Incidence of Common Preleukemic Gene Fusions in Umbilical Cord Blood in Slovak Population

Abstract

The first event in origination of many childhood leukemias is likely the presence of preleukemic clone (transformed hematopoietic stem/progenitor cells with preleukemic gene fusions (PGF)) in newborn. Thus, the screening of umbilical cord blood (UCB) for PGF may be of high importance for developing strategies for childhood leukemia prevention and treatment. However, the data on incidence of PGF in UCB are contradictive. We have compared multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (RT qPCR) in neonates from Slovak National Birth Cohort. According to multiplex PCR, all 135 screened samples were negative for the most frequent PGF of B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To explore the prevalence of prognostically important TEL-AML1, MLL-AF4 and BCR-ABL (p190), 200 UCB were screened using RT qPCR. The initial screening showed an unexpectedly high incidence of studied PGF. The validation of selected samples in two laboratories confirmed approximately ¼ of UCB positive, resulting in ∼4% incidence of TEL-AML1, ∼6.25% incidence of BCR-ABL1 p190, and ∼0.75% frequency of MLL-AF4. In most cases, the PGF presented at very low level, about 1–5 copies per 105 cells. We hypothesize that low PGF numbers reflect their relatively late origin and are likely to be eliminated in further development while higher number of PGF reflects earlier origination and may represent higher risk for leukemia.

Low numbers of pre-leukemic fusion genes are frequently present in umbilical cord blood without affecting DNA damage response

“…The quantitative analysis of summary data confirmed the tendency observed in the first 200 samples, namely an extremely low level of studied PFG in few positive samples, reaching usually < 6 copies per 100,000 cells. Our data clearly show that the amount of PFG in the majority of PFG + UCB samples is very close to the threshold of RT-qPCR sensitivity, which was reached at approximately 1–3 copies per 100,000 cells in our hands [14]. This fact stresses the sensitivity limitations of the screening method and suggests that only an approximate estimation of PFG incidence can be made if the obtained data are only slightly above the sensitivity level.…”

“…In their pioneer study, the Greaves’ group reported ~1.06% incidence of TEL-AML1/ETV6-RUNX1, the most common ALL-associated PFG, in umbilical cord blood (UCB) samples from England and Italy [10]. These data were further confirmed by other groups by screening UCB of newborns from Czech Republic, Japan, USA, and Slovakia [10, 13, 14] but challenged by the Danish group reporting that the incidence of ETV6-RUNX1 is the same as the incidence of ETV6-RUNX1 related ALL [15]. The purported coincidence of overt ALL and related PFG suggested an effective approach for early ALL diagnostics by screening UCB of newborns.…”

IFN‐γ directly inhibits murine B‐cell precursor leukemia‐initiating cell proliferation early in life

“…The incidence of pediatric ALL peaks at 3–5 years of age, but in many cases the initiating events occur in utero, with greater than 70% of patients having detectable leukemia‐initiating cells (LICs) at birth . The size of the early preleukemic cell population has been reported to correlate with time to diagnosis in some studies , so the quantity of preleukemic cells, rather than simply their presence, may be a relevant marker for ALL risk .…”

Combined multiplex and monoplex RT-PCR as a reliable and cost-effective method for molecular diagnostics of pediatric acute lymphoblastic leukemia

“…We have not estimated the sensitivity of monoplex RT-PCRs which is expected to reach at least the sensitivity of multiplex RT-PCR. The sensitivity of RT qPCR is significantly higher, reaching in our laboratory maximum level of ~ 1-3 x 10 -5 [16] which may be considered as too high providing no benefit for primary diagnostics. Extremely high sensitivity of RT qPCR can be utilized for monitoring of minimal residual disease during leukemic patient's treatment.…”

“…The sensitivity of multiplex RT-PCR we used in this study was estimated to be approx. 5 x 10 -3 using standard plasmids with cloned gene fusions in pCR II TOPO vector as template [16]. The real sensitivity of our multiplex RT-PCR might be slightly lower because our sensitivity test assay did not take into consideration the efficiency of cDNA synthesis which is integral part of the RT-PCR method.…”