Additional Information Offered By Snpa in Myelodysplastic Syndromes with Excess Blasts (MDS-EB) and Future Perspectives

Introduction: The detection of chromosomal abnormalities in myelodysplastic syndromes (MDS) supports the diagnosis, classification, prognostic stratification, therapy option, treatment monitoring and better understanding of the biology of disease. The main chromosomal abnormalities of MDS are losses and gains of genetic material, and these changes are among the most important prognostic parameters in IPSS-R. As a consequence, major advances have been achieved in the treatment and survival of patients. However, nearly half of the patients present a normal karyotype, which prevents their further characterization. Therefore, there is the desire to increase the abnormalities detection rate by other methods. Single nucleotide polymorphisms array (SNPa), also referred to as chromosomal microarray, is a sensitive technology used to perform high-resolution genome-wide DNA losses, gains and copy-neutral loss of heterozygosity (CN-LOH). The genomic DNA is hybridized to polymorphic probes which are SNP markers and non-polymorphic probes which are copy number markers, providing information about CN-LOH and copy number alteration (CNA), respectively. Previous studies have shown that the addition of the SNPa to karyotype (KT) increases by 28% the detection rate of cytogenetic abnormalities.

Objective: To substantiate this idea we describe two cases of MDS to whom SNPa added valued information to karyotype.

Methods: DNA was extracted from bone marrow cells for genomic clonal evaluation. The DNA was digested, amplified, fragmented, labeled and hybridized to the chip (Affymetrix - CytoScan HD^®) containing the probes. The chip was scanned to detect the signals' intensity emitted by the hybridized probes which were further analyzed by a software (ChAS) that allows visualization of CNA and CN-LOH. CNA´s analysis relies on the comparison of the obtained signals to a reference diploid DNA signal, and the difference encountered is characterized as loss or gain. CN-LOH analysis is based on two possible nucleotide signals (A or B), which are evaluated to discriminate three genotypes: AA, AB and BB. CN-LOH occurs when one allele is lost and duplicate another, resulting in genotypes AA or BB.

Results: Case 1, 64y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II fibrosis. KT: 46,XY,del(5)(q15q33),del(17)(p11.2)[16] / 46,XY[4]. SNPa: 3p21.31p21.2 CN-LOH; 5q21.1q35.3 loss (CN:1.00); -7; +8; 12p13.33p12.3, 12p12.1p11.22, 12q22q23.3 loss (CN:1.00); -16; 17p13.3p11.2 mosaic loss (CN: 1.50) and -Y.

Case 2, 74y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II/lll fibrosis. KT: 46,XY[15]. SNPa: 21q21.1q22.3 CN-LOH.

Discussion and Conclusion: The SNPa has the advantage of detecting genomic alterations regardless of the cell cycle, even when the cell is quiescent or growth is defective. It also enables the identification of CN-LOH (also known as uniparental disomy, UPD), submicroscopic amplifications and deletions that are not detected by KT. On the other hand, SNPa does not allow the identification of balanced translocations and polyploidy. In case 1, after SNPa analysis, some chromosomal abnormalities (−7, +8, −16 and −Y) were found in sporadic metaphases during the KT reanalysis, but had not initially been described because they did not meet the criterion to be considered as a cytogenetic clone. The risk-stratification (IPSS-R) for this patient was intermediate, but the addition of the SNPa results the risk-stratification could be changed to very poor. Seven months after diagnosis the patient developed acute myeloid leukemia and died. In case 2, CN-LOH detected by SNPa could be responsible for homozygosity of mutations in critical genes located in the 21p region, such as RUNX1 that encodes a protein, which is a transcription factor critical in hematopoiesis. Indeed, sequencing of candidate genes in CN-LOH regions should be considered a priority in the search of driver mutations of MDS. Twenty-four months after diagnosis the patient died due to other non-hematologic causes. In Summary, SNPa analysis may add value to KT non-informative results and occasionally reveal cryptic abnormalities not recognized by karyotyping. However, SNPa analysis should be viewed as a complimentary tool.

Acknowledgment: The SNPa test was supported by Grupo Fleury