”)  known cell lineage, differentiation stage, etc (“class prediction”)  genes involved in sensitivity / resistance  risk factors / prognostic groups  novel molecular targets for therapy

FOLLICULAR LYMPHOMA: MORPHOLOGY & PHENOTYPE Grogan, LYMPHOMAS, 1998

FOLLICULAR LYMPHOMA: MOLECULAR GENETICS Ig genesrearranged; IgV genes extensively mutated (intraclonal heterogeneity) KARYOTYPE GENE FREQUENCY FEATURES t(14;18) (q32;q21) BCL2 80% anti-apoptosis sole abnormality in ~ 10% 6q % deletions of ? suppressor genes at q21,q23,q25-27 most common 2 nd abnormality in cells with t(14;18) 3q27 BCL6 transcriptional repressor 40% 5‘ mutations 15% translocations p15, p16 deletions, mutations 17pp53 15% deletions, mutations progression to DLBCL +7, %

FOLLICULAR LYMPHOMA: EXPRESSION PROFILING 6 FL (relapsed; no Rx for prior 6 months) vs. GC B-cells (immunomagnetic bead separation) from 6 tonsils Clontech microarray (588 cDNA’s) microarrayRQ-PCR verification 37 genes   genes   8 Husson et al, Blood, 2002

FOLLICULAR LYMPHOMA: DIFFERENTIALLY EXPRESSED GENES UPREGULATED GENES cell cycle control: CDK10, p120, CDKNIA(p21), CDK2A (p16) transcription factors: PAX5, ID2 (B cell differentiation) cell-cell interaction: TNF, IL-2R , IL-4R  signal transduction: MLK3 DOWNREGULATED GENES cell adhesion / communication: MRP 8/14, CD40, thymosin  10

FOLLICULAR LYMPHOMA : CHROMOSOMAL LOCALIZATION OF DIFFERENTIALLY EXPRESSED GENES GENES INCREASEDLOCUS BCL218q21 CDKNIAC (p21)6p21 TNF6p21 JUN1p32-p31 HSF18q24 XPB2q21 MLK311q13 HSP277q PAX59p13 GENES DECREASED S-100 A 8/9 (MRP 8/14)1q12-q22 PAGA1p34

DIFFUSE LARGE B CELL LYMPHOMA: MORPHOLOGY

DIFFUSE LARGE B CELL LYMPHOMA : MOLECULAR GENETICS Ig genes rearranged; IgV genes mutated KARYOTYPE GENE FREQUENCY FEATURES 3 q 27 BCL6 POZ/zinc finger transcriptional repressor required for - GC formation - Ab affinity maturation - TH 2 -dependent immune response 70-75% mutations ( 5' regulatory sequences ) 35% translocations multiple partners ( 5' region truncated, juxtaposed to heterologous promoters) Dalla-Favera et al, 1996, 2000

BCL 6: PROMISCUOUS TRANSLOCATIONS Gaidano et al, 2000

DIFFUSE LARGE B CELL LYMPHOMA : MOLECULAR GENETICS KARYOTYPE GENE FREQUENCYFEATURES t (14;18) (q32;q21) BCL-225%anti-apoptosis transformed FL unfavorable prognosis 17p p53mutations, deletions transformed FL p15, p16mutations, deletions hypermethylation REL, NF  B2transcription factors 20% amplification REL: extranodal lymphoma

DIFFUSE LARGE B CELL LYMPHOMA: GENE PROFILES Alizadeh et al, Nature, 2000

DIFFUSE LARGE B CELL LYMPHOMA: EXPRESSION PROFILING 2 distinct groups identified by microarray analysis 5-year OS  germinal center B-like 76%  activated peripheral B-like 16% Note: prognostic significance independent of IPI residual clinical variability within each type NF-  B pathway constitutively active in “activated peripheral” type ? therapeutic approach to poor prognosis group Alizadeh et al, Nature, 2000 Davis et al, J Exp Med, 2001

MICROARRAY EXPRESSION PROFILING : ISSUES / PROBLEMS  Quality of tumor banking, RNA retrieved  Type of platform: spotted (cDNA), Affymetrix  Standardization / validation of results  Data analysis / management: - large data sets generated on small sample numbers - different clustering algorithms - bioinformatics capabilities - selection of endpoints  Intercurrent variables: therapies, etc  Discrimination of primary from secondary events, epiphenomena, spurious results (false + / – )  Genomics vs proteomics Experience suggests that diseases will continue to be defined by combination of parameters: clinical, morphologic, phenotypic, genotypic