Next Generation Sequencing Bologna, 11 Maggio 2012 NEXT GENERATION SEQUENCING: I PROGRESSI NELLA CARATTERIZZAZIONE DELLA LEUCEMIA LINFATICA CRONICA Davide Rossi, M.D., Ph.D Division of Hematology Department of Translational Medicine Amedeo Avogadro University of Eastern Piedmont Novara-Italy
TP53 disruption does not fully account for fludarabine-refractoriness in CLL CLL2H trial (GCLLSG)= Alemtuzumab s.c. 100 75 Overall survival Median: 19.1 mo (n=103) TP53 44/ 99 ( 40%)??? 99 ( 60%) 50 25 0 0 6 12 18 24 30 36 42 48 54 Time (months) Courtesy of T. Zenz Zenz T, et al, Blood 2009 Stilgenbauer S, et al. J Clin Oncol 2009 Need to search for other genetic determinants of fludarabine-refractoriness in CLL
Disease model: high risk CLL Fludarabine-refractory CLL without evidence of DLBCL transformation Richter syndrome histologically proven DLBCL LN BM PB
Identification of novel candidate genes in fludarabinerefractory CLL and Richter s. by whole exome sequencing Discovery Whole Exome Sequencing N=10 paired N/T samples from 10 fludarabine-refractory CLL N=9 paired CLL/DLBCL samples from 9 clonally related RS Identification of candidate genes involved in fludarabine-refractory CLL and RS Assessment of recurrence Extension PCR amplification and sequencing of coding exons and consensus splicing sites of candidate genes in independent extension panels of: i) 49 fludarabine-refractory CLL and 24 clonally related RS Filter for: i) known polymorphisms (dbsnp database; Ensembl; The 1000 Genome Project); ii) variants present in matched germline DNA; iii) synonimous changes; iv) recurrent variants, unless confirmed to be of somatic origin Assessment of recurrence in other clinical phases of CLL Super extension PCR amplification and sequencing of coding exons and consensus splicing sites of candidate genes in: i) 63 MBL ii) 301 CLL at diagnosis
Whole exome capture and sequencing approach Exon capture SureSelect Human All Exon Agilent NimbleGen Array 3µg of human genomic DNA (N=10 paired N/F-ref CLL) (N=9 paired CLL/RS) DNA shearing linker ligation-pcr Hybridization Magnetic beads selection Magnetic beads selection Sequencing 454 GS FLX Roche Amplified target pool LM-PCR amplification Enriched target DNA Wash beads Digest RNA Magnetic beads selection SOliD 3 Plus Applied biosystems HiSeq2000 Illumina High Throughput Sequencing
3 novel pathways in CLL NOTCH Splicing NF-κB
NOTCH1 Maps on 9q34.3 Class I transmembrane glycoprotein Functions as a ligand-activated transcription factor Activated by mutations in 60% T-ALL MYC activation PI3K-AKT-mTOR signaling CCND3, CDK4, and CDK6 activation p21 and p27 inactivation NF-κB signaling
NOTCH1 mutations in newly diagnosed CLL and RS 454 N vs CLL Mean depth ~12x Frequency (%) 20 15 10 5 0 NOTCH1 TP53 PLEKHG5 TGM7 BIRC3 SOliD CLL vs DLBCL Mean depth ~30x N of mutations acquured at RS transformation 49 mutations in 45 genes (2 recurrent in >1 case) TP53 TP53 NOTCH1 NOTCH1 TP53 TP53 Fabbri G, et al. J Exp Med 2011; 208:1389-401
NOTCH1 mutations occur in ~10% CLL at diagnosis and their prevalence increases with CLL aggressiveness NOTCH1 mutations in pivotal CLL studies Frequency (%) N=7/133 N=31/255 N=10/120 Sportoletti P, et al. Br J Haematol 2010 Puente XS, et al. Nature 2011 Fabbri G, et al. J Exp Med 2011 NOTCH1 mutations across different CLL clinical phases Frequency (%) Fabbri G, et al. J Exp Med 2011; 208:1389-401 Rossi D, et al. Blood 2012; 119:521-9 Rasi S, et al. Haematologica 2012; 97:153-4 50% 40% 30% 20% 10% 0% P<0.001 *** P<0.05 ** N=2/134 N=2/63 ** *** N=60/539 ** *** N=10/48 de novo DLBCL MBL CLL diagnosis Chemorefractory CLL N=18/58 Richter syndrome
Experimental strategy Training series (n=309) Consecutive series of 309 newly diagnosed CLL who presented for initial evaluation at a single center (Diagnostic criteria: 2008 IWCLL-NCI) Validation series (n=230) Multi-institutional retrospective series of 230 newly diagnosed CLL (Diagnostic criteria: 2008 IWCLL-NCI) NOTCH1 analysis PCR amplification and Sanger sequencing of the NOTCH1 mutation hotspot in CLL (exons 26, 27 and 34; RefSeq NM_017617.2) Filter for: i) known polymorphisms (dbsnp database build 133; Ensembl; 1000 Genome project); ii) variants present in matched germline DNA; iii) synonimous changes; Identification of mutated patients Statistical analysis Primary endpoint: OS (Secondary endpoints: TFS; time to RS) Cox multivariate analysis for OS Covariates: NOTCH1 mutations, age, gender, Rai stage, IGHV identity, +12, del11q22-q23,tp53 disruption Internal boostrapping validation in the training series External validation in the validation series REMARK criteria (McShane LM, et al. J Clin Oncol 2005) Rossi D, et al. Blood 2012;119:521-9
Prevalence and distribution of NOTCH1 mutations in the CLL training series NOTCH1 M NOTCH1 GL 11.0% N=34/309 del13q14 +12 NOTCH1 M del11q22-q23 TP53 dis Training series (n=309) p<.001 NOTCH1 GL NOTCH1 M Frequency p=.006 p=.002 p<.001
In the training series, NOTCH1 mutations identify a subgroup of CLL characterized by poor outcome Training series (n = 309) NOTCH1 31 3 TP53 30 NOTCH1 GL NOTCH1 M p<.001 GL NOTCH1 M TP53 DIS NOTCH1 M vs GL NOTCH1 M vs TP53 DIS TP53 DIS vs GL p<.001 p=.685 p<.001 No. at Risk NOTCH1 GL 275 133 39 13 2 1 0 NOTCH1 M 34 8 0 0 0 0 0 Events Total Median 95% CI NOTCH1 GL 62 275 13.9 10.5-17.3 NOTCH1 M 16 34 3.5 0-7.3 No. at Risk GL 245 124 37 13 2 1 0 NOTCH1 M 31 8 0 0 0 0 0 TP53 DIS 33 9 2 0 0 0 0 Events Total Median 95% CI GL 48 245 15.6 13.1-18.1 NOTCH1 M 13 31 7.8 0.4-15.2 TP53 DIS 17 33 4.6 1.8-7.3 Rossi D, et al. Blood 2012;119:521-9
NOTCH1 mutations are an independent predictor of OS in the CLL validation series NOTCH1 M NOTCH1 GL p=.002 NOTCH1 GL NOTCH1 M 11.3% NOTCH1 25 1 TP53 26 N=26/230 GL NOTCH1 M TP53 DIS NOTCH1 M vs GL NOTCH1 M vs TP53 DIS TP53 DIS vs GL p<.001 p=.646 p<.001 NOTCH1 M TP53 dis External validation in the validation series Hazard ratio 0.5 1 2 3 4 5 6 7 8 9 10 Age >60y Rai III-IV Male HR 70% CI 80% CI 90% CI 95% CI del11q22-q23 +12 IGHV UM Rossi D, et al. Blood 2012;119:521-9
TP53 disruption and MYC activation contribute to a dual hit mechanism of transformation in Richter syndrome Frequency (%) Clonally related Richter syndrome N=30/54 N=11/51 N=18/58 CLL RS NOTCH1 M MYC abnormalities TP53 disruption Clonally related Richter syndrome paired clonal M subclonal M WT Fabbri G, et al. J Exp Med 2011; 208:1389-401 Rossi D, et al. Blood 2011; 117:3391-401 Rossi D, et al. Blood 2012;119:521-9
NOTCH1 Maps on 9q34.3 Class I transmembrane glycoprotein Functions as a ligand-activated transcription factor Activated by mutations in 60% T-ALL MYC activation PI3K-AKT-mTOR signaling CCND3, CDK4, and CDK6 activation p21 and p27 inactivation NF-κB signaling
TP53 disruption and MYC activation contribute to a dual hit mechanism of transformation in Richter syndrome MYC translocation/a mplification NOTCH1 mutations MYC activation TP53 disruption transformation CLL DLBCL (Richter)
Risk of Richter transformation according to NOTCH1 mutation status at CLL diagnosis NOTCH1 wt NOTCH1 M NOTCH1 wt & no IGHV4-39 NOTCH1 wt & IGHV4-39 NOTCH1 M & no IGHV4-39 NOTCH1 M & IGHV4-39 p<.001 p<.001 p<.001 No. at Risk NOTCH1 wt 531 279 92 31 11 3 1 0 0 NOTCH1 M 74 28 8 1 0 0 0 0 0 No. at Risk NOTCH1 wt & no IGHV4-39 519 273 90 30 11 3 1 0 0 NOTCH1 wt & IGHV4-39 12 12 12 12 0 0 0 0 0 NOTCH1 M & no IGHV4-39 67 27 8 1 0 0 0 0 0 NOTCH1 M & IGHV4-39 7 1 0 0 0 0 0 0 0 Events Total 5-year risk 95% CI NOTCH1 wt 18 531 3.9% 2.0-5.8 NOTCH1 M 12 74 18.6% 7.3-29.9 Events Total 5-year risk 95% CI NOTCH1 wt & no IGHV4-39 18 519 4.0% 2.1-5.9 NOTCH1 wt & IGHV4-39 0 12 0 NOTCH1 M & no IGHV4-39 8 67 12.5% 2.9-22.1 NOTCH1 M & IGHV4-39 4 7 75.0% 32.5-100
~80% NOTCH1 mutations in CLL are represented by a recurrent c.7544_7545delct deletion 5 3 1 TM EGF repeats (1-36) LNR HD RAM Ankyrin TAD PEST 2556 Training series Validation series Missense Nonsense Frameshift N=60 TAD PEST 2155 2556 NOTCH 1 EX34: c.7544_7545delct p.p2515fs*4 Rossi D, et al. Blood 2012;119:521-9
ARMS PCR is a diagnostic tool for detecting the NOTCH1 c.7544_7545delct mutation ForC ForMUT Rev Mutated allele CT 5 3 284bp 183 bp Sanger sequencing vs ARMS for c.7544_7545delct mutation detection ARMS Sanger Pos Neg Total Pos 26 0 26 Sensitivity=100% Neg 0 283 283 Specificity=100% Total 26 283 309 WildType allele 5 3 PPV=100% NPV=100% Κ=1 284bp ARMS neg ARMS pos CLL samples p<.001 300bp ARMS was calibrated to detect a mutation present in >10% alleles 200bp Events Total Median 95% CI ARMS neg 66 283 13.0 10.1-15.9 ARMS pos 12 26 7.8 0.8-14.7 Rossi D, et al. Blood 2012;119:521-9 No. at Risk ARMS neg 283 134 39 12 2 1 0 ARMS pos 26 7 0 0 0 0 0
3 novel pathways in CLL NOTCH Splicing NF-κB
WES identifies SF3B1 as a recurrently mutated gene in fludarabine-refractory CLL Ilumina N vs CR-CLL Mean depth ~100x 160 mutations in 150 genes (2 genes recurrently mutated in >2 case) N of mutated genes SF3B1 21 TP53 20 19 18 SF3B1 17 16 TP53 15 TP53 13 SF3B1 11 9 Rossi D, et al. Blood 2011;118: 6904-8
Emerging role of the spliceosome in hematologic malignancies RNA splicing Spliceosome SF3B1 mutations in pivotal CLL studies Luke et al, Mol cell Biol 1996 David et al, Genes Dev 2010 Kaida et al, Nat Chem Biol 2007 Corrionero et al, Genes Dev 2011 Yoshida et al, Nature 2011 Rossi D, et al. Blood 2011 Quesada V, et al. Nat Genet 2012 Wang L, et al. N Engl J Med 2011
SF3B1 is recurrently mutated in 17% fludarabine-refractory CLL Fludarabine-refractory CLL (N=59) *p<.05 17% Frequency * * * SF3B1 mutations N=10/59 SF3B1 mutations TP53 disruption NOTCH1 mutations ATM deletion Rossi D, et al. Blood 2011 Fludarabine-refractory CLL (n=59)
SF3B1 mutation prevalence increases with CLL aggressiveness p=.002 p=.200 N=10/59 (17%) Frequency N=1/63 (1%) N=17/301 (5%) N=2/33 (6%) MBL CLL diagnosis Fludarabine refractory CLL Richter syndrome SF3B1 mutations TP53 disruption NOTCH1 mutations ATM deletion Rossi D, et al. Blood 2011 CLL at diagnosis (n=301)
At diagnosis, SF3B1 mutations identify a subgroup of CLL characterized by poor outcome SF3B1 M SF3B1 GL 5% SF3B1 16 1 TP53 30 N=17/301 SF3B1 wt SF3B1 M N=301 SF3B1 & TP53 wt SF3B1 M TP53 DIS N=301 p=.011 SF3B1 M vs SF3B1 & TP53 wt TP53 DIS vs SF3B1 & TP53 wt SF3B1 M vs TP53 DIS p.=002 p=.002 p=.708 No. at Risk SF3B1 wt 284 107 21 8 2 1 0 SF3B1 M 17 3 0 0 0 0 0 Events Total Median 95% CI SF3B1 wt 71 284 nr - SF3B1 M 6 17 2.5 1.7-3.3 No. at Risk SF3B1 & TP53 wt 255 102 21 8 2 1 0 SF3B1 M 17 3 0 0 0 0 0 TP53 DIS 29 5 0 0 0 0 0 Events Total Median 95% CI SF3B1 & TP53 wt 55 255 nr - SF3B1 M 6 17 2.5 1.7-3.3 TP53 DIS 16 29 2.9 0.1-5.77
Mutations cluster in the HEAT3-4-5 repeats of SF3B1 and recurrently target codons 662, 666 and 700 5 3 p14 binding domain HEAT repeats 1 2 3 4 5 6 7 8 9 10 11 1 223 491 529 604 718 763 801 843 881 1010 1090 1122 1201 1034 Missense In frame deletion Homo sapiens A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K P. troglodytes A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K C. familiaris A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K B. taurus A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K M. musculus A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K R. norvegicus A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K G. gallus A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K D. rerio A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L R S L V E I I E H G L V D E Q Q K V R T I S A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R Q H R G K D. melanogaster A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L K A L V E I I E H G L V D E Q Q K V R T I T A L A I A A L A E A A T P Y G I E S F D S V L K P L W K G I R T H R G K A. gambiae A R A F A V V A S A L G I P S L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q I A I L M G C A I L P H L K S L V E I I E H G L V D E Q Q K V R T I T A L A L A A L A E A A T P Y G I E S F D S V L K P L W K G I R T H R G K C. elegans A R A F A V V A S A L G I P A L L P F L K A V C K S K K S W Q A R H T G I K I V Q Q M A I L M G C A V L P H L K A L V D I V E S G L D D E Q Q K V R T I T A L C L A A L A E A S S P Y G I E A F D S V L K P L W K G I R M H R G K S. pombe A R A F S V V A S A L G V P A L L P F L K A V C R S K K S W Q A R H T G V R I I Q Q I A L L L G C S I L P H L K N L V D C I G H G L E D E Q Q K V R I M T A L S L S A L A E A A T P Y G I E A F D S V L K P L W S G V Q R H R G K M. oryzae A R A F A V V A S A L G I P A L L P F L Q A V C R S K K S W Q A R H T G V K I V Q Q I P I L M G C A V L P H L K R L V D C I G P N L N D E Q T K V R T V T S L A I A A L A E A A N P Y G I E S F D D I L N P L W T G A R K Q R G K N. crassa A R A F A V V A S A L G I P A L L P F L R A V C R S K K S W Q A R H T G V K I V Q Q I P I L M G C A V L P H L K Q L V D C I G P N L N D E Q T K V R T V T S L A I A A L A E A S N P Y G I E S F D D I L N P L W T G A R K Q R G K A. thaliana A R A F S V V A S A L G I P A L L P F L K A V C Q S K R S W Q A R H T G I K I V Q Q I A I L I G C A V L P H L R S L V E I I E H G L S D E N Q K V R T I T A L S L A A L A E A A A P Y G I E S F D S V L K P L W K G I R S H R G K O. sativa A R A F S V V A S A L G T P A L L P F L K A V C Q S K K S W Q A R H T G I K I V Q Q I A I L M G C A V L P H L K S L V E I I E H G L S D E N Q K V R T I T A L S L A T L A E A A A P Y G I E S F D T V L K P L W K G I R S H R G K 6666666666666666666666666666666666666666666666666666 6 666666666666666666777777777777777777777777777777777777777777 Codons 2333333333344444444445555555555666666666677777777778 8 888888889999999999000000000011111111112222222222333333333344 9012345678901234567890123456789012345678901234567890 1 234567890123456789012345678901234567890123456789012345678901
SF3B1 is a component of the U2 snrnp complex of the major spliceosome U1 U4 Intron Ex5 SS Ex3 SS pre-mrna U1 U6 U4 U5 U2 U6 U2 U5 U6 U2 U5 mrna U2 snrnp SF3b complex SF3B1 mutations U2 snrna SF3B1 SF3b145 SF3b130 SF3b49 SF3b14b SF3b10 SF3b14a/p14 A B SF3a120 SF3a 66 SF3a 60 1 1034 p14 p14 binding site HEAT 1-2-3-4 repeats pre-mrna Golas MM, et al. Science 2003 Will CL & Lührmann R. Cold Spring Harb Perspect Biol 2011
3 novel pathways in CLL NOTCH Splicing NF-κB
The NF-κB pathway is an attractive candidate in fludarabine-refractory CLL Kern C, et al. Blood 2004 Endo T, et al. Blood 2007 Hewamana S, et al. Blood 2008 Buggins AG, et al. Cancer Res 2010 Herishanu Y, et al. Blood 2011 Compagno M, et al. Nature 2009 NF-κB activation provides pro-survival signals to CLL cells through the upregulation of a number of anti-apoptotic genes, and correlates with both survival and enhanced fludarabine resistance of CLL cells NF-κB activation is generally viewed as a consequence of specific interactions between CLL cells and protective microenvironmental niches NF-κB signaling may be activated in B-cell neoplasia through an array of molecular lesions affecting genes at different levels of the pathway
BIRC3 mutations in newly diagnosed CLL and RS 454 N vs CLL Mean depth ~12x Frequency (%) 20 15 10 5 0 NOTCH1 TP53 PLEKHG5 TGM7 BIRC3 SOliD CLL vs DLBCL Mean depth ~30x N of mutations acquured at RS transformation 49 mutations in 45 genes (2 recurrent in >1 case) BIRC3 Fabbri G, et al. J Exp Med 2011; 208:1389-401
BIRC3 is a negative regulator of non-canonical NF-κB signaling Basal CD40 BAFFR RANK LTBR CD40 BAFFR RANK LTBR Stimulated TRAF3 TRAF2 TRAF3 MAP3K14 Ub Ub Ub Ub Ub TRAF2 BIRC3 MAP3K14 P IKK BIRC3 RelB p100 p52 RelB MAP3K14 degradation Non canonical NF-kB activation
BIRC3 is recurrently disrupted in 24% fludarabine-refractory CLL p<.001 p<.001 n=12/49 (24%) BIRC3 disruption frequency n=0/63 n=13/306 (4%) n=0/68 n=0/33 12/32 (37%) TP53 WT fludarabine refractory CLL Clinical MBL CLL at diagnosis Fludarabine Fludarabine refractory CLL sensitive CLL Richter syndrome TP53 disruption TP53 disruption BIRC3 disruption BIRC3 disruption SF3B1 mutations SF3B1 mutations NOTCH1 mutations NOTCH1 mutations CLL diagnosis (n=306) Fludarabine-refractory CLL (n=49) Rossi D, et al. Blood 2012; doi:10.1182/blood-2011-12-395673
At diagnosis, BIRC3 disruption identifies CLL patients characterized by poor outcome BIRC3 M BIRC3 GL 4% BIRC3 13 TP53 31 N=13/306 WT BIRC3 dis WT TP53 dis BIRC3 dis p<.001 BIRC3 dis vs WT TP53 dis vs WT BIRC3 dis vs TP53 dis p<.001 p<.001 p=.543 No. at Risk WT 293 111 19 8 2 1 0 BIRC3 dis 13 1 0 0 0 0 0 Events Total Median 95% CI WT 69 293 nr - BIRC3 dis 9 13 3.1 0-6.7 No. at Risk WT 262 106 19 8 2 1 0 TP53 dis 31 5 0 0 0 0 0 BIRC3 dis 13 1 0 0 0 0 0 Events Total Median 95% CI WT 52 262 nr - TP53 dis 17 31 2.9 0.6-5.3 BIRC3 dis 9 13 3.1 0-6.7
BIRC3 is targeted by inactivating lesions in CLL BIRC3 Chr 11q 101700 kb 101720 kb 101740 kb 101760 kb 101780 kb 101800 kb 101820 kb 101840 kb RING CARD UBA BIR3 BIR2 BIR1 1 29 96 169 235 255 322 377 423 439 529 557 592 604 09-396 09-872 09-142 09-361 Uiquitin E3 ligase activity Frameshift Non-sense Missense BIRC3 BIRC2 TMEM123 Chr11 A B Frameshift Non-sense Deletion BIRC3 del BIRC3 mut BIRC3 6550 5610 3410 10649 7561 3802 4848 5092 5175 JJN3 KMS-12PE 09-361 BIRC3 ATM 11q22.2 Rossi D, et al. Blood 2012; doi:10.1182/blood-2011-12-395673 Relative BIRC3 expression Actin
BIRC3 disruption might activate NF-κB by inducing MAP3K14 accumulation Basal CD40 BAFFR RANK LTBR CD40 BAFFR RANK LTBR Stimulated TRAF3 TRAF2 TRAF3 MAP3K14 Ub Ub Ub Ub Ub TRAF2 BIRC3 MAP3K14 P IKK BIRC3 RelB p100 p52 RelB MAP3K14 degradation Non canonical NF-kB activation
BIRC3 mutations activate non-canonical NF-kB signaling in CLL Rossi D, et al. Blood 2012; doi:10.1182/blood-2011-12-395673 BIRC3 deletion BIRC3 mutation 6550 5610 3410 10649 7561 3802 4848 5092 5175 JJN3 KMS-12PE Mutated Deleted wt p100 NFKB2 p52 Actin
BIRC3 might represent a second tumor suppressor in the 11q22-q23 region Rossi D, et al. Blood 2012; doi:10.1182/blood-2011-12-395673 BIRC3 dis ATM del Chromosome 11 Chr 11q22.2 BIRC3 ATM WT ATM del BIRC3 dis BIRC3 dis & ATM del BIRC3 dis vs WT p=.001 ATM del vs WT p=.013 BIRC3 dis & ATM del vs WT p<.001 ATM del vs BIRC3 dis p=.397 ATM del vs BIRC3 dis & ATM del p=.281 BIRC3 dis vs BIRC3 dis & ATM del p=.137
A model for understanding the molecular basis of fludarabine-refractory CLL N=49 TP53 disruption BIRC3 disruption SF3B1 mutation NOTCH1 mutation Fludarabine-refractory CLL (n=49)
Open issues Integrate NOTCH1, SF3B1 and BIRC3 mutations and cytogenetic lesions in a new hierarchical model for CLL outcome prediction Assess the clonal evolution of NOTCH1, SF3B1 and BIRC3 mutations during disease course Validate the prognostic impact of NOTCH1, SF3B1 and BIRC3 mutations within prospective studies Define the role of new agents in overcoming the poor impact of NOTCH1, SF3B1 and BIRC3 mutations
Methods of screening to identified mutational prevalence of candidate gene Sanger sequencing ABI Prism 3100 DNA sequencer 96 samples in a single run Amplimer based sequencing (broad sequencing) 600 samples in a single run GS Junior 454 System
NOTCH1 mutations associate with trisomy 12 del13q14 +12 del11q22-q23 TP53 disruption BIRC3 disruption SF3B1 mutation NOTCH1 mutation CLL at diagnosis (n=306) All NOTCH1 SF3B1 BIRC3 del13q14 +12 TP53 del11q22-q23 Del Giudice I, et al. Haematologica 2012;97:437-41
Putative pattern of transformation Initial CLL clone CLL clone evolution Sequential evolution from a secondary CLL subclone Direct evolution from the initial CLL clone RS RS Rossi D, et al. Int J Cancer 2011
Backtracking in the CLL phase of candidate gene mutations identified in the RS phase Amplicon library preparation GS Junior 454 System EmulsionPCR amplification Ultra-deep next generation sequencing (~7000 reads per amplicon) Subclonal variant identification in the CLL phase (BackTracker algorithm) Fabbri G, et al. J Exp Med 2011
The genetic lesions of RS may be detectable at subclonal levels in the initial CLL clone NOTCH 1 EX34: c.7544_7545delct p.p2515fs*4 (heterozygous) TP53 EX7: g.13353 A>C p.n239t (heterozygous) 5.6% 58% 0.39% 43% CLL 30 months CLL 62 months Reads (%) Fabbri G, et al. J Exp Med 2011
Silvia Rasi Alessio Bruscaggin Carmela Ciardullo Stefania Cresta Clara Deambrogi Lorenzo De Paoli Rosella Famà Marco Fangazio Sara Monti Valeria Spina Gianluca Gaidano Laura Pasqualucci Giulia Fabbri Riccardo Dalla Favera Francesco Bertoni Vladimir Trifonov Hossein Khiabanian Raul Rabadan CRO AVIANO Pietro Bulian Valter Gattei Monica Messina Sabina Chiaretti Ilaria Del Giudice Anna Guarini Robin Foà Francesco Forconi Luca Laurenti Grant support: UNIVERSITA DEGLI STUDI DI MODENA E REGGIO EMILIA Roberto Marasca Tiziana Vaisitti Silvia Deaglio
Selection criteria of germline DNA samples for WES Total amount 4-5 μg of DNA High molecular weight DNA No contamination by tumor DNA
Source of germline DNA in CLL: saliva vs urina Tumor DNA contamination 1 2 3 4 5 6 T S U T S U T S U T S U T S U T S U 500 bp Prevalence of tumor DNA contamination 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 5% Saliva (CLL in CR) 14% 14% 17% Saliva (CLL active) Urine (CLL in CR) Urine (CLL active) 50% PB G (CLL in CR) 100% PB G (CLL active)
Source of germline DNA in CLL: saliva vs urina DNA yield DNA from saliva DNA from urine Frequency Frequency DNA yield (μg) DNA yield (μg) DNA yield (μg) Huma DNA (%)
Source of germline DNA in CLL: saliva vs urina ii) DNA quality High molecular weight DNA from saliva (97% samples) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 10 Kb High molecular weight DNA from urine (100% samples) 1 2 3 4 5 6 7 8 9 10 10 Kb
Saliva is a reliable source of germline DNA for genome-wide studies in CLL DNA samples passing QC for SNP array DNA samples passing QC for WES 100% 100% 90% 80% 75% 83% 90% 80% 70% 70% 70% 60% 60% 50% 40% 50% 40% 37% 30% 30% 20% 20% 10% 10% 0% Saliva Urine 0% Saliva Urine Criteria: total amount >500 ng purity (OD 260/280 ratio: 1.6-2.1), high molecular weight non-human DNA contamination <20% absence of tumor DNA contamination suitability for PCR amplification Criteria: total amount >3 μg purity (OD 260/280 ratio: 1.6-2.1), high molecular weight non-human DNA contamination <20% absence of tumor DNA contamination suitability for PCR amplification
BIRC3 mutations are selectively restricted to CLL and SMZL across mature B-cell tumors (n=317) Alterations of BIRC3 and other NF-κB genes in SMZL BIRC3 mutation frequency N=2/20 N=6/101 N=0/30 N=0/38 N=0/20 N=0/68 N=0/18 N=0/22 CLL SMZL DLBCL BL FL EMZL HCL^ MM Rossi D, et al. Blood 2011;118:4930-4
SF3B1: summary SF3B1 mutations occur in 17% fludarabine-refractory CLL and distribute in a mutually exclusive fashion with TP53 disruption, thus representing alternative mechanisms contributing to chemorefractoriness At CLL diagnosis, SF3B1 mutations occur at a low rate and identify patients with a poor survival similar to that associated with TP53 abnormalities SF3B1 mutations might identify a therapeutic target for SF3b inhibitors, a new class of anti-cancer drugs that are currently under pre-clinical development
BIRC3: summary BIRC3 disruption recurrently and selectively associates with 37% fludarabinerefractory but TP53 wild type cases, thus representing an alternative mechanism contributing to chemorefractoriness At CLL diagnosis, BIRC3 disruption occurs at low rate and identifies patients with a poor survival similar to that associated with TP53 abnormalities The poor prognosis associated with BIRC3 disruption might be a consequence of the constitutive NF-κB activation observed in these cases NF-κB signaling may represent a new therapeutic target in CLL
NOTCH1: summary NOTCH1 mutations occur in ~10% newly diagnosed CLL, cluster with cases harboring trisomy 12 and tend to be mutually exclusive with TP53 disruption At CLL presentation, NOTCH1 mutations identify a high risk subgroup of patients showing poor survival similar to that associated with TP53 abnormalities The poor prognosis predicted by NOTCH1 mutations is due to an increased risk of RS transformation NOTCH1 mutations might provide a therapeutic target for NOTCH1 inhibitors that are currently under development in other clinical contexts