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The patient developed flu-like symptoms including severe body pain, fatigue and night sweats. Upon worsening of the symptoms and admission to the emergency room, blood work revealed pancytopenia. The patient was subsequently admitted to the City of Hope National Medical Center for further workup and diagnosis.

29 yrs

M

Pancytopenia

75

B-cell ALL

Flow cytometry confirmed the presence of an abnormal immature B-cell population that was positive for CD10, CD19, CD24, CD34, CD38, HLA-DR, and TdT.

N/A

A bone marrow exam showed B-lymphoblastic leukemia involving a hypercellular marrow with 75% blasts and moderate reticulin fibrosis. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) analysis of the bone marrow showed no evidence of BCR/ABL1 fusion transcripts. A bone marrow sample was submitted to the cytogenetics laboratory for evaluation and risk stratification.

N/A

B-lymphoblastic leukemia

07-2017

The patient was induced with a pediatric ALL regimen and achieved a minimal residual disease-negative remission by day 28 and continues on interim maintenance therapy.

N/A

A

06-2018

11+

Bone Marrow

24

GTG

66,XY,+X,+1,+2,+5,+6,+8,+9,+10,+11,+12,+14,+16,+der(19)t(1;19)(q23;p13.3),+21,+21,+21,+21,+21,+21,+21[16/20]

N/A

FISH

FISH studies confirmed trisomy for chromosomes 9, 10 and 11, nine copies of chromosome 21 and no evidence of the BCR/ABL1 translocation. However, the FISH probes targeting the TCF3/19p13.3 locus produced two normal signal patterns, ruling out involvement of TCF3 in the supernumerary der(19)t(1;19) or any deletion in this region (Figure 2B-C).

Cytogenomic Microarray Analysis (CMA)

These confounding results prompted us to further investigate potential causes for such drastic differences between CMA and CC/FISH analyses. Upon review of the available materials, it was decided to perform the initial analysis of the data using an alternative method. Therefore, the Normal Diploid Analysis (NDA) workflow was utilized in lieu of the routinely used Single Sample Analysis (SSA). Interestingly, conversion of the same data file using the NDA workflow resulted in remarkably concordant results that fully matched the CC and FISH with no discrepancies between copy number calls and SNP/BAF tracks, confirming a hyperdiploid pattern (Figure 1B). Moreover, analysis of the SNP patterns enabled us to verify the presence of retained heterozygosity in the disomic chromosomes which, along with the absence of any tetrasomic chromosomes, ruled out any potential doubling event. Curiously, analysis of the TCF3 locus revealed a copy number of 2 with a corresponding ~7 Mb terminal region of copy neutral loss of heterozygosity. Additionally, although present in 3 copies, PBX1 was located outside the breakpoint region on chromosome 1.

We explored upfront utilization of NDA using several other oncology specimens and side- by-side comparison using both the SSA and NDA workflows was performed (data not shown). The results indicated that the use of the NDA workflow produced identical results to SSA in the absence of massive chromosomal abnormalities. Consequently, use of NDA does not appear to adversely affect the results of tumor specimens if they are indeed negative for massive hypo- or hyperdiploidy. Dry-lab validation for implementation of NDA could be completed by each laboratory in a short amount of time using previously-tested specimens.

A challenging aspect in the cytogenetic workup ALL is due to a biological phenomenon reported in this condition whereby a near haploid or hypodiploid genome undergoes doubling without concomitant cell division, resulting in the generation of a pseudohyperdiploid clone. Distinction between pseudo- and true hyperdiploidy may therefore be a problem, particularly in cases with atypical patterns and result in uncertainty with regard to the prognostic implications. By virtue of SNP probes and B-allele frequency (BAF), CMA can solve this problem. The retention or loss of heterozygous allelic loci on disomic chromosomes would indicate true- or pseudo-diploidy, respectively. However, as cautioned by ACMG guidelines (Cooley LD et al, 2013), overall ploidy status may affect the normalization of array data, in which case the resultant copy number gain and loss calls might be altered. This case underscores the clinical utility of rapid and low cost integration of NDA in cancer CMA analyses where the CytoScan HD platform is routinely used.