The Evolution of Genetic Aberrations in CML Patients Harboring a Ph-Negative Clone: A 6-Year Follow-up Study of 52 Patients
Authors: Baer Constance , Kern Wolfgang , Haferlach Torsten , Haferlach Claudia 
Published: October 2019

Abstract

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: To determine the pattern of molecular mutations and their evolution 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: 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. Molecular mutations are significantly more frequent in CCA/Ph- than in non-CCA/Ph-. 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. 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.

Disclosures
Baer: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.