In children with ALL attended at public hospitals in Mexico City, the identification of common fusion gene translocations associated with the prognosis of the disease is not routinely performed, but is available through research projects such as the present study. The purpose of this study was to identify the prevalence of ETV6-RUNX1, KMT2A-AFF1, TCF3-PBX1, and BCR-ABL1p190 fusion genes using a one-step TaqMan RT-qPCR assay and present the technical details of this assay. In Mexico, as in other countries, several groups have published different protocols for the detection of leukemia-specific fusion transcripts using conventional RT-PCR, and valuable prognostic information on human leukemias has been provided [16, 19–21]. To improve the specificity and timeliness of the detection of common fusion gene transcripts, the abovementioned assay was developed and validated in the present study. All one-step RT-qPCR assays for each fusion gene showed good sensitivities and specificities. The sensitivity could be explained, at least in part, by the elimination of the cDNA synthesis step using reverse transcription prior to PCR. Currently, a different enzyme with a higher efficiency is available in one-step kits. In our work, we compared different one-step kits and found that QuantiTect Probe RT-PCR (QIAGEN) showed high sensitivity for detecting the four fusion genes assessed.
Another important aspect of the one-step RT-qPCR assay was the precision obtained. It was estimated by the coefficient of variation, which reflects the day-to-day differences and variations of operators with different levels of experience. This coefficient was acceptably low to propose the use of this assay. Our results also showed high repeatability and reproducibility, and all cross-reactivity assays demonstrated that the primers and probes were specific for each fusion gene. The results of the interlaboratory comparison showed an acceptable concordance of results.
While commercially available multiple fusion gene transcript detection kits are relatively expensive, when considering the importance of detecting the four most common fusion gene transcripts associated with prognosis, the developed one-step real-time RT-PCR assay may be more cost-effective than QuanDx. This is supported by the fact that major problems for the determination of common fusion genes in Mexican health institutions are the lack of resources and specialized personnel.
Prevalence and prognostic impact of common fusion genes
It is well recognized that the B-lineage ALL (B-ALL) subtype has large genetic heterogeneity. Of importance, the fusion gene transcripts evaluated here are relevant for stratifying the risk of children with this disease (Table 2, Supplementary Fig. 3).
The frequencies of ETV6-RUNX1, TCF3-PBX1, KMT2A-AFF1 and BCR-ABL1p190 were 10.3%, 7.5%, 2.8% and 3.6%, respectively, similar to a previous study conducted by our research group where a comparable number of clinical samples were analyzed . Of note, the prevalence of these four fusion gene transcripts was 24.2%, which is similar to the 20% detection reported in a recent study carried out in southern Mexico using an in vitro diagnostic test (HemaVision-Q28, DNA Diagnostic) . Nonetheless, it has a low prevalence compared with studies conducted in other countries, particularly developed nations [24, 25]. This highlights the importance of discovering new molecular determinants possibly contributing to the high incidence and mortality of ALL in the Mexican population.
ETV6-RUNX1 is the most prevalent fusion gene transcript reported in children with ALL from developed countries (~ 25%), and it is associated with a favorable prognosis of the disease, currently reaching survival rates higher than 90% at 5 years of follow-up . However, in the present study, the analysis revealed that 26 of 253 cases (10.3%) presented the ETV6-RUNX1 fusion; most were male (69.2%), with a < 50,000 WBC count (84.6%) in peripheral blood at diagnosis. It was present at all ages but most commonly observed in children under 10 years of age (Table 2). The low prevalence of ETV6-RUNX1 is consistent with previous Mexican reports using different detection methodologies: 8.7% , 8.5% , 7.4% , 6.9% , 13.5% , and 14.9% . These results are similar to those reported in Hispanics living in other countries 4.5% in Guatemalan and 14% in Hispanic residing in the Northern California [30, 31]. This finding is probably due to the genetic background of these populations .
However, it also raises concerns about whether this finding is due to a misdiagnosis. In this regard, we used RT-qPCR, which is considered a highly sensitive method to detect fusion gene transcripts. In addition, it is well known that fusion genes may have different breakpoints, making them undetectable using standard methods; however, this phenomenon occurs infrequently. Notably, the full identification of fusion gene transcripts is possible using next-generation sequencing (NGS) technology . In this context, in a previous study by our research group using RNA-Seq analysis of 24 bone marrow samples of children with ALL, only one patient was ETV6-RUNX1 positive, which was consistent with the prevalence found in the present study. Conversely, we found a novel fusion gene, ETV6-NUFIP1 and ETV6-SNUPN, that interrupts the ETV6 gene with break sites in the first and second exons, respectively; therefore, it is possible that these novel fusions play a causal role in tumorigenesis . Nevertheless, although the prevalence of genetic fusion in the Mexican population is low, several groups have reported other genetic alterations related to ETV6 and AML1 genes, such as deletions or duplications. Perez-Vera, 2005 reported 35% alterations in AML1 copy numbers in LLA, and structural changes in ETV6 increased the number of ETV6 and AML1 consequences of polysomy of chromosomes 12 and 21, which play significant roles in disease progression .
The TCF3-PBX1 t(1;19)(q23;pl3.3) translocation results in the expression of a protein that combines the transactivation domains of TCF3 with the DNA binding homeodomain region of PBX1. TCF3-PBX1 fusion can promote cell transformation both in vitro and in vivo. Interestingly, the existence of a loss-of-function allele of TCF3 as a consequence of translocations in this gene has also been proposed. It reduces the levels of wild-type TCF3, which contributes to the development of leukemia . Because TCF3 plays an important role as a mediator of B cell differentiation, TCF3 functions as a negative regulator of tumorigenesis . The prevalence reported for TCF3-PBX1 fusion gene transcripts varies between 3–5% in childhood ALL; however, in this study, the prevalence was 7.5% (19 of 253 patients). TCF3-PBX1 patients were distributed across all age groups, particularly in children with < 50,000 WBCs at diagnosis. One child relapsed, and another died early during treatment (16.2 and 17.7 months after diagnosis, respectively). TCF3-PBX1-positive ALL has been related to an intermediate disease prognosis [29, 38]. Nonetheless, currently, with the increase in the intensity of the subgroup of E2A-PBX1-positive patients, they have a similar survival as those patients classified as standard risk . In the present study, we observed a DFS of 89% in patients with TCF3-PBX1, which correlates with previous reports in children with ALL and TCF3-PBX1 positivity. Importantly, in public hospitals in Mexico City, the TCF3-PBX1 group showed significant improvements in survival rates over time (data in preparation to publish) after the detection of this rearrangement through research project funding. In particular, the adverse prognostic value of these rearrangements has been overcome by using contemporary treatments such as Berlin-Frankfurt-Münster (BFM) [40, 41] and CCLG-ALL2008, a risk-based intensified treatment trial .
BCR-ABL1 p 190
The t(9;22)(q34;q11) reciprocal translocation encodes a tyrosine kinase and is considered a pathogenetic driver that can be therapeutically targeted. There are several breakpoints in BCR that produce distinct BCR-ABL1 isoforms in children with ALL. In patients with chronic myeloid leukemia, the most common is the BCR-ABL1 p220 isoform, while in the ALL subtype, it is the p190 isoform associated
with a dismal prognosis . However, treatment with tyrosine kinase (TK) inhibitors has improved the overall survival of patients with this rearrangement . In the present study, the BCR-ABL1p190 translocation was identified in 3.6% of patients, with the majority being over 10 years of age at the time of diagnosis confirmation, which is similar to that reported in other populations. The survival analysis showed low survival rates (DFS: 86% and OS: 63%) in patients with this translocation, but they are similar to those reported in other studies . Several mechanisms have been related to relapse and death among patients with BCR-ABL1. Among these mechanisms is resistance to imatinib due to mutations within the ABL1 (tyrosine kinase) domain of BCR-ABL1 (T315I). Another important mechanism is secondary chemotherapy resistance for different reasons. Therefore, it is necessary to study all the possible causes in our population, such as long-term adverse effects, resistance to TKIs, effects of other chemotherapy drugs and treatment noncompliance. In addition, a periodic follow-up of patients would be important to know the proportion of children with resistance to imatinib and to propose alternative treatments.
The KMT2A-AFF1 t(4;11) fusion was observed in this study in children between 4–16 years of age. This frequency highlights that this rearrangement is not exclusive to infants with ALL, as has been previously reported . Patients with ALL and the t(4;11) translocation have a poor prognosis . In the present investigation, KMT2A-AFF1 patients had a poor prognosis with a high frequency of relapse, which was reflected in a low DFS (56%) (Fig. 3).
Mexico is a country with limited financial resources where the detection of fusion rearrangements is not routinely performed in all hospitals due to high cost and lack of trained personnel in molecular biology. This assay could be used routinely to detect common fusion genes in children with ALL and may contribute significantly to clinical outcomes.