TP53 pathway analysis in paediatric Burkitt lymphoma reveals increased MDM4 expression as the only TP53 pathway abnormality detected in a subset of cases

The TP53 (p53) pathway can be inhibited by TP53 mutation or deletion or by MDM2 overexpression. Both occur in Burkitt lymphoma (BL), but many cases lack either abnormality. Expression patterns of the TP53 inhibitor MDM4 have not been reported in BL, and increased MDM4 could deregulate the TP53 pathway in cases without TP53 or MDM2 abnormalities. We investigated TP53 pathway disruption in paediatric BL patient samples (n = 30) by studying MDM4, MDM2, and CDKN1A (p21) protein and mRNA expression; TP53 mutations; TP53 protein expression; and gene copy number abnormalities. MDM4 protein was expressed in 30/30 tumours, and MDM2 protein was weakly expressed in 7/30 (23%). All cases were negative for CDKN1A protein, and CDKN1A mRNA levels were decreased. TP53 mutations were detected in 5/28 (18%) cases and confirmed by sequencing. TP53 protein was expressed in 15/30 (50%) cases, including 7/8 with TP53 genetic alterations. MDM2 protein and mRNA expression levels did not correlate with lack of TP53 genetic changes or TP53 protein expression; however, there was an inverse relationship between detectable TP53 protein expression and MDM4 copy number gains and mRNA expression. The TP53 pathway is deregulated in paediatric BL cases, and increased MDM4 expression may be the primary mechanism in some cases.     

SOX11 and TP53 add prognostic information to MIPI in a homogenously treated cohort of mantle cell lymphoma – a Nordic Lymphoma Group study

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma, where survival has been remarkably improved by use of protocols including high dose cytarabine, rituximab and autologous stem cell transplantation, such as the Nordic MCL2/3 protocols. In 2008, a MCL international prognostic index (MIPI) was created to enable stratification of the clinical diverse MCL patients into three risk groups. So far, use of the MIPI in clinical routine has been limited, as it has been shown that it inadequately separates low and intermediate risk group patients. To improve outcome and minimize treatment‐related morbidity, additional parameters need to be evaluated to enable risk‐adapted treatment selection. We have investigated the individual prognostic role of the MIPI and molecular markers including SOX11, TP53 (p53), MKI67 (Ki‐67) and CCND1 (cyclin D1). Furthermore, we explored the possibility of creating an improved prognostic tool by combining the MIPI with information on molecular markers. SOX11 was shown to significantly add prognostic information to the MIPI, but in multivariate analysis TP53 was the only significant independent molecular marker. Based on these findings, we propose that TP53 and SOX11 should routinely be assessed and that a combined TP53/MIPI score may be used to guide treatment decisions.     

Mutation and genomic deletion status of ataxia telangiectasia mutated (ATM) and p53 confer specific gene expression profiles in mantle cell lymphoma

Although mantle cell lymphoma (MCL) frequently harbors inactivated ataxia telangiectasia mutated (ATM) and p53 alleles, little is known about the molecular phenotypes caused by these genetic changes. We identified point mutations and genomic deletions in these genes in a series of cyclin D1-positive MCL cases and correlated genotype with gene expression profiles and overall survival. Mutated and/or deleted ATM and p53 alleles were found in 56% (40/72) and 26% (21/82) of the cases examined, respectively. Although MCL patients with inactive p53 alleles showed a significant reduction in median overall survival, aberrant ATM status did not predict for survival. Nevertheless, specific gene expression signatures indicative of the mutation and genomic deletion status of each gene were identified that were different from wild-type cases. These signatures were comprised of a select group of genes related to apoptosis, stress responses, and cell cycle regulation that are relevant to ATM or p53 function. Importantly, we found the molecular signatures are different between cases with mutations and deletions, because the latter are characterized by loss of genes colocalized in the same chromosome region of ATM or p53. This information on molecular phenotypes may provide new areas of investigation for ATM function or may be exploited by designing specific therapies for MCL cases with p53 aberrations.     

Uniparental disomies, homozygous deletions, amplifications, and target genes in mantle cell lymphoma revealed by integrative high-resolution whole-genome profiling

Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. However, only a limited number of target genes have been identified. We have studied 10 MCL cell lines and 28 primary tumors with a combination of a high-density single-nucleotide polymorphism array and gene expression profiling. We detected highly altered genomes in the majority of the samples with a high number of partial uniparental disomies (UPDs). The UPD at 17p was one of the most common, and it was associated with TP53 gene inactivation. Homozygous deletions targeted 4 known tumor suppressor genes (CDKN2C, BCL2L11, CDKN2A, and RB1) and 6 new genes (FAF1, MAP2, SP100, MOBKL2B, ZNF280A, and PRAME). Gene amplification coupled with overexpression was identified in 35 different regions. The most recurrent amplified regions were 11q13.3-q13.5, 13q31.3, and 18q21.33, which targeted CCND1, C13orf25, and BCL2, respectively. Interestingly, the breakpoints flanking all the genomic alterations, including UPDs, were significantly associated with genomic regions enriched in copy number variants and segmental duplications, suggesting that the recombination at these regions may play a role in the genomic instability of MCL. This integrative genomic analysis has revealed target genes that may be potentially relevant in MCL pathogenesis.     

Totipotent stem cells bearing del(20q) maintain multipotential differentiation in Shwachman Diamond syndrome

SBDS /7q11 gene mutations underlie the congenital Shwachman Diamond syndrome (SDS), characterized by bone marrow failure and high risk of haematological malignancies. In two cases of SDS with bone marrow failure and isolated del(20q) interphase fluorescence in situ hybridization (I-FISH) found no abnormalities in FHIT /3p14.2, IKZF1 /7p13, D7S486/7q31, PTEN /10q23.3, WT1 /11p13, ATM /11q23, D13S25/13q14, TP53 /17p13, NF1 /17q11, SMAD2 /18q21, RUNX1 /21q22. Fluorescence immunophenotype combined with I-FISH found del(20q) in a totipotent haematopoietic stem cell (CD34⁺, CD133⁺) and downstream myelocyte (CD33⁺, CD14⁺, CD13⁺), erythrocyte (Glycophorin A⁺) and lymphocyte lineages (CD19⁺, CD20⁺, CD3⁺, CD7⁺). These findings and clinical follow-ups confirm the benign course of SDS with isolated del(20q).     

Ataxia telangiectasia mutated-deficient B-cell chronic lymphocytic leukemia occurs in pregerminal center cells and results in defective damage response and unrepaired chromosome damage.

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease involving more than one molecular mechanism that leads to the transformation of CD5(+) B cells at either the pregerminal or postgerminal center stage of differentiation. It was previously demonstrated that ataxia telangiectasia mutated (ATM) gene mutations can occur in B-CLL and cause a defect in the p53 pathway. Here the role of ATM mutations in the pathogenesis of B-CLL is addressed. Of 50 B-CLL tumors with fully analyzed ATM and TP53, 16 had ATM mutations. Six of 50 B-CLLs showed mutations in TP53 and the remaining 28 tumors had wild-type ATM or TP53. No tumor had both ATM and TP53 mutations. Remarkably, all 16 ATM mutant B-CLLs showed the absence of somatic variable region heavy chain hypermutation indicating a pregerminal center cell origin and a common pathogenesis for these tumors. Furthermore, in 5 of the 16 B-CLLs, ATM mutation preceded the transformation stage of differentiation. At the cellular level, ATM mutant tumors exhibited a deficient ATM-dependent p53 response to gamma irradiation, failure to up-regulate TRAIL-R2, a downstream target that links irradiation-induced p53 response with apoptosis, and an inability to repair induced chromosome breaks. Mantle cell lymphoma (MCL) is also of pregerminal center origin and ATM mutations are frequent in this malignancy. It is concluded that ATM is likely to play an important role at the pregerminal center stage and a model is proposed where loss of ATM function during B-cell ontogeny drives B-CLL tumorigenesis in pregerminal B cells by a dual defect in p53 damage response and repair of chromosome breaks.     

p16INK4A gene homozygous deletions in human acute leukaemias with alterations of chromosome 9

Acute leukaemias are characterized by non-random chromosomal aberrations which are often strictly related to the inactivation of tumour suppressor genes (TSGs). Alterations at the short arm of chromosome 9 have been reported in a remarkable percentage of acute lymphoblastic leukaemias (ALL) and have been suggested to cause the loss of activity of the putative TSG, p16^INK4A (MTS1/CDKN2) gene. In order to evaluate the correlation between this gene inactivation and visible cytogenetic abnormalities, we have investigated p16^INK4A homozygous gene deletions in 10 paediatric acute leukaemias of different cell lineages which demonstrated karyotype aberrations involving chromosome 9. Moreover, the dimension of the genetic alteration was evaluated by studying the loss of heterozygosity of two highly polymorphic markers of chromosome 9p, namely α-interferon (IFNA) and D9S104, and the deletion of 5'-methylthioadenosine phosphorylase (MTAPase) gene. Finally, the deletion of a gene belonging to p16^INK4A family, the p18 gene, was analysed in these acute leukaemias. Our results demonstrated that: (i) the biallelic loss of p16^INK4A gene is strictly related to a specific immunophenotype, namely ALL of T-cell lineage; (ii) no significant correlation exists between alterations at chromosome 9p level and the homozygous deletions of p16^INK4A gene; and (iii) p18 gene was not deleted in the examined cases. These findings suggest a possible correlation between the T-lymphocyte phenotype and the expression of p16^INK4A gene. Moreover, the absence of MTAPase activity seems to be a valuable marker of p16^INK4A gene inactivation, thus indicating that the deleted chromosomal area on 9p21 very frequently involves the MTAPase gene.     

Oligonucleotide microarrays demonstrate the highest frequency of ATM mutations in the mantle cell subtype of lymphoma

Mutations have been described in the ataxia telangiectasia mutated (ATM) gene in small numbers of cases of lymphoid neoplasia. However, surveys of the ATM mutation status in lymphoma have been limited due to the large size (62 exons) and complex mutational spectrum of this gene. We have used microarray-based assays with 250,000 oligonucleotides to screen lymphomas from 120 patients for all possible ATM coding and splice junction mutations. The subtypes included were diffuse large B cell, mantle cell, immunoblastic large B cell, follicular, posttransplant lymphoproliferative disorder, and peripheral T cell lymphoma. We found the highest percentage of ATM mutations within the mantle cell (MCL) subtype (43%, 12 of 28 cases), followed by a lower level (10% of cases) in the other subtypes. A frame-shift ATM mutation was found in one peripheral T cell lymphoma patient. In six MCL cases examined, four ATM variants were due to somatic mutation in the tumor cells whereas two others seemed to be germ-line in origin. There was no difference in p53 mutation status in the ATM mutant and wild-type groups of MCL. There was no statistically significant difference in the median overall survival of patients with wild-type vs. mutated ATM in MCL. Additional mutational and functional analyses are needed to determine whether ATM mutations contribute to the development and progression of MCL or are just the consequence of genomic instability in MCL.     

Impact of Single or Combined Genomic Alterations of TP53, MYC,and BCL2 on Survival of Patients With Diffuse Large B-Cell Lymphomas: A Retrospective Cohort Study

MYC and BCL2 translocations as well as TP53 deletion/mutation are known risk factors in diffuse large B-cell lymphoma (DLBCL) but their interplay is not well understood.In this retrospective cohort study, we evaluated the combined prognostic impact of TP53 deletion and mutation status, MYC and BCL2 genomic breaks in tumor samples of 101 DLBCL patients. The cohort included 53 cases with MYC rearrangements (MYC+).TP53 deletions/mutations (TP53+) were found in 32 of 101 lymphomas and were equally distributed between MYC+ and MYC− cases (35.8% vs. 27.1%). TP53+ lymphomas had lower responses to treatment than TP53− (complete remission 34.4% vs. 60.9%; P = 0.01). TP53 alteration was the dominant independent prognostic factor in multivariate analysis (P = 0.01). Overall survival (OS) varied considerably between subgroups with different genomic alterations: Patients with sole MYC translocation, and interestingly, with triple MYC+/BCL2+/TP53+ aberration had favorable outcomes (median OS not reached) similar to patients without genomic alterations (median OS 65 months). In contrast, patients with MYC+/BCL2+/TP53− double-hit lymphomas (DHL) (28 months), MYC+/BCL2−/TP53+ lymphomas (10 months) or sole TP53 mutation/deletion (12 months) had a poor median OS. Our findings demonstrate differences in OS of DLBCL patients depending on absence or presence of single or combined genetic alterations of MYC, BCL2, and TP53. Cooccurrence of TP53 and BCL2 aberrations ameliorated the poor prognostic impact of single TP53+ or BCL2+ in MYC positive patients.This pilot study generates evidence for the complex interplay between the alterations of genetic pathways in DLBCL, which goes beyond the concept of DHL. The variable survival of DLBCL patients dependent on single or combined alterations in the TP53, MYC, and BCL2 genes indicates the need for comprehensive genomic diagnosis.     

Advances in the understanding of mantle cell lymphoma

Mantle cell lymphoma (MCL) is a well-defined lymphoid neoplasm characterized by a proliferation of mature B lymphocytes expressing CD5 that may show a spectrum of morphological and phenotypic features broader than initially described. Although some patients may follow an indolent clinical evolution, in most of them the tumour has an aggressive behaviour with poor response to conventional chemotherapy. The genetic hallmark is the t(11;14)(q13;q32) translocation leading to the overexpression of cyclin D1, which is considered the initial oncogenic event. In addition to this translocation, MCL may carry a high number of secondary chromosomal and molecular alterations that target regulatory elements of the cell cycle machinery and senescence (BMI1/INK4/ARF/CDK4/RB1), DNA damage response pathways (ATM/CHK2/p53), and cell survival signals. The knowledge of these mechanisms and their influence on the behaviour of the tumour are facilitating the development of prognostic models with a more precise prediction of the clinical evolution of the patients. This information coupled with the availability of a new generation of innovative drugs targeting basic molecular process of the tumour cells, should facilitate the design of new therapeutic protocols able to overcome the resistance of this aggressive lymphoma to conventional treatments and improve the life expectancy of the patients.     

GeneChip analyses point to novel pathogenetic mechanisms in mantle cell lymphoma

The translocation t(11;14)(q13;q32) is the genetic hallmark of mantle cell lymphoma (MCL) but is not sufficient for inducing lymphomagenesis. Here we performed genome-wide 100K GeneChip Mapping in 26 t(11;14)-positive MCL and six MCL cell lines. Partial uniparental disomy (pUPD) was shown to be a recurrent chromosomal event not only in MCL cell lines but also in primary MCL. Remarkably, pUPD affected recurrent targets of deletion like 11q, 13q and 17p. Moreover, we identified 12 novel regions of recurrent gain and loss as well as 12 high-level amplifications and eight homozygously deleted regions hitherto undescribed in MCL. Interestingly, GeneChip analyses identified different genes , encoding proteins involved in microtubule dynamics, such as MAP2 , MAP6 and TP53, as targets for chromosomal aberration in MCL. Further investigation, including mutation analyses, fluorescence in situ hybridisation as well as epigenetic and expression studies, revealed additional aberrations frequently affecting these genes. In total, 19 of 20 MCL cases, which were subjected to genetic and epigenetic analyses, and five of six MCL cell lines harboured at least one aberration in MAP2, MAP6 or TP53 . These findings provide evidence that alterations of microtubule dynamics might be one of the critical events in MCL lymphomagenesis contributing to chromosomal instability.     

Mantle-cell lymphoma genotypes identified with CGH to BAC microarrays define a leukemic subgroup of disease and predict patient outcome

To identify recurrent genomic changes in mantle cell lymphoma (MCL), we used high-resolution comparative genomic hybridization (CGH) to bacterial artificial chromosome (BAC) microarrays in 68 patients and 9 MCL-derived cell lines. Array CGH defined an MCL genomic signature distinct from other B-cell lymphomas, including deletions of 1p21 and 11q22.3-ATM gene with coincident 10p12-BMI1 gene amplification and 10p14 deletion, along with a previously unidentified loss within 9q21-q22. Specific genomic alterations were associated with different subgroups of disease. Notably, 11 patients with leukemic MCL showed a different genomic profile than nodal cases, including 8p21.3 deletion at tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor gene cluster (55% versus 19%; P = .01) and gain of 8q24.1 at MYC locus (46% versus 14%; P = .015). Additionally, leukemic MCL exhibited frequent IGVH mutation (64% versus 21%; P = .009) with preferential VH4-39 use (36% versus 4%; P = .005) and followed a more indolent clinical course. Blastoid variants, increased number of genomic gains, and deletions of P16/INK4a and TP53 genes correlated with poorer outcomes, while 1p21 loss was associated with prolonged survival (P = .02). In multivariate analysis, deletion of 9q21-q22 was the strongest predictor for inferior survival (hazard ratio [HR], 6; confidence interval [CI], 2.3 to 15.7). Our study highlights the genomic profile as a predictor for clinical outcome and suggests that "genome scanning" of chromosomes 1p21, 9q21-q22, 9p21.3-P16/INK4a, and 17p13.1-TP53 may be clinically useful in MCL.     

Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage

The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase. However, these modifications alone may not account in full for p53 stabilization. p53's stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner. This phosphorylation is independent of p53 and the DNA-dependent protein kinase. Furthermore, MDM2 is directly phosphorylated by ATM in vitro. These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.     

Mutational features of myelodysplastic syndromes with Auer rods reveal them are more akin to acute myeloid leukemia

Survival for patients diagnosed with mantle cell lymphoma (MCL) has improved drastically in recent years. However, patients carrying mutations in tumour protein p53 (TP53) do not benefit from modern chemotherapy-based treatments and have poor prognosis. Thus, there is a clinical need to identify missense mutations through routine analysis to enable patient stratification. Sequencing is not widely implemented in clinical practice for MCL, and immunohistochemistry (IHC) is a feasible alternative to identify high-risk patients. The aim of the present study was to investigate the accuracy of p53 as a tool to identify patients with TP53 missense mutations and the prognostic impact of overexpression and mutations in a Swedish population-based cohort. In total, 317 cases were investigated using IHC and 255 cases were sequenced, enabling analysis of p53 and TP53 status among 137 cases divided over the two-cohort investigated. The accuracy of predicting missense mutations from protein expression was 82%, with sensitivity at 82% and specificity at 100% in paired samples. We further show the impact of p53 expression and TP53 mutations on survival (hazard ratio of 3·1 in univariate analysis for both), and the association to risk factors, such as high MCL International Prognostic Index, blastoid morphology and proliferation, in a population-based setting.     

Differential alterations of the genes in the CDKN2A-CCND1-CDK4-RB1 pathway are associated with the development of head and neck squamous cell carcinoma in Indian patients

Purpose The aim of this study was to analyse the alterations of the genes in the CDKN2A/CCND1/CDK4/RB1 pathway in the G1-S phase of the cell cycle during development of head and neck squamous cell carcinoma (HNSCC).Methods The alterations of these genes were analysed in 22 dysplastic lesions, 26 stage-I/II and 33 stage-III/IV HNSCC tumours of Indian patients.Results The alterations [mutation, hypermethylation, homozygous deletion and loss of heterozygosity/microsatellite size alteration (LOH/MA)] in the CDKN2A were found to be highest in 57% of the samples, followed by CCND1 amplification and LOH/MA at the RB1 locus in 14% and 8.5% of the samples, respectively. No dominant CDK4 Arg24Cys mutation was seen in our samples. Comparatively high frequency of CDKN2A alterations (except homozygous deletion) was found in dysplastic head and neck lesions and remained almost constant or increased during progression of the tumour, whereas the homozygous deletion of CDKN2A and the alterations in CCND1 and RB1 genes were seen mainly in the later stages of the tumour.Conclusions Our study suggested that mutation/hypermethylation/allelic alterations (LOH/MA) of CDKN2A were associated with the development of dysplastic head and neck lesions. All the other alterations might provide some cumulative effect during progression of later stages of the tumour to have selective growth advantages.     

Molecular characterization of 11q deletions points to a pathogenic role of the ATM gene in mantle cell lymphoma.

Deletions involving the long arm of chromosome 11 (11q) have been recently found as recurrent chromosome aberrations in mantle cell lymphoma (MCL). In the current study, the incidence and molecular extent of 11q deletions were analyzed in a series of 81 MCL by fluorescence in situ hybridization with probes from a contiguous set of yeast artificial chromosomes (YACs). Loss of chromosome 11 material was observed in 37 of 81 cases (46%). The minimally deleted segment comprised YAC 801e11 containing the ATM gene. To further narrow the minimal region of loss, P1-derived artificial chromosomes mapping to the critical region were isolated and used as probes in cases without aberrations detectable with YACs. This allowed the identification of an ATM deletion that was beyond the resolution of YAC probes. The identification of a minimally deleted segment affecting ATM suggests a pathogenic role of ATM as a tumor suppressor gene in MCL.     

Mutations in p53 as potential molecular markers for human breast cancer.

Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors, we investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. We examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, over-expression of protein, and point mutations in the p53 gene in 50% of the cell lines tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus. Multiplex PCR of genomic DNA showed that up to 36% of primary tumors contained aberrations in the p53 locus. Mutations in exons 5-9 of the p53 gene were found in 10 out of 59 (17%) of the primary tumors studies by single-stranded conformation polymorphism analysis. We conclude that, compared to amplification of HER2/NEU, MYC, or INT2 oncogene loci, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer related to a single gene. Correlated to disease status, p53 gene mutations could prove to be a valuable marker for diagnosis and/or prognosis of breast neoplasia.