The Evolution of Genetic Aberrations in CML Patients Harboring a Ph-Negative Clone: A 6-Year Follow-up Study of 52 Patients

Background: Clonal chromosome aberrations in Philadelphia chromosome-negative metaphases (CCA/Ph-) occur in a subset of chronic myeloid leukemia (CML) patients. A shorter survival was reported for "non -Y" CCA/Ph- cases (Issa et al. Blood 2017). Besides -Y, the most frequent CCA/Ph- is +8, but a broad spectrum of other abnormalities can be found, including 7q-/-7, which is a typical aberration of myelodysplastic syndromes (MDS).

In previous analyses we had shown an increased number of molecular genetic aberrations in CCA/Ph- compared to non-CCA/Ph- patients (Schnittger et al. ASH 2013, 2014). However, the clinical impact and the evolution of the mutation pattern is largely unknown. Here we extended follow-up and genetic characterization of the initial CCA/Ph- cohort.

Aim:

1. To determine the pattern of molecular mutations and their evolution

2. To determine if mutations are part of the Ph+ or Ph- clone

Patients and Methods: We included 52 CCA/Ph- patients (female: 25; male 27), with a median age of 58 [33-81] years, and a median BCR-ABL1/ABL1 ratio of 4.322% [0-58.088%] (N.A. for 4 pts.) at the time of initial CCA/Ph- detection. The following CCA/Ph- were present: trisomy 8 (n=26), other trisomies (n=4), -Y (n=7), del(7q)/-7 (n=4), others (n=7), two CCA/Ph- (n=4). We performed sequencing of myeloid gene panels on follow-up samples (1 to 3 per patient) on Illumina's MiSeq and NextSeq instruments (library preparation: 29-gene panel Thunderstorm RainDance [Lexington, MA] or 28-gene panel TruSeq [Illumina, San Diego, CA]). Data was analyzed with SeqNext (JSI Medical Systems, Kippenheim, Germany). Detected mutations were monitored on additional time points to determine variant allele frequency (VAF: mutated/all reads) development. A reference cohort of 47 patients with no sign of CCA/Ph- after MMR achievement was presented as part of our initial study (Schnittger et al. ASH 2014).

Results:

Cytogenetic monitoring was available over a median period of 31 [0-126] months for the CCA/Ph- and 26 [12-85] months for the reference cohort. Of the CCA/Ph- patients, 5/52 (10%) acquired additional typical aberrations as CCA/Ph- clone (incl. one -7), while in the reference cohort only one of 47 (2%) patients developed a -Y clone (n.s.).

On the molecular level, we conducted a median follow-up of 72 [9-150] months for the CCA/Ph- cohort (mutations and BCR-ABL1/ABL1 ratio). Following the CCA/Ph- detection, somatic mutations were found in 30/52 (58%) patients (up to 4 per patient): ASXL1 (n=13), DNMT3A (n=10), TET2 (n=6), NRAS (n=3), RUNX1 (n=3), non-recurrent (n=8).

The VAF of 7 mutations was strongly correlated to the BCR-ABL1 ratio and thus most likely present in the Ph+ clone. Mutations in ASXL1 were present in the Ph+ clone in five patients, of whom four never reached MMR, while 6/8 patients with ASXL1 mutations in Ph-independent clones achieved MMR under first- or second-line TKI therapy.

Molecular genetic aberrations in Ph- cells were found in 23/52 (42%) CCA/Ph- patients, but only in 2 of 47 (4%) cases of the non-CCA/Ph- cohort (p<0.001). Importantly, in the reference cohort only TET2 and DNMT3A mutations were identified, which is the typical pattern in age related clonal hematopoiesis (ARCH) and also found in older individuals without a hematological malignancy. In the CCA/Ph- cohort, eight of 23 (35%) showed only TET2 or DNMT3A mutations. However, a highly predictive mutation signature for development of a myeloid malignancy (according to Malcovati et al. Blood 2017) was found in 8/23 (35%). In addition, of three NRAS positive CCA/Ph- cases, one was diagnosed with MDS/MPN overlap (also CALR positive) and two developed s-AML during the follow-up period and the NRAS VAF had increased to ≥35% at our last monitoring time point.

Conclusions:

1. ASXL1 mutations can occur in the Ph- as well as in the Ph+ clone and are associated with a poor TKI response, if present in the Ph+ clone.

2. Molecular mutations are significantly more frequent in CCA/Ph- than in non-CCA/Ph-.

3. While the mutation pattern in non-CCA/Ph- resembles ARCH, the spectrum in CCA/Ph- includes a higher risk pattern for development of a myeloid malignancy.

4. Further prospective studies are required to evaluate the clinical impact of mutations acquired during the course of CML in order to determine which mutation pattern is related to a higher incidence of secondary myeloid malignancies.