Clonal Progression during First Course of Chemotherapy in Acute Myeloid Leukemia Suboptimal Responders Heralds Reinduction Failure and Early Death

INTRODUCTION: Optimal response to induction chemotherapy for acute myeloid leukemia (AML) is achievement of complete remission (CR) and minimal residual disease (MRD) negativity. Suboptimal responses include achievement of CR but MRD positivity (CR_MRD+ patients) as well as failure to achieve CR. The prospect is bleak, especially in the latter group, but intensified treatment can cure some of these. Careful deliberation of patient fitness, drug toxicities and level of residual disease is required prior to choice of reinduction therapy.

HYPOTHESIS: We hypothesized that by studying clonal architecture changes during AML treatment, subclonal development or progression would add another dimension to the chemotherapy response assessment beyond that of merely a quantitative reduction in blasts or a given MRD marker.

PATIENTS AND METHODS: 25 uniformly treated ("3+7" like) AML patients were included, 7 CR_MRD+ (flow cytometry-determined MRD levels between 0.02% and 1%), and 18 who did not achieve CR after the first course of chemotherapy, of whom 17 received reinduction chemotherapy. Median time from diagnosis to CR evaluation after first course of chemotherapy: 33 days (range: 20-74 days)

For the CR_MRD+ patients; MRD cells from bone marrow samples taken after 1^st treatment course were isolated using a FACSAria III based on the appropriate leukemia associated phenotype determined at diagnosis.

All samples were analyzed using a 26-gene panel (ASXL1, BCOR, CALR, CBL, CSNK1A1, DNMT3A, ETV6, EZH2, FLT3-TKD, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2) . Furthermore, CSFR3, GATA1, PTPN11 and WT1 were analyzed in the group who did not achieve CR. The TruSeq Custom Amplicon kit was used for library prep and samples were sequenced using the MiSeq instrument.

RESULTS: In the CR_MRD+ group clonal architecture in unsorted diagnosis samples were compared to clonal architecture in the FACS products. In 3 informative cases, a development in the clonal architecture was seen. In two cases, a subclone positive for TET2 only persisted even if the fully mutated clones of the patients (one TET2 +/ NRAS +, one TET2 +/ NPM1 +/ IDH2 +) disappeared. Both patients relapsed. In the third patient; a IDH2 + SRSF2 + clone persisted, a IDH2 + SRSF2 + BCOR + clone disappeared. In the remaining 4 patients no mutations were detected in the FACS product.

For the group of patients not in CR clonal architecture development between diagnosis and regeneration after first course of chemotherapy could be studied in 16/18 cases. Two cases were negative for all aberrations tested. 9/16 cases were clonally stable, displaying a reduction in mutation load comparable to leukemic burden reduction. In the remaining 7 cases, clonal development was seen: In 4 cases, one or more subclones were selectively eradicated leaving the more resistant clones. NPM1 was the only marker that was eradicated in more than one clone (n=2) and TET2 was the only marker that persisted in more than one clone (n=2). In 3/7 cases subclones emerged during chemotherapy; a WT1 + NRAS + clone in a NRAS + background, a SETBP1 + TET2 + ASXL1 + RUNX1 + clone in an RUNX1 + background and a DNMT3A + NPM1 + clone in a NPM1 + background. The 2-year survival of patients who received further chemotherapy was significantly different in the three groups (Clonally stable (n=9): 0.58, clonal architecture development but no emerging subclones (n=4): 0.25, AMLs with emerging subclones (n=3): 0 (last patient died after 224 days), P = 0.01 (Log-rank test)).

CONCLUSION: This study provides proof of the concept of subclonal architecture changes even in patients with minute amounts of residual leukemia (CR_MRD+ group) and we were able to study the subclonal architecture changes in three of these patients.

In patients not reaching CR, clonal architecture development was a rather common occurrence (7/16 cases). In three patients we observed that not only did a subclone resist chemotherapy, it grew during treatment. Even in this small study, this finding proved to be a significant marker of ultimate treatment failure.

If corroborated in larger studies, we envisage that an approach like the one applied here can assist physicians' decisions in the choice of further treatment in patients experiencing suboptimal response to induction chemotherapy.