In this study, cytogenetic abnormalities were analyzed using high-density SNP-A in a large AML cohort. Previous studies show the superb resolution of SNP-A in detecting novel genomic abnormalities in other myeloid and lymphoid disorders.16,18
In AML without balanced chromosomal abnormalities, we demonstrated that SNP-A is superior to MC; new defects in both non-CBF and CBF AML may allow for substratification of these groups.
The higher detection rate of chromosomal defects seen in SNP-A is likely due to its ability to detect lesions even when cells are not actively dividing or its capacity to detect small CN changes and CN-LOH. However, unlike MC, SNP-A cannot detect balanced chromosomal lesions and cannot resolve clonal mosaicism.27
The high-resolution level of SNP-A may lead to false-positive findings related to artifacts, normal CNVs, and germline LOH. Consequently, SNP-A results must be carefully analyzed. We examined a large number of control samples and used internal and public CNV databases to distinguish germline variants from somatic defects, excluding all known CNVs from our evaluation. We have excluded most small regions of homozygosity, as observations made in controls suggest that they represent autozygosity or germline UPD. Through this method, large regions of germline UPD were found in a small proportion of controls, whereas large, often invariantly occurring areas of UPD were found at much higher frequency in patients, suggesting that these regions are of somatic origin. Whenever possible, we confirmed relevant clonal lesions by parallel analysis of nonclonal cells or serial samples derived from the same patients. In addition, fidelity of 250K SNP-A results was reaffirmed by application of 6.0 arrays in a significant number of cases. The application of a stringent algorithm was useful for excluding lesions that are most likely nonsomatic; however, it does have the limitation of leading to false-negative results (eg, true somatic lesions overlapping with CNVs). Our analyses produced plausible results which, if avoided, may improve the significance level of differences found. AS-UPD is a common and clinically important lesion in AML, accounting for a significant proportion of newly detected SNP-A defects in patients with pAML/sAML. Our results concur with findings of prior studies involving smaller patient numbers studied using 10K SNP-A.20,29
Previously, description of AS-UPD9p in MPD paved the way for further studies, leading to the discovery of JAK2
V617F mutations in MPD, followed shortly by NF-1
mutation in UPD17q11.2 in juvenile myelomonocytic leukemia.31,32
By analogy, in AML, AS-UPD of other chromosomal regions may indicate the presence of mutations in a homozygous constellation, for example, association of UPD13q with Flt-3
mutation, UPD19q in CEBPA
, or biallelic AML1/RUNX1
Examples of other putative homozygous mutations include UPD1p (c-Mpl
and 11q (MLL, c-CBL
In AML, SNP-A abnormalities demonstrate prognostic information beyond that of MC or Flt3
mutational status. Previously, we and others have shown the clinical correlation of SNP-A karyotypic results in MDS.18,19
New lesions detected by SNP-A negatively affected OS, RFS, RD, and EFS in our AML cohort including individual subsets, like normal MC pAML and abnormal MC pAML/sAML. The absence of survival difference in the normal MC sAML group may be related to the small number of patients analyzed and a more aggressive disease process. Importantly, our results indicate that CN-LOH is a defect that confers worse outcome similar to that of deletions. The early relapse observed in two patients with CBF AML with SNP-A lesions may be an important observation, because additional lesions detected by MC do not influence relapse in this AML type.39
There is evidence that c-KIT
mutants in CBF AML predict early relapse, but both patients do not carry this mutation.40
This suggests that other genes may be responsible, and it would be reasonable to investigate this issue in future studies.
Molecular diagnostics may supplant the more subjective morphologic prognostic factors. Mutational analysis for Flt3
provide important prognostic information in AML. However, the impact of mutational status may depend on coexisting chromosomal lesions (eg, t(8;21)) or other genomic features.7,42
In agreement with previous reports, we found that patients with Flt3
-TKD and NPM-1
mutations are primarily associated with pAML rather than sAML. The higher frequency of Flt3
-TKD and NPM-1
mutations in patients without SNP-A defects compared with those with SNP-A lesions may partially explain the better survival seen in these patients and vice versa. We demonstrated that SNP-A–detected lesions can further risk-stratify patients with and without Flt3
mutations. This finding is important because in our cohort of patients, univariate (with the exception of NPM-1
for OS and EFS) and multivariate analysis (with the exception of NPM-1
for EFS) did not reveal that these mutations have significant effects on outcomes.
Previously validated prognostic markers of unfavorable outcome in AML include older age,3,4,44
high WBC count,45–48
chemotherapy exposure or MDS,3,8,49
presence of certain cytogenetic abnormalities,2,4
and mutational status.7,41
Univariate analysis confirmed the important role of some of these factors in predicting outcomes. Multivariate analysis showed that new lesions detected by SNP-A, sAML, and abnormal chromosomal changes detected by MC were predictors of worse OS. Patients with age ≥ 60 years and sAML portend shorter remission duration, and new SNP-A–detected lesions, age ≥ 60 years, sAML, poor-risk karyotype by MC, and NPM-1
wild-type status predicted for worse EFS in our cohort. The inferior OS in the absence of worse RFS and RD seen in patients with AML with new SNP-A defects suggests a higher rate of resistant disease, a more aggressive clinical course, and higher treatment-related mortality at the time of relapse. A similar mechanism, in addition to higher treatment-related failure, is likely responsible for the worse EFS seen in patients with new SNP-A defects.
In summary, our study demonstrates the diagnostic utility of SNP arrays in AML. New SNP-A–detected defects, including AS-UPD, provide important prognostic information in AML and may reveal new pathogenic mechanisms that may serve as novel targets for new molecular treatments.