Convergent TP53 loss and evolvability in cancer.

Cancer cell populations evolve by a stepwise process involving natural selection of the fittest variants within a tissue ecosystem context and as modified by therapy. Genomic scrutiny of patient samples reveals an extraordinary diversity of mutational profiles both between patients with similar cancers and within the cancer cell population of individual patients. Does this signify highly divergent evolutionary trajectories or are there repetitive and predictable patterns?Major evolutionary innovations or adaptations in different species are frequently repeated, or convergent, reflecting both common selective pressures and constraints on optimal solutions. We argue this is true of evolving cancer cells, especially with respect to the TP53 gene. Functional loss variants in TP53 are the most common genetic change in cancer. We discuss the likely microenvironmental selective pressures involved and the profound impact this has on cell fitness, evolvability and probability of subsequent drug resistance.

Evolutionary determinants of curability in cancer.

The emergence of drug-resistant cells, most of which have a mutated TP53 gene, prevents curative treatment in most advanced and common metastatic cancers of adults. Yet, a few, rarer malignancies, all of which are TP53 wild type, have high cure rates. In this Perspective, we discuss how common features of curable cancers offer insights into the evolutionary and developmental determinants of drug resistance. Acquired loss of TP53 protein function is the most common genetic change in cancer. This probably reflects positive selection in the context of strong ecosystem pressures including microenvironmental hypoxia. Loss of TP53's functions results in multiple fitness benefits and enhanced evolvability of cancer cells. TP53-null cells survive apoptosis, and tolerate potent oncogenic signalling, DNA damage and genetic instability. In addition, critically, they provide an expanded pool of self-renewing, or stem, cells, the primary units of evolutionary selection in cancer, making subsequent adaptation to therapeutic challenge by drug resistance highly probable. The exceptional malignancies that are curable, including the common genetic subtype of childhood acute lymphoblastic leukaemia and testicular seminoma, differ from the common adult cancers in originating prenatally from embryonic or fetal cells that are developmentally primed for TP53-dependent apoptosis. Plus, they have other genetic and phenotypic features that enable dissemination without exposure to selective pressures for TP53 loss, retaining their intrinsic drug hypersensitivity.

IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia.

Cytokine Receptor-Like Factor 2 (CRLF2) overexpression occurs in 5-15% of B-cell precursor acute lymphoblastic leukaemia (B-ALL). In ∼50% of these cases, the mechanisms underlying this dysregulation are unknown. IKAROS Family Zinc Finger 1 (IKZF1) is a possible candidate to play a role in this dysregulation since it binds to the CRLF2 promoter region and suppresses its expression. We hypothesised that IKZF1 loss of function, caused by deletions or its short isoforms expression, could be associated with CRLF2 overexpression in B-ALL. A total of 131 paediatric and adult patients and 7 B-ALL cell lines were analysed to investigate the presence of IKZF1 deletions and its splicing isoforms expression levels, the presence of CRLF2 rearrangements or mutations, CRLF2 expression and JAK2 mutations. Overall survival analyses were performed according to the CRLF2 and IKZF1 subgroups. Our analyses showed that 25.2% of patients exhibited CRLF2 overexpression (CRLF2-high). CRLF2-high was associated with the presence of IKZF1 deletions (IKZF1del, p = 0.001), particularly with those resulting in dominant-negative isoforms (p = 0.006). Moreover, CRLF2 expression was higher in paediatric samples with high loads of the short isoform IK4 (p = 0.011). It was also associated with the occurrence of the IKZF1 plus subgroup (p = 0.004). Furthermore, patients with CRLF2-high/IKZF1del had a poorer prognosis in the RELLA05 protocol (p = 0.067, 36.1 months, 95%CI 0.0-85.9) and adult cohort (p = 0.094, 29.7 months, 95%CI 11.8-47.5). In this study, we show that IKZF1 status is associated with CRLF2-high and dismal outcomes in B-ALL patients regardless of age.

CRLF2 expression associates with ICN1 stabilization in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy with few molecular alterations showing a consensual prognostic value. CRLF2 overexpression was recently identified in high-risk T-ALL patients. For these cases, no genomic abnormality was found to be associated with CRLF2 overexpression. IKZF1 has been recently shown to be a direct transcriptional regulator of CRLF2 expression. Moreover, it is known that NOTCH1 antagonizes IKZF1 in T-ALL. In light of these pieces of evidence, we reasoned that IKZF1 binding perturbation and CRLF2 upregulation could be associated in T-ALL. We evaluated two independent series of pediatric T-ALL cases (PHOP, n = 57 and TARGET, n = 264) for the presence of common T-ALL molecular abnormalities, such as NOTCH1/FBXW7 mutations. We also assessed CRLF2 and IKZF1 gene expression. CRLF2 overexpression was observed in 14% (PHOP) and 16% (TARGET) of T-ALL patients. No correlation was found between mRNA expression of CRLF2 and IKZF1 in both cohorts. Interestingly, we show that patients with mutations affecting NOTCH1-PEST domain and/or FBXW7 had higher CRLF2 expression (P = .04). In summary, we demonstrate for the first time that only mutations resulting in ICN1 (intracellular domain of NOTCH1) stabilization are associated with CRLF2 overexpression.

A novel PAX5 rearrangement in TCF3-PBX1 acute lymphoblastic leukemia: a case report.

BACKGROUND: Chromosome translocations are a hallmark of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Additional genomic aberrations are also crucial in both BCP-ALL leukemogenesis and treatment management. Herein, we report the phenotypic and molecular cytogenetic characterization of an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated chromosome translocations: t(1;19)(q23;q13.3) and t(9;17)(p13;q11.2). Of note, we described a new rearrangement between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located. This rearrangement seems to disrupt PAX5 similarly to a PAX5 deletion. Furthermore, a distinct karyotype between diagnosis and relapse samples was observed, disclosing a complex clonal evolution during leukemia progression. CASE PRESENTATION: A 16-year-old boy was admitted febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly, low white blood cell count and 92% lymphoblast. He was diagnosed with pre-B ALL and treated according to high risk GBTLI-ALL2009. Twelve months after complete remission, he developed a relapse in consequence of a high central nervous system and bone marrow infiltration, and unfortunately died. CONCLUSIONS: To our knowledge, this is the first report of a rearrangement between PAX5 and SPECC1. The presence of TCF3-PBX1 and PAX5-rearrangement at diagnosis and relapse indicates that both might have participated in the malignant transformation disease maintenance and dismal outcome.

A Novel Assay for the Identification of NOTCH1 PEST Domain Mutations in Chronic Lymphocytic Leukemia.

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions.

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

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.

Impact of complex NOTCH1 mutations on survival in paediatric T-cell leukaemia.

BACKGROUND: Molecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether NOTCH1 mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases. METHODS: T-ALL subtypes, status of SIL-TAL1 fusion, ectopic expression of TLX3, and mutations in FBXW7, KRAS, PTEN and NOTCH1 were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test. RESULTS: The frequencies of mutations were 43.5% for NOTCH1, while FBXW7, KRAS and PTEN exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between NOTCH1 mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. SIL-TAL1 was identified as an adverse prognostic factor. NOTCH1 mutation status was not associated with outcome, while the presence of NOTCH1 complex mutations (indels) were associated with a longer overall survival (p = 0.031) than point mutations. CONCLUSION: NOTCH1 mutations alone or in combination with FBXW7 did not impact T-ALL prognosis. Nevertheless, complex NOTCH1 mutations appear to have a positive impact on OS and the SIL-TAL1 fusion was validated as a negative prognostic marker in our series of T-ALL.

Genetic variability in N-acetyltransferase 2 gene determines susceptibility to childhood lymphoid or myeloid leukemia in Brazil.

Seven single nucleotide polymorphisms (SNPs) were genotyped in 535 Brazilian children (158 with acute lymphoblastic leukemia [ALL], 74 with acute myeloid leukemia [AML] and 303 controls). The subjects were classified as fast or slow acetylators based on their genotypic variants. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals. N-acetyltransferase 2 (NAT2) SNP 341T > C frequency was higher among both leukemia subtypes compared to controls. There was also a significant difference in the frequency of SNP 590G > A in AML (OR, 1.57, 1.07-2.30). The haplotypes *14A, *5A and *5C conferred an increased risk in cases of ALL, while *14E, *6B and *6F conferred an increased risk for AML. An age-dependent analysis demonstrated that the NAT2 slow-acetylators conferred an increased risk association with leukemia in children ≤ 1 year old (OR, 7.91, 3.87-16.16) and also in older children (1 ≥ 10 years old) (OR, 1.53, 1.01-2.31). However, in this latter group the magnitude was reduced. The results demonstrate that the different NAT2 haplotypes contribute to the risk of either ALL or AML.

T-cell lymphoblastic leukemia in early childhood presents NOTCH1 mutations and MLL rearrangements.

T-cell acute lymphoblastic leukemia (T-ALL) may affect children in very early age. However, the critical events leading to this brief latency is still unclear. We used standard methods to explore NOTCH1 mutations and other specific molecular markers in 15 early childhood T-ALL cases. Most of them consisted of immature differentiation subtype. Despite being found in a lower frequency than that described for overall pediatric T-ALL, NOTCH1 alterations were the most frequent ones. Other alterations included MLL(+) (n=4), SIL-TAL1(+) (n=3), FLT3 mutation (n=1) and HOX11L2(+) (n=1). Our results suggest that NOTCH1 and MLL abnormalities are primary leukemogenic hits in early T-ALL.

SIL-TAL1 fusion gene negative impact in T-cell acute lymphoblastic leukemia outcome.

SIL-TAL1 fusion gene and the ectopic expression of HOX11L2 are common molecular abnormalities in T-cell acute lymphoblastic leukemia (T-ALL). To verify their influence on outcome, we analyzed a Brazilian pediatric T-ALL series of cases. One hundred and ninety two children, age ranged 0-21 years old, were consecutively diagnosed and treated. Reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to identify the molecular alterations. Kaplan-Meyer method was applied to estimate overall survival. The most frequent maturation stage was T-IV (40.1%), and 30.7% of cases were CD10(+). SIL-TAL1(+) and HOX11L2(+) accounted for 26.7% and 10.3% of the cases, respectively. The overall survival (OS) was 74% in 80-month follow-up. HOX11L2(+) was not predictive factor for outcome. Considering patients younger than nine years-old, those with SIL-TAL1(+) presented a poorer outcome (p = 0.02). The results of this study suggest that in the Brazilian population only the presence of SIL-TAL1 can predict outcome in a restricted group of patients.

Mutações do gene notch1 e outras alterações moleculares relacionadas à etiopatogênese das leucemias linfoblásticas agudas de linhagem t (lla-t)

As leucemias linfoblásticas agudas de célula T (LLAs-T) apresentam alta prevalência em países em desenvolvimento, justificando a realização de estudos exploratórios na população brasileira. Análises das alterações genético-moleculares têm permitido a identificação de elementos envolvidos na leucemogênese. A fusão SIL-TAL1, a expressão do gene HOX11L2, e as mutações do NOTCH1 são alterações genéticas comuns nas LLAs-T. Com o objetivo de testar a influência destas anormalidades no prognóstico das LLAs-T de pacientes brasileiros, nós analisamos uma série de 170 crianças e adultos jovens com LLA-T, com idades entre 1-21 anos, sendo 39 meninas e 131 meninos, diagnosticados entre 2001-2007. A metodologia incluiu imunofenotipagem para a definição do subtipo celular e para análise do status do CD10 e CD1a; RT-PCR para detecção do SIL-TAL1e do HOX11L2, PCR, DHPLC e seqüenciamento para a identificação de mutações no NOTCH1. O método de Kaplan-Meyer foi usado para estimar a sobrevida dos casos em 36 meses de acordo com o perfil dos marcadores analisados. Nossos resultados mostraram 36,4 % das amostras com CD10+ e 28,8 % com CD1a+. As freqüências do SIL-TAL1 e do HOX11L2 foram 24,4 % e 11,5 % respectivamente. Sobre as mutações do NOTCH1, o domínio PEST estava mutado em 8,8 % dos casos; o HD representou 16,2 % das mutações, enquanto a análise de ambos os domínios juntos demonstrou 57,4 % das mutações. Interessantemente, duas irmãs com LLA-T (não-gemelares) foram analisadas para o NOTCH1, vimos que ambas apresentaram mutações nos domínios HD e PEST nas mesmas regiões. A análise das células do SP dos pais destas irmãs não demonstrou mutações no NOTCH1. Com estes resultados, nós podemos afirmar que estas mutações são somáticas. O impacto de cada marcador na sobrevida não foi estatisticamente significante. Através destes resultados vê-se que é necessário continuar este estudo com um número maior de casos analisados para o NOTCH1, para que, assim, possamos avaliar melhor o impacto destes marcadores na patogênese das LLAs-T.

T acute lymphoblastic leukemias (T-ALL) subtype presents higher prevalence in developing countries, justifying exploring studies in a Brazilian population. The analyses of genetic-molecular alterations have allowed the identification of elements involved in leukemogenesis. The SIL-TAL1 fusion gene, the expression of HOX11L2 gene, and NOTCH1 mutations are the common genetic alterations found in T-ALL. In order to test the influence of such abnormalities on outcome of T-ALL in Brazilian patients, we carried out on analysis of a series of 170 children and young adults with T-ALL, age range 1-21 years old, being 39 girls and 131 boys consecutively diagnosed between 2001-2007. Methodology included immunophenotyping for subtype definition and analyses of CD10 and CD1a status; RT-PCR for molecular analyses of SIL-TAL1and HOX11L2, PCR, DHPLC and sequencing for NOTCH1 mutations detections. Kaplan-Meyer survival methodology was used to estimate the 36-months survival rate of T-ALL cases according to the markers analyzed. Our results show that CD10 was positive in 36.4 % of the samples and CD1a in 28.8 %. The frequency of SILTAL1 fusion gene and HOX11L2 were 24.4 % and 11.5 % respectively. Regarding NOTCH1mutations, the domain PEST was mutated in 8.8 % of the cases; the HD domain demonstrated 16.2 % of mutations, whereas the analysis of both domains together presented 57.4 % of mutations. Interesting, T-ALL in two sisters (non-twins) included in these analyses, demonstrated that they both had mutation in HD and PEST domains in the same region. The analysis of PB cells of both parents demonstrated any mutation in NOTCH1. With these results, we can confirm that NOTCH1 mutations are somatic. The impact of all markers in survival was not significant statistically. From these results it is necessary to continue the study with a larger number of cases analyzed to NOTCH1, so we can better evaluate the impact of each marker with the pathogenesis of T-ALLs.