CLONAL ¡EVOLUTION ¡IN ¡THERAPY-‑RELATED ¡NEOPLASMS ¡ E. ¡Fabiani ¡ 1 , ¡G. ¡Falconi ¡ 1 , ¡L. ¡Fianchi 2 , ¡M. ¡Criscuolo 2 , ¡T. ¡O9one 1 , ¡L. ¡Cicconi 1 , ¡ ¡ S. ¡Hohaus 2 , ¡S. ¡Sica 2 , ¡M. ¡Postorino 1 , ¡A. ¡Neri 3 , ¡M. ¡LioneB 3 , ¡ ¡ G. ¡Leone 2 , ¡F. ¡Lo-‑Coco 1 ¡and ¡MT. ¡Voso 1 ¡ 1 Department ¡of ¡Biomedicine ¡and ¡Preven3on, ¡Universita’ ¡Tor ¡Vergata, ¡ ¡Rome, ¡Italy, ¡ ¡ 2 Department ¡of ¡Hematology, ¡Universita’ ¡CaBolica ¡Sacro ¡Cuore, ¡Rome, ¡Italy ¡ ¡ 3 Department ¡of ¡Clinical ¡Sciences ¡and ¡Community ¡Health, ¡Università ¡degli ¡studi ¡di ¡Milano, ¡Italy ¡ ¡
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS Therapy-related myeloid neoplasms are characterized by high incidence of complex karyotypes, frequent abnormalities of chromosome 7 and/or 5 High prevalence of the t(4;11)(q21;q23) translocation have been commonly reported in t-ALL. Italian ¡Registry ¡on ¡Secondary ¡Leukemias. ¡Fianchi ¡et ¡al., ¡Am ¡J ¡Hematol ¡2015 ¡
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS Prevalence of Somatic Mutations in t-MN = ¡ ≠ ¡ OK ¡et ¡al., ¡Leuk ¡Res ¡2015. ¡Muta3on ¡hotspots ¡of ¡53 ¡genes ¡ TP53 ¡ Voso ¡et ¡al., ¡Leukemia ¡2013 ¡and ¡Fabiani ¡et ¡al., ¡Haematologica ¡2014. ¡
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS Patient Characteristics
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 1. Methods Mutational screening of common somatic mutations and other gene alterations in therapy-related leukemia patients Sanger Sequencing Next Generation Sequencing IDH1 R132 TP53 IDH2 R140/R172 Epigenetic enzymes DNMT3A R882 ASXL1 Q-RT-Polymerase chain reaction t(4;11)(q21;q23) SF3B1 ex 13-16 Spliceosome machinery KMT2A/AFF1 SRSF2 P95 U2AF1 S34 Pyrosequencing SETBP1 SKI Domain Validation & quantification N-RAS of the mutated clone K-RAS
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 2. Methods Mutations identified in the t-MN sample were then tracked backwards in samples collected during follow-up of the primary tumor, using high sensitivity techniques High-throughput Next Generation Sequencing ² Specific homemade designed primers were linked to specific flags ² Average coverage was >120000x for all corresponding specific amplicon ² Variant allele frequencies in % (alternative allele count/total depth x100)
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS Screening of genetic changes in therapy-related neoplasms $"%& !"# IDH1 R132 IDH2 R172 IDH2 R140 DNMT3A R882 U2AF1 SF3B1 13-16 SRSF2 SETBP1 N-RAS K-RAS ASXL1 ! "# "# "# "# "# "# "# "# "# "# "# $%&!' ( $(()* "# "# "# "# "# "# "# "# "# "# "# + "# "# "# "# "# "# "# "# "# "# "# ,-.&' / "# "# "# "# "# "# "# "# "# "# "# 0-&+' % "# "# "# "# "# "# "# "# "# "# "# "# - "# "# "# "# "# "# 12))3 "# "# "# "# "# 2 "# "# "# "# "# "# "# "# "# "# "# "# . "# "# "# "# "# "# "# "# "# "# "# "# & "# 0!+(4 "# "# "# "# "# 5&%4 "# "# "# "# !) "# "# "# "# "# "# "# "# "# "# "# "# !! "# "# "# "# "# "# "# "# "# "# "# "# !( "# "# "# "# "# "# "# "# "# "# "# "# !+ "# "# "# "# "# "# "# "# 6.2)0 "# "# "# ² We identified 8 mutations ( IDH1 R132H, SRSF2 P95H, SF3B1 K700E, SETBP1 G870R, TP53 Y220C, ASXL1 Y591*, ASXL1 S689* and ASXL1 R693* ) in 7 of 13 t-MN patients ² UPN 9 carried two mutations ( IDH1 R132H and SRSF2 P95H) ² UPN 14, a t-ALL patient, was KMT2A-AFF1 -positive
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS Quantification of the identified variants at the time of t-MN diagnosis ² Pyrosequencing analyses, specifically designed to quantify the mutations confirmed the data obtained from Sanger sequencing in all cases. ² Mutations identified in epigenetic regulators, spliceosome enzymes and SETBP1 were detectable at a variant allele frequency (VAF) of 20 to 42% at the time of t-MN diagnosis ² TP53 mutation was detectable at a lower rate (6,75%) ¡ ¡ ² UPN9 carried two mutations ( IDH1 R132H and SRSF2 P95H), at a similar VAF (38% and 35%, respectively) suggesting that the two mutations were present in the same leukemic clone. ² Quantitative evaluation of the KMT2A-AFF1 transcript in UPN 14 revealed strong positivity for the chimeric transcript at the time of t-ALL diagnosis (3500 copies /10 4 ABL)
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS Disease markers and clonal evolution Ultra-deep NGS reveals at least two different patterns of clonal evolution First ¡scenario ¡ Second ¡scenario ¡ Somatic mutations Somatic mutations characterizing the t-MN were detectable at clone were not detectable primary cancer at primary cancer diagnosis, prior to any diagnosis and appeared cytotoxic treatment after cytotoxic treatment
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 1. ¡First ¡scenario ¡ Somatic mutations characterizing the t-MN clone were not detectable at primary cancer diagnosis and appeared after cytotoxic treatment MICMA/PB-SCT RTX F) UPN6: SF3B1 K700E G) UPN13: SETBP1 G870R D) UPN6: SF3B1 K700E E) UPN13: SETBP1 G870R 40 25 25 Variant allele frequency (%) Variant allele frequency (%) 35.0 35 20.0 20 20 30 25 15 15 20 10 10 15 10 5 5 5 0 0.0 0.0 0.0 0 0 B-ALL CR t-MN DG AML BC DG t-MN DG DG (23 mos) (32 mos) DG (53 mos) (83 mos) GMALL 05/93 AML-12 Trial CTH + RT Fabiani et al., submitted
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 2. ¡First ¡scenario ¡ Somatic mutations characterizing the t-MN clone were not detectable at primary cancer diagnosis and appeared after cytotoxic treatment A) UPN4: ASXL1 R693* &#" 35 30.0 Variant allele frequency (%) 30 &!" 25 %#" 20 %!" $#" 15 13.0 10 $!" 5 #" 0.0 0.0 0.0 0.0 0 !" NHL (21 mos) (30 mos) (43 mos) t-MN DG (120 mos) DG (113 mos) CHOP/MICMA/ Fludarabin RTX HD-CTX CTX MICMA/PB-SCT RTX Fabiani et al., submitted
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 3. ¡First ¡scenario ¡ Somatic mutations characterizing the t-MN clone were not detectable at primary cancer diagnosis and appeared after cytotoxic treatment B) UPN3: ASXL1 S689* 46.0 45 Variant allele frequency (%) 40 35.0 34.0 35 30 29.0 25 19.0 20 15 10 5 0.0 0.0 0.0 0.0 0 NHL (3 mos) (9 mos) (14 mos) (20 mos) (23 mos) (49 mos) (78 mos) t-MN DG DG (86 mos) CHOP R-MICMA PB-SCT R-Vel-dex Lenalidomide Fabiani et al., submitted
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 4. ¡First ¡scenario ¡ Genomic alteration characterizing the t-ALL clone was not detectable at primary cancer diagnosis and appeared after cytotoxic treatment C) UPN14: KMT2A/AFF1 5000 4390 4000 Copies/10 4 ABL 3522 3000 2000 962 1000 58 0 0 0 0 0 0 APL CR CR CR t-ALL DG (21 mos) Allo-SCT (31 mos) (20 mos) DG (11 mos) (13 mos) (15 mos) (18 mos) (24 mos) AIDA 2000 (induction, consolidation Clofarabine Vincristine Endoxan & manteinance) Daunorubicin L-Asparaginase Fabiani et al., submitted
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 1. Second scenario Somatic mutations were detectable at primary cancer diagnosis, prior to any cytotoxic treatment A) UPN2: TP53 Y220C 7.0 6.00 Variant allele frequency (%) 6.0 5.0 4.0 3.0 2.01 2.03 2.0 1.0 0.10 0 APL CR CR APL t-MN DG DG (14 mos) (20 mos) (30 mos) AIDA 2000 Fabiani et al., submitted
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 2. Second scenario Somatic mutations were detectable at primary cancer diagnosis, prior to any cytotoxic treatment B) UPN1: ASXL1 Y591* 45 42.0 Variant allele frequency (%) 40.0 40 35 30 25 20 15 10 7.0 5 0.3 0 NHL (31 mos) (36 mos) t-MN DG DG (83 mos) Pro MACE Cytabom/RT Fabiani et al., submitted
CLONAL EVOLUTION IN THERAPY-RELATED NEOPLASMS 3. Second scenario Somatic mutations were detectable at primary cancer diagnosis, prior to any cytotoxic treatment C) UPN9 40 38.0 IDH1 R132H Variant allele frequency (%) 35.0 35 35.0 30 25 20 SRSF2 P95H 15 10 5 0.0 0.0 0 NHL Follow-up t-MN DG DG (1 mos) (100 mos) Chlorambucil/Rituximab/RT Fabiani et al., submitted
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