Clonal evolution in therapy-related neoplasms

Abstract

Therapy-related myeloid neoplasms (t-MN) may occur as a late effect of cytotoxic therapy for a primary malignancy or autoimmune diseases in susceptible individuals. We studied the development of somatic mutations in t-MN, using a collection of follow-up samples from 14 patients with a primary hematologic malignancy, who developed a secondary leukemia (13 t-MN and 1 t-acute lymphoblastic leukemia), at a median latency of 73 months (range 18-108) from primary cancer diagnosis.Using Sanger and next generation sequencing (NGS) approaches we identified 8 mutations (IDH1 R132H, ASXL1 Y591*, ASXL1 S689*, ASXL1 R693*, SRSF2 P95H, SF3B1 K700E, SETBP1 G870R and TP53 Y220C) in seven of thirteen t-MN patients (54%), whereas the t-ALL patient had a t(4,11) translocation, resulting in the KMT2A/AFF1 fusion gene. These mutations were then tracked backwards in marrow samples preceding secondary leukemia occurrence, using pyrosequencing and a NGS protocol that allows the detection of low variant allele frequencies (≥0.1%).Somatic mutations were detectable in the BM harvested at the primary diagnosis, prior to any cytotoxic treatment in three patients, while they were not detectable and apparently acquired by the t-MN clone in five patients.These data show that clonal evolution in t-MN is heterogeneous, with some somatic mutations preceding cytotoxic treatment and possibly favoring leukemic development.

Keywords: NGS; clonal evolution; mutation; therapy-related neoplasms.

Figure 1. Genetic changes present in t-MN were not detectable at primary cancer diagnosis

A. In UPN4, the ASXL1 R693* mutation detected in the t-MN sample was undetectable at the time of NHL diagnosis (113 months prior to t-MN onset). B. In UPN3, the ASXL1 S689* mutation was undetectable at the time of primary cancer diagnosis (NHL) but was detectable for the first time during NHL follow-up (at 20 months) and increased until the time of t-MN diagnosis (at 86 months). Of note, the mutation was first detected after high-dose therapy and PB-SCT. C. Similarly, the KMT2A-AFF1 fusion identified in UPN 14 at t-ALL diagnosis was, as expected, undetectable in the primary APL diagnostic sample, and was detected at low levels by Q-RT-PCR thirteen months after achievement of complete molecular remission of APL, using the AIDA 2000 protocol [17]. Notably, a constant increase in the transcript copy number was evident from first identification (58 copies/10⁴ ABL) to t-ALL onset (3522 copies/10⁴ ABL). The fusion transcript became undetectable only after re-induction treatment according to the LAL0904 protocol [27] and resulted to date undetectable (54+ months) after ASCT. The variant allele frequency (VAF) is indicated. Legend. NHL DG: non-Hodgkin lymphoma diagnosis; t-MN DG: therapy-related myeloid neoplasm diagnosis; APL DG: acute promyelocitic leukemia diagnosis; CR: complete remission; t-ALL DG: therapy-related acute lymphoblastic leukemia diagnosis; Allo-SCT: allogeneic stem cell transplantation; CHOP: cyclophosphamide, adriblastin, vincristine, prednisone; MICMA: mitoxantrone, carboplatin, cytarabine, methylprednisolone; PB-SCT: peripheral blood stem cell transplantation; RTX: radiotherapy; CTX: cyclophosphamide; HD-CTX: high-dose cyclophosphamide; R-MICMA: mitoxantrone, carboplatin, cytosine arabinoside, and methylprednisolone; R-Vel-dex: lenalidomide, bortezomib, dexametasone; AIDA: ATRA, idarubicine.

Figure 2. Somatic mutations were present prior to any cytotoxic treatment

A. A TP53 Y220C mutation was identified in UPN2 in all the available BM-MNC specimens, from APL diagnosis to complete remission, and expanded in the t-MN clone. B. Similarly, in UPN1 the ASXL1 Y591* mutation was identified in all the available BM-MNC specimens, from NHL diagnosis to t-MN. Notably, this mutation was detected at very low levels in the BM NHL specimen (0.3%), significantly increased at 31 months follow-up (7%) and reached the highest VAF at t-MN diagnosis (42%). C. In UPN9, the IDH1 R132H mutation was originally present at a high allele frequency at NHL diagnosis, 9 years before t-MN onset, when the patient was 72 years old, indicating that this mutation could have occurred as a pre-leukemic event. The SRSF2 P95H mutation was acquired later, at the time of t-MN diagnosis. Legend: APL DG: acute promyelocitic leukemia diagnosis; CR: complete remission; NHL DG: non-Hodgkin lymphoma diagnosis; t-MN DG: therapy-related myeloid neoplasm diagnosis; AIDA: ATRA, idarubicine; RT: radiotherapy; ProMACE-CytaBOM: cyclophosphamide, doxorubicin, etoposide, bleomycin, vincristine, methotrexate and prednisone.