Molecular Characterization of Leukemia Evolving from Paroxysmal Nocturnal Hemoglobinuria
Authors: Awada Hassan , Rahman Shafia , Kerr Cassandra M , Durrani Jibran , Adema Vera , Kishtagari Ashwin , Kongkiatkamon Sunisa , Carraway Hetty E. , Sekeres Mikkael A. , Maciejewski Jaroslaw P. , Visconte Valeria 
Published: November 2019

Myeloid malignancies can evolve from prior hematologic disorders, most commonly AML evolving from MDS. AA and PNH are not malignant conditions but they can evolve to myeloid malignancies with a lower frequency compared to MDS and other myeloid and myeloproliferative diseases. PNH can evolve to myeloid disorders with an incidence rate of 4-10%. Here we aim to revisit the subject of malignant evolution of PNH to myeloid disorders by analyzing the molecular background of PNH using modern NGS technologies.
Clinical characteristics, demographics and mutational profiles of patients were collected at The Cleveland Clinic Foundation. In total a cohort of 243 patients evaluated for hemolytic PNH (n=83), AA (39) and AA/PNH (n=121) followed for a period of 22 years was evaluated. Inclusion criteria were: complete flow cytometric panels of PNH cells, patients without antecedent AA (pPNH, 45), patients evolving from AA with PNH clone >20% (sPNH, 38), karyotype (at diagnosis of PNH and transformation), clinical parameters including time to malignant progression, and molecular characterization resulting from NGS performed using various library preparation systems (TruSeq, TruSight, Nextera), comparison of molecular mutations with control cohorts (AA, 160; MDS, 835).
The incidence rate of myeloid disorders in our PNH cohort was 3% (7/243). Among hemolytic PNH patients, 7 patients progressed to AML (n=1), MDS (n=5) or myelofibrosis (n=1). Median age was 48 yr (range, 24-80); M/F, 5/2. Median PNH clone size was 71% (range, 29-99). Three progressors were in the pPNH and 4 in the sPNH group. Time to malignant diagnosis was <1-22 yr: 4 yr for 3 patients, 5 yr for 1 patient, <1 yr for 2 patients and 22 years for 1 patient. At the time of transformation, combination of karyotypic abnormalities and molecular lesions showed that 4 cases had abnormal karyotype (+8, -7, del13q) and 5 cases carried myeloid mutations (ASXL1, BCOR, NPM1, TET2, U2AF1, WT1). Normal karyotype was seen in 3 patients with ASXL1+U2AF1, U2AF1 and BCOR+NPM1+WT. In 2 patients, del13q and -7 were associated with BCOR and TET2. In 2 others, del(13q) and +8 were the only detected aberrancies.
A total of 45 somatic mutations equally distributed in the 2 groups (pPNH, 22; sPNH, 23) and with similar VAFAVG (pPNH vs. sPNH 38% vs. 33%) were found. PNH showed a higher proportion of individuals with mutations compared to AA/PNH+AA (42 vs. 22% of cases with ≥1 mutation; P=.002) with a median VAF percentage significantly higher in PNH vs. AA/PNH+AA (40 vs. 19%; P<.0001). FLT3 (n=2), JAK2 (n=1), LUC7L2 (n=1), NPM1 (n=1), SRSF2 (n=1) and ZRSR2 (n=2) were exclusively mutated in PNH with the presence of some of them (FLT3, NPM1) possibly representing an early onset of clonal evolution. BCOR/BCORL1 core was more mutated in PNH as compared to AA/PNH+AA patients (11 vs. 3%; P=.01) as did TET2 and U2AF1. This pattern of mutations had a strong similarity with that of MDS. VAF comparison between myeloid mutations and PIGA mutations showed that accessory mutations in patients with PNH were secondary hits, with PIGA mutations being the founder lesions. We then analyzed the clonal hierarchy of mutations using VAF-adjusted for X-chromosomal loci in males and inferred mutational rank (founder/ co-dominant) vs. secondary (sub-clonal). PIGA mutations were founder lesions in 70% of the cases, co-dominant in 2% and secondary in 28% suggesting that additional mutations represented mainly cooperative events without over representing PIGA. In fact, comparison of VAF and PNH clonal size showed that 70% of the hits were present in the same clone (VAFSUM >55), 17% were equivocal (possibly hits biclonal with a VAFSUM between 45-55), while 13% were more likely a result of clonal chimerism (hits present in different clones; VAFSUM <45).
Clonal evolution of hemolytic PNH to MDS/AML is rare, but still occurs and it is accompanied by mutations in typical myeloid genes (BCOR, NPM1, TET2, U2AF1) which in permissive circumstances are capable to change the cell's fate favoring clonal evolution. Our results suggest that most PNH cases can carry additional mutations in the same clone and these mutations can be secondary hits, with PIGA mutations being the founder lesions. However even when mutations in myeloid genes are dominant, the phenotype of the patients is inferred by PIGA. This observation supports the nature of PNH as a monogenic disease with clinical manifestations resulting by PIGA mutations rather than by myeloid genes.

Disclosures
Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.