Bruno de Almeida Lopes

Functional analysis of the two reciprocal fusion genes MLL-NEBL and NEBL-MLL reveal their oncogenic potential.

MLL gene aberrations are frequently diagnosed in infant acute myeloid leukemia (AML). We previously described the MLL-NEBL and NEBL-MLL genomic fusions in an infant AML patient with a chromosomal translocation t(10;11)(p12;q23). NEBL was the second Nebulin family member (LASP1, NEBL) which was found to be involved in MLL rearrangements. Here, we report on our attempts to unravel the oncogenic properties of both fusion genes. First, RT-PCR analyses revealed the presence of the MLL-NEBL and NEBL-MLL mRNAs in the diagnostic sample of the patient. Next, expression cassettes for MLL-NEBL and NEBL-MLL were cloned into a sleeping beauty vector backbone. After stable transfection, the biological effects of MLL-NEBL, NEBL-MLL or the combination of both fusion proteins were investigated in a conditional cell culture model. NEBL-MLL but also co-transfected cells displayed significantly higher growth rates according to the data obtained by cell proliferation assay. The focus formation experiments revealed differences in the shape and number of colonies when comparing MLL-NEBL, NEBL-MLL- and co-transfected cells. The results obtained in this study suggest that the reciprocal fusion genes of the Nebulin gene family might be of biological importance.

Subclonality and prenatal origin of RAS mutations in KMT2A (MLL)-rearranged infant acute lymphoblastic leukaemia.

KMT2A (previously termed MLL) rearrangements (KMT2Ar) constitute the most frequent aberration in infant lymphoblastic leukaemia (iALL). Mutations in the RAS genes have previously been recognized as an important somatic abnormality in KMT2A-r-iALL (Taketani et al, 2004; Liang et al, 2006; Driessen et al, 2013; Prelle et al, 2013). Certainly, KMT2A-r in iALL are present at birth (Greaves et al, 2003). On the other hand, RAS mutations (RASmut) are suggested to be secondary and subclonal hits in the leukaemogenic pathway. In this context, our study aimed to (i) quantitatively evaluate the association between RASmut at diagnosis and the main clinical features of KMT2A-r-iALL, and (ii) analyse the mutations in matched samples [neonatal blood spots, also known as Guthrie cards (Gc), remission and/or relapse]. KRAS/NRASmut were screened by direct sequencing, and mutated cases were also subjected to pyrosequencing in order to quantify the percentage of blasts harbouring the mutation. The detailed methods performed in this study are described in the Supporting Information (Data S1). Ninety-two KMT2A-r-iALL cases were included. RASmut were found in 21 7% of cases [KRAS (14 1%) and NRAS (7 6%)]. In childhood ALL, RASmut have been diagnosed in 6–21% of patients (Liang et al, 2006; Wiemels et al, 2010). Recent studies in large cohorts of KMT2A-r cases have reported frequencies of 14–15% RASmut, with an even higher frequency in t(4;11) infants (c. 25%) (Driessen et al, 2013; Prelle et al, 2013). These previous data are therefore concordant with our study. The clinical-laboratory features of KMT2A-r-iALL according to RAS status are shown in Table SI. Sixty percent of RASmut patients were ≤6 months old, 60% harboured AFF1 as the KMT2A-translocation partner gene (TPG), and 80% presented both the KMT2A-AFF1 and AFF1-KMT2A transcripts at mRNA level. Although these differences were not statistically significant, these analyses are in agreement with previous data, such as a higher frequency of RASmut among younger and t(4;11) infants (Driessen et al, 2013; Prelle et al, 2013). Furthermore, KRAS (P = 0 033) but not NRAS (P = 0 415) mutations were associated with high white blood cell (WBC) counts. The quantitative association between RASmut at diagnosis and clinical features was analysed by pyrosequencing the mutated clones. The median percentage of mutated clones did not differ according to age group (31% vs. 27%; Fig S1A; P = 0 43) or KMT2A-TPG (28% vs. 33%; Fig S1C; P = 0 89). Significantly higher percentages of mutated clones were found in cases with WBC counts >300 x 10/l (Fig S1B; P = 0 01). The correlation of RAS-mutated clones with higher WBC count may indicate that RASmut play a role in aggressiveness. Accordingly, the presence of RASmut was an independent prognostic factor in a previous cohort (Driessen et al, 2013). Despite recent attempts, neither the role of a second-hit nor the subclonality of RASmut in KMT2A-riALL has been demonstrated (Driessen et al, 2013; Prelle et al, 2013). Nevertheless, according to pyrosequencing analyses, we checked for a putative subclonality of RASmut and found that, in 19/ 20 cases, the percentage of mutated alleles at diagnosis was lower than expected for a clonal alteration. The RASmut were monoallelic (50% in a single cell). Taking the percentage of blast cells into account, if the alteration is clonal, the percentage of mutated alleles is expected to be half of the blast count for each patient (Table SII). Then, subclonality was observed in this present study. Regarding the analysis of matched samples, 17 samples of patients who achieved remission were analysed, but no mutations were found. Among the relapse samples (n = 12), five patients lost RASmut at relapse and seven patients were wildtype at both diagnosis and relapse. Concerning the two Gc samples, in both cases the RAS-mutated allele ‒ present at diagnosis ‒ was also detected at birth (Table SII). One of these infants (Patient 13) was diagnosed with ALL 17 d after birth, with high WBC, KMT2A-MLLT10 gene fusion (Fig 1A) and KRASmut (Fig 1B) detected at birth. The intrapatient’s quantitative comparison between samples showed that the Gc sample had a higher percentage of KRASmutated clones than the diagnostic sample (42% vs. 33%, Fig 1C, D). Finding the KRASmut in this infant’s Gc sample was an expected result as the baby was presumably born with the disease and presented with 97% blasts in the peripheral blood. The other infant (Patient 15) had an NRASmut (Fig 2A) at 7-months old, with 96% peripheral blood blasts and KMT2A-AFF1. The percentage of NRAS mutated clones was 50% in both the Gc and the diagnostic sample (Fig 2C, D). This high percentage of mutated cells within the Gc sample may explain the backtracking success. The presence of RASmut at birth clearly argues against a secondary role in a two-hit model for this subgroup of leukaemias. Although former studies performed in hyperdiploid childhood leukaemia strongly indicate that RASmut are secondary hits (Case et al, 2008; Davidsson et al, 2010), this might not be a correspondence

Polymorphisms in CYP1B1, CYP3A5, GSTT1, and SULT1A1 Are Associated with Early Age Acute Leukemia.

Based on observational studies, early age leukemia (EAL) was associated with maternal hormone exposure during pregnancy. We studied the association between genetic polymorphisms of estrogen metabolism and EAL. Using data from the Brazilian Collaborative Study Group of Infant Acute Leukemia (2000–2012), 350 cases and 404 age-matched controls and 134 mothers of cases and controls were genotyped to explore polymorphisms in genes of the estrogen metabolism pathway: CYP1B1 (c.1294C>G, rs1056836), CYP3A4 (c.-392A>G, rs2740574), CYP3A5 (c.219-237G>A, rs776746), GSTM1/GSTT1 deletions, and SULT1A1 (c.638G>A, rs9282861; and c.667A>G, rs1801030). Logistic regression was used to calculate the odds ratios (OR) with 95% confidence intervals (CIs), and unconditional logistic regression was used to estimate adjusted odds ratios (aORs) by ethnicity. Because of multiple testing, p values < 0.01 were significant after Bonferroni correction. SULT1A1 (c.638G>A) was associated to infant acute lymphoblastic leukemia and acute myeloid leukemia (AML) risk in males (additive model: aOR = 0.52; 95% CI: 0.29–0.95, p = 0.03; dominant model: aOR = 2.18; 95% CI: 1.17–4.05, p = 0.01, respectively). CYP1B1 polymorphism was associated with a decreased risk of AML either for non-white or female children (additive model: OR = 0.24; 95% CI: 0.08–0.76, p < 0.01; additive model: aOR = 0.27; 95% CI: 0.08–0.89, p = 0.03, respectively). Since polymorphisms of Cytochrome P450 genes presented gender-specific risk associations, we also investigated their expression. CYP1B1 was not expressed in 57.1% of EAL cases, and its expression varied by genotype, gender, and leukemia subtype. Maternal-fetal GSTT1 null genotype was associated with risk of EAL. This study shows that polymorphisms in genes of estrogen metabolism confer genetic susceptibility to EAL, mainly in males, and maternal susceptibility genes modify the risk for developing EAL in newborns.

Impact of mutations in FLT3, PTPN11 and RAS genes on the overall survival of pediatric B cell precursor acute lymphoblastic leukemia in Brazil

Abstract We analyzed mutations in four genes (FLT3, KRAS/NRAS and PTPN11) that might disrupt the RAS/mitogen activated protein kinase (MAPKinase) signaling pathway, to evaluate their prognostic value in children younger than 16 years old with B-cell precursor acute lymphoblastic leukemia (Bcp-ALL). The overall survival (OS) was determined with the Kaplan–Meier method. MAPKinase genes were mutated in 25.4% and 20.1% of childhood and infant Bcp-ALL, respectively. Children with hyperdiploidy were more prone to harboring a MAPKinase gene mutation (odds ratio [OR] 3.18; 95% confidence interval [CI] 1.07–9.49). The mean OS of all cases was 54.0 months. FLT3 and PTPN11 mutations had no impact on OS. K/NRAS mutations were strongly associated with MLL–AFF1 (OR 5.78; 95% CI 1.00–33.24), and conferred poorer OS (p = 0.034) in univariate analysis.

COBL is a novel hotspot for IKZF1 deletions in childhood acute lymphoblastic leukemia

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in childhood B-cell precursor acute lymphoblastic leukemia. Because of its clinical importance, we previously mapped breakpoints of intragenic deletions and developed a multiplex PCR assay to detect recurrent intragenic ΔIKZF1. Since the multiplex PCR was not able to detect complete deletions (IKZF1 Δ1-8), which account for ~30% of all ΔIKZF1, we aimed at investigating the genomic scenery of IKZF1 Δ1-8. Six samples of cases with IKZF1 Δ1-8 were analyzed by microarray assay, which identified monosomy 7, isochromosome 7q, and large interstitial deletions presenting breakpoints within COBL gene. Then, we established a multiplex ligation-probe amplification (MLPA) assay and screened copy number alterations within chromosome 7 in 43 diagnostic samples with IKZF1 Δ1-8. Our results revealed that monosomy and large interstitial deletions within chromosome 7 are the main causes of IKZF1 Δ1-8. Detailed analysis using long distance inverse PCR showed that six patients (16%) had large interstitial deletions starting within intronic regions of COBL at diagnosis, which is ~611 Kb downstream of IKZF1, suggesting that COBL is a hotspot for ΔIKZF1. We also investigated a series of 25 intragenic deletions (Δ2–8, Δ3–8 or Δ4–8) and 24 relapsed samples, and found one IKZF1-COBL tail-to-tail fusion, thus supporting that COBL is a novel hotspot for ΔIKZF1. Finally, using RIC score methodology, we show that breakpoint sequences of IKZF1 Δ1-8 are not analog to RAG-recognition sites, suggesting a different mechanism of error promotion than that suggested for intragenic ΔIKZF1.

A new complex rearrangement in infant ALL: t(X;11;17)(p11.2;q23;q12).

We present a case of an infant who developed pro-B acute lymphoblastic leukemia with a rare and complex MLL-translocation. Cytogenetic analysis of bone marrow cells at diagnosis showed a 46,XY,t(X;11)(p11.2;q23)[13]/46,XY[7] karyotype. Fluorescence in situ hybridization analysis using a break apart specific probes showed a split in the MLL gene. Long distance inverse-PCR and next generation sequencing analysis depicted a complex rearrangement t(X;11;17)(p11.2;q23;q12) involving MLL, MLLT6 and the genomic region Xp11.23, 41 bases upstream of the WDR45 gene. WDR45 encodes a beta-propeller protein essential for autophagocytosis. MLL rearrangements with involvement of Xp have not been previously described.

Molecular approaches identify a cryptic MECOM rearrangement in a child with a rapidly progressive myeloid neoplasm

Myeloid neoplasms are a heterogeneous group of hematologic disorders with divergent patterns of cell differentiation and proliferation, as well as divergent clinical courses. Rare recurrent genetic abnormalities related to this group of cancers are associated with poor outcomes. One such abnormality is the MECOM gene rearrangement that typically occurs in cases with chromosome 7 abnormalities. MECOM encodes a transcription factor that plays an essential role in cell proliferation and maintenance and also in epigenetic regulation. Aberrant expression of this gene is associated with reduced survival. Hence, its detailed characterization provides biological and clinical information relevant to the management of pediatric myeloid neoplasms. In this work, we describe a rare karyotype harboring three copies of MECOM with overexpression of the gene in a child with a very aggressive myeloid neoplasm. Cytogenetic studies defined the karyotype as 46,XX,der(7)t(3;7)(q26.2;q21.2). Array comparative genomic hybridization (aCGH) revealed a gain of 26.04 Mb in the 3q26.2-3qter region and a loss of 66.6 Mb in the 7q21.2-7qter region. RT-qPCR analysis detected elevated expression of the MECOM and CDK6 genes (458.5-fold and 35.2-fold, respectively). Overall, we show the importance of performing detailed molecular cytogenetic analysis of MECOM to enable appropriate management of high-risk pediatric myeloid neoplasms.

EPHX1 rs1051740 T>C (Tyr113His) is strongly associated with acute myeloid leukemia and KMT2A rearrangements in early age

Experimental and epidemiological data have shown that acute myeloid leukemia in early-age (i-AML) originates prenatally. The risk association between transplacental exposure to benzene metabolites and i-AML might be influenced by genetic susceptibility. In this study, we investigated the relationship between genetic polymorphisms in CYP2E1, EPHX1, MPO, NQO1, GSTM1 and GSTT1 genes, and i-AML risk. The study included 101 i-AMLs and 416 healthy controls. Genomic DNA from study subjects was purified from bone marrow or peripheral blood aspirates and genotyped for genetic polymorphisms by real-time PCR allelic discrimination, Sanger sequencing and multiplex PCR. Crude and adjusted odds ratios (OR, adjOR, respectively) with 95% confidence intervals (95% CI) were assessed using unconditional logistic regression to estimate the magnitude of risk associations. EPHX1 rs1051740 T>C was associated with i-AML risk under the co-dominant (adjOR 3.04, P = 0.003) and recessive (adjOR 2.99, P = 0.002) models. In stratified analysis, EPHX1 rs1051740 was associated with increased risk for i-AML with KMT2A rearrangement (adjOR 3.06, P = 0.045), i-AML with megakaryocytic differentiation (adjOR 5.10, P = 0.008), and i-AML with type I mutation (adjOR 2.02, P = 0.037). EPHX1 rs1051740-rs2234922 C-G haplotype was also associated with increased risk for i-AML (adjOR 2.55, P = 0.043), and for i-AML with KMT2A rearrangement (adjOR 3.23, P = 0.034). Since EPHX1 enzyme is essential in cellular defense against epoxides, the diminished enzymatic activity conferred by the variant allele C could explain the risk associations found for i-AML. In conclusion, EPHX1 rs1051740 plays an important role in i-AML’s genetic susceptibility by modulating the carcinogenic effects of epoxide exposures in the bone marrow.

IKZF1 gene in childhood B cell precursor acute lymphoblastic leukemia: interplay between genetic susceptibility and somatic abnormalities

SNPs in IKZF1 are associated with inherited susceptibility to B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Besides, somatic copy number abnormalities (CNA) in genes related to lymphopoiesis (e.g., IKZF1, CDKN2A/B, BTG1) impact patients outcome. Therefore, this study aimed to investigate an association between germline susceptibility and CNAs in BCP-ALL. The IKZF1 SNPs (rs11978267 and rs4132601) were genotyped in 276 cases and 467 controls. Bone marrow samples were used to determine the presence of somatic abnormalities. The IKZF1 transcript levels were quantified and associated with the SNPs and CNAs. Categorical variables were compared by χ2 test. ORs were estimated with unconditional logistic regression with 95% confidence interval (CI). The variant allele of IKZF1 rs4132601 conferred increased risk of BCP-ALL (OR, 2.09; 95% CI, 1.16–3.74). Individuals with either rs11978267 or rs4132601 had an increased risk for harboring IKZF1 deletion (OR, 2.80; 95% CI, 1.25–6.23 and OR, 2.88; 95% CI, 1.24–6.69, respectively). Increased risks were observed for individuals harboring both IKZF1 and BTG1 deletions (OR, 4.90; 95% CI, 1.65–14.55, rs11978267 and OR, 5.80; 95% CI, 1.94–17.41, rs4132601). Germline genetic variation increases the risk for childhood ALL in general, but also acts as a susceptibility factor bound for risk of specific somatic alterations. These findings provide new insight into the development of childhood ALL regarding causal variants and the biological basis of the risk association, offering the opportunity for future tailored research. Cancer Prev Res; 10(12); 738–44. ©2017 AACR.