Evidence-based genomic diagnosis characterized chromosomal and cryptic imbalances in 30 elderly patients with myelodysplastic syndrome and acute myeloid leukemia

The identification of recurrent chromosomal abnormalities in various leukemias and the understanding of molecular defects and pathogenic mechanisms underlying these abnormalities have made cytogenetic analysis valuable in providing diagnostic and prognostic parameters for disease stratification and treatment evaluation [1]. With an average resolution of 6-10 megabases (Mb) on a 300-500 G-band level, conventional karyotyping has been the current standard for screening chromosomal abnormalities on metaphases from direct and cultured bone marrow (BM) and leukemic blood (LB) cells. This approach requires mitotic active cells and frequently encounters difficulties due to the low mitotic index and poor chromosome morphology of leukemic cells. Fluorescence in situ hybridization (FISH) tests using targeted probes to detect gene/locus-specific rearrangements have enhanced the analytical resolution to 300-800 kilobases (Kb) and extended conventional metaphase analysis into interphase cells. Current cytogenetic analysis for patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) involves cell-based conventional chromosomal analysis and FISH assays using a panel of targeted probes [2, 3]. We have previously validated a DNA-based genome-wide oligonucleotide array comparative genomic hybridization (aCGH) for clinical diagnosis of constitutional chromosomal abnormalities and genomic disorders in pediatric patients with mental retardation and developmental delay [4]. The clinical utility of this aCGH based on Agilent's 44K design (CGH4410B) has demonstrated an average analytical resolution of 300-500 Kb and an improved abnormal detection rate from 5-7% by conventional chromosome and FISH analyses to 12% by aCGH [5]. Evidence-based guidelines to interpret genomic findings in the pediatric patients have been proposed [6, 7]. Recently, genome-wide analyses using BAC-clone aCGH, oligonucleotide aCGH and SNP array have been applied in a research or an exploratory setting to profile the genomic alterations in patients with MDS and AML [8–15]. To evaluate the diagnostic value of aCGH in detecting somatic chromosomal and segmental copy number alterations (CNAs), we have performed aCGH analysis on 30 MDS and AML cases with different clonal abnormalities. The results further characterized the genomic complexity of recurrent chromosomal deletions, duplications, amplifications and cryptic aberrations. Despite its inherent limitation in detecting recurrent balanced reciprocal translocations and low level secondary clonal abnormalities, the aCGH analysis provides detailed genomic features of simple and complex chromosomal abnormalities and cryptic aberrations otherwise not detectable by conventional G-band and FISH assays. Integrated chromosome and genomic analyses and evidence-based interpretation should be a standardized cytogenomic procedure for patients with MDS and AML.

The detected clonal chromosomal abnormalities of the 30 patients were listed in Table 1. All recurrent chromosomal deletions and translocations were confirmed by FISH tests using targeted probes. The aCGH analysis detected all main clone numerical chromosomal abnormalities and chromosomally-observed segmental deletions and duplications. The observed numerical abnormalities included gains of chromosomes 1, 4, 5, 6, 7, 8, 9, 11, 13, 14, 18, 19, 20 and 22, and losses of chromosomes Y, 5, 7, 13 and 18. No genomic aberration was noted in two patients with a balanced translocation (cases #10 and #21, Table 1). In five cases (cases #21, #26-27, #29-30, Table 1), sideline clones with unbalanced chromosomal abnormalities were not detected by aCGH. A total of 113 CNAs with size ranging from 0.257 to 102.519 Mb were found in 23 patients (an average of 4.8 CNAs per case and of 20.241 Mb per CNA, see Additional file 1, Table S1). The genome-wide distribution of chromosomal and genomic copy number alterations was plotted using the Nexus5 software (Figure 1). Cases sharing similar cytogenetic abnormalities, such as the 5q deletion or monosomy 5 (n = 9), 7q deletion or monosomy 7 (n = 3), trisomy 8 (n = 3), 20q deletion (n = 2), loss of Y (n = 3) and other abnormalities (n = 10), were grouped together to estimate the frequency of other associated aberrations. It was noted that 5q deletions showed many associated recurrent aberrations, especially the 12p deletion (5/9 cases), 17p deletion (3/9) and 17q deletion (2/9). The size distribution and percentage of detected CNAs listed in Table 2 indicated that conventional chromosome analysis with a resolution of 5~10 Mb can miss a significant proportion (44%, 50 CNAs < 10 Mb out of 113 CNAs) of genomic aberrations.

The comparison between chromosomal abnormalities and genomic CNAs further delineated the breakpoints and gene contents involved in both simple and complex chromosomal rearrangements. For example, of the complex karyotype of case #4, the chromosomally observed 5q deletion was actually a result of an 80.5474 Mb deletion of 5q14.3-q34 followed by a fusion of structurally rearranged 17q (segmental deletions and duplication); the 7p deletion was a result of a 20.596 Mb deletion of 7p14.3-p11.2 and a 17.391 Mb deletion of 7p22.1-p15.3; the 9p had a 3.050 Mb microdeletion of 9p23; and what was denoted as additional material onto a 22q and a marker chromosome was most likely a result of a 22q/22q translocation with a 15.235 Mb duplication and a 11.694 Mb triplication of 22q11.2-q12.3 and a 5.199 Mb deletion of 22q13.31-q13.33 (Figure 2A, Additional file 1, Table S1). The case-by-case comparison of chromosomal and genomic findings not only defined the chromosomal abnormalities, but also demonstrated the genomic heterogeneity of recurrent chromosomal abnormalities. Chromosomally observed recurrent deletions of 5q, 7q, 9q, 12p, and 20q were all characterized by aCGH and confirmed in ten, three, two, six, and three cases, respectively. Of the 10 cases (cases #1~9 and #13 in Table 1) with a 5q deletion or monosomy 5, a simple deletion of various sizes was seen in seven cases and compound deletions of two or three segments were found in three cases (Additional file 1, Table S1). The deletion of the ETV6 gene at 12p13.2, a gene essential in hematopoiesis and frequently encountered in translocations in acute leukemias and MDS, was noted in five of the six cases with the 12p deletion, including in case #13 a microdeletion of 1.154 Mb confirmed by FISH using the ETV6 probe. Segmental rearrangements involving the TP53 gene by either the whole arm loss or a large deletion of 17p were noted in four cases (#1, #7-8 and #17). A chromosomally undetected deletion of 10.981 Mb at 9p21.3-p21.1 involving the CDKN2A and CDKN2B genes was noted in case #16. Chromosomal analysis recognized two unrelated clones in case #26 featuring a 20q deletion and trisomy 8, respectively. The aCGH detected a 14.920 Mb deletion of 1p36.21-p35.3 and a 20.202 Mb deletion of 20q11.23-q13.3 but not the unrelated clone with trisomy 8. This result suggested that the 1p deletion region may harbor haploinsufficient candidate genes relevant to the etiology or evolution of MDS and AML.

Microdeletions of 1.510 to 3.615 Mb encompassing the NF1 gene at 17q11.2 were found in three cases (cases #4, #5 and #22). FISH analysis using BAC clone probes for the NF1 and RHOT1 genes confirmed these deletions (Figure 2B). It was interesting to observe a transition pattern in case #4 with deletions of NF1 alone, NF1 and RHOT1, and RHOT1 alone in 11%, 12.5% and 34% of cells, respectively. In case #13, a deletion involving the RHOT1 gene but not the NF1 gene was noted and confirmed by FISH. These results suggested that the 17q11.2 region may contain hot spots for initiating deletions from either one or both proximal and distal orientations.

The dmin chromosomes observed in case #19 were further defined as a 4.277 Mb amplification of 8q24.13-q24.21 containing genes TRIB1, MYC and CCDC26. From the log2 ratio of 2.53 for this amplified region, it was estimated that there were average four dmin chromosomes in each leukemic cell. Compound deletions, triplications or quadruplications for 11q were noted in cases #1 and #13, which resulted in complex intrachromosomal rearrangements carrying amplified segments of the MLL gene (Figure 2B). The derivative chromosome 6, der(6)t(3;6)(q21.3;p22.2), in case #12 was further defined with breakpoints involving the RAB43 gene at 3q21.3 and the KIAA0319 gene at 6p22.2; and the isodicentric chromosome of Xp in case #27 was resulted from break and fusion distal to the PHKA1 gene at Xq13.1 (Additional file 1, Table S1).

Cytogenetics has played a major role in both diagnosis and prognosis in patients with hematological malignancies. The newly revised WHO classification of acute myeloid leukemias categorizes AML into subgroups with recurrent genetic abnormalities, with MDS features, treatment related and "not otherwise specified"; the last one is by far the largest category [1]. Cytogenetics is key components of both the international prognostic scoring (IPSS) and the WHO classification-based prognostic scoring systems (WPSS) for the myelodysplastic syndromes [17, 18]. However, within all categories prognostic variation is observed, likely among others due to low sensitivity of conventional cytogenetics [12]. Our data demonstrated that aCGH can detect chromosomal, segmental and cryptic aberrations in LB and BM cells from MDS and AML patients. As summarized in Table 2, the aCGH detected an average of 4.8 CNAs per case ranging from 0 to 22, of which an approximately 44% were less than 10 Mb and not evident by conventional cytogenetic analysis. It is noteworthy while on one hand some relatively large deletions and duplications (> 10 Mb) were missed or unresolved by conventional chromosomal analysis, on the other hand chromosomally detected sideline clones were missed by aCGH. Therefore, an integrated cytogenomic approach using chromosome analysis, FISH assay and aCGH would definitely improve the analytical resolution and abnormality detection rate for MDS and AML patients.

The interpretation of the clinical significance of somatic CNAs has been challenge especially for cases with numerous genomic aberrations. An evidence-based approach for interpreting constitutional genomic aberrations could be adopted for somatic CNAs with the classification of research evidence into four levels [6, 7]. Level I evidence is derived from recognized disease-causing clonal chromosomal abnormalities in the WHO classification [1] or from well-designed systematic studies; level II evidence comes from series of diagnostic studies; level III evidence is derived from cohort design observational studies or basic research of gene functions and disease mechanisms on in-vitro systems and/or model animals, and level IV is based on case reports or expert's opinions. Genomic findings without evidence were documented as unknown significance and uninterpretable in the laboratory's database but not presented in the diagnostic report. We propose a workflow chart to illustrate the integration of chromosome, FISH and aCGH procedures and the algorithm for interpreting results (Figure 3).

Basic researches toward the understanding of gene functions and disease-causing mechanisms or case reports with unique clinical and genomic findings could provide level III and IV evidence for reporting genomic alterations. Detailed genomic analyses of the malignant cells in MDS and AML patients is likely to yield specific types or regions of recurrent chromosomal and genomic abnormalities providing further evidence for disease association and allowing identification of to date unknown candidate genes. Eventually, further classification of disease and targeted treatment can potentially result from such knowledge. The 5q deletion is possibly the best understood recurrent chromosomal deletion in myeloid malignancies. Two common deletion regions (CDR) have been identified conferring either a good (CDR1) or a poor (CDR2) prognosis, but usually changes are more complex [19]. All 5q deletions in our 10 cases and in another study of 12 MDS cases with an isolated 5q deletion by Evers C et al. [20] involved the loss of the RPS14 gene at 5q33.1, which support the causal role of RPS14 haploinsufficiency and were specified in the diagnostic reports [21]. Other haploinsufficiency genes or miR-145 and miR146a at 5q could also have causal or modifying effects [22–24], and should also be referred in the report. A der(6)t(3;6)(q21;p22) in a patient with AML at relapse and a t(1;6)(q21;p22) in another patient were reported [25]. We reported here a der(6)t(3;6)(q21.3;p22.2) likely caused by fusion of the RAB43 gene at 3q21.3 and the KIAA0319 gene at 6p22.2. Analysis of additional cases with similar 6p22 translocations could clarify if this is a recurrent primary or secondary rearrangement in AML. Monosomy 7 and 7q deletions portend a particularly poor prognosis in myeloid malignancies. The candidate genes within the recurrent 7q deletion are still under investigation and a recent study suggested that the SAMD9, SAMD9L and Miki (LOC253012) as the candidate genes for 7q21.3 [26]. Two out of the three cases with 7q deletions in our patients had the 7q21.3 deletion. As reported in the literature [16, 27, 28] and also shown from our cases #1, #13, # 19 and #23, genomic analysis has been used effectively to define dmin chromosomes of 8q24 and complex 11q rearrangements with or without the MLL gene amplification in simple or complex karyotype. The presence of cytogenetic unresolved marker chromosome in our case #23 and the reported hidden abnormalities associated with t(15;17) justified further genomic analysis for this obvious balanced rearrangements [29, 30]. Microdeletions at 17q11.2 involving the NF1 gene are considered to be recurrent cryptic alterations in three of our cases (#4, #5 and #22) and also documented in several reports [9, 11–13, 31]. The presence of clustered flanking repetitive sequences of the NF1 locus is a likely explanation for recurrence of both constitutional and the somatic deletions [32]; further characterization of the noted transition pattern for the somatic 17q11.2 deletion could lead to better understanding of its mutagenesis mechanism.

In conclusion, our current diagnostic application of aCGH and accumulated evidence from previous studies support an integrated cytogenomic approach with evidence-based interpretation in MDS and AML patients with 1) > 20% blast cells with or without clonal chromosomal abnormality, 2) with complex chromosomal abnormalities, and 3) with simple and balanced rearrangements or a normal karyotyope but suspected cryptic abnormalities. This cytogenomic approach may not only provide a better diagnostic scheme to delineate breakpoints and gene contents of chromosomal and cryptic abnormalities in patients with MDS and AML, but hopefully allow identification of disease-causing or modifying candidate genes, and eventually lead to improved prognostification and treatment of patients with MDS and AML.