Clinical Relevance of Normal Diploid Workflow in Microarray Analysis of Massively Aneuploid Genomes: Lessons from a Case of B-Lymphoblastic Leukemia

S Abbas Padeganeh, Joo Song, Patricia Aoun, Matthew Mei, Haris Ali, Dennis D. Weisenburger, Joyce L. Murata-Collins  

Cancer Cytogenetics Laboratory, Department of Pathology, City of Hope National Medical Center, Duarte, California (AP, JMC);; Department of Pathology, City of Hope National Medical Center, Duarte, California (JS, PA, DDW); Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010 (MM, HA);

Previous history

Malignant disease
Inborn condition
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.

Clinics case report

29 yrs

Blood data

Bone marrow

Cyto path

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.
Rearranged ig tcr
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.
Electron microscopy
Precise diagnosis
B-lymphoblastic leukemia

Survival data

Date diagnosis
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.
Date last follow


Bone Marrow
Culture time
Karyotype relapse
Mol cytogenet technics
Mol cytogenet results
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).

Other molec studies

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.

Other findings

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.


Atlas Image
figure 1: Whole genome-views of SSA and NDA workflows assignment of copy number calls. The top, middle and bottom panels in A, B, C and D show log2 ratio/smooth signal, SNP patterns and BAF, respectively A. dashed boxes indicate examples of discrepancies between log2-based copy numbers and corresponding allele tracks in SSA. B. Solid boxes indicate concordance between log2 and allele patterns in NDA. C. Admixture of genomic DNA with an unrelated specimen to recapitulate a chimeric state, results in same copy number calls using SSA, with SNP and BAF patterns serving a less effective role in recognition of an error in normalization. D. Same experiment as in C with NDA of the data, resulting in correct normalization despite an overall noisy background due to SNP mismatch.
Atlas Image
figure 2: Conventional cytogenetics and FISH results consistent with atypical hyperdiploidy. A. Karyogram showing extra copies of chromosomes X, 1, 2, 5, 6, 8, 9, 10, 11, 12, 14, 16, a supernumerary der(19)t(1;19) and nine copies of chromosome 21. B. Interphase FISH showing three copies of ETV6 (green) at 12p13.2 and nine copies of RUNX1 (red) at 21q22.12. C. FISH probes targeting the TCF3 telomeric (green) and centromeric (red) sequences at 19p13.3 indicating an intact signal pattern.

Comments section

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.


Pubmed IDLast YearTitleAuthors
90782851997Acute lymphoblastic leukemia and chromosome 21.Berger R et al
236192742013American College of Medical Genetics and Genomics technical standards and guidelines: microarray analysis for chromosome abnormalities in neoplastic disorders.Cooley LD et al
270332382016New and emerging prognostic and predictive genetic biomarkers in B-cell precursor acute lymphoblastic leukemia.Moorman AV et al
279035302017Near-haploid and low-hypodiploid acute lymphoblastic leukemia: two distinct subtypes with consistently poor prognosis.Safavi S et al
257295752015A (1;19) translocation involving TCF3-PBX1 fusion within the context of a hyperdiploid karyotype in adult B-ALL: a case report and review of the literature.Tirado CA et al


S Abbas Padeganeh, Joo Song, Patricia Aoun, Matthew Mei, Haris Ali, Dennis D. Weisenburger, Joyce L. Murata-Collins

Clinical Relevance of Normal Diploid Workflow in Microarray Analysis of Massively Aneuploid Genomes: Lessons from a Case of B-Lymphoblastic Leukemia

Atlas Genet Cytogenet Oncol Haematol. 2018-07-01

Online version: