Inactivation of CDKN2A in Childhood Acute Lymphoblastic Leukemia (ALL) Occurs Principally by Deletion and Is Strongly Correlated with Cytogenetic Subgroups.

Abstract Genetic alterations play a key role in the leukemogenesis of childhood ALL. Inactivation of CDKN2A (p16), a tumour suppressor gene located at 9p21, can occur by deletion, methylation or mutation. Published reports are inconsistent in terms of incidence and mode of inactivation. We report a comprehensive analysis of CDKN2A inactivation in 1230 diagnostic and 101 relapse samples, including 46 matched diagnostic and relapse pairs, from 1285 children with ALL. Using data from cytogenetics (CC) (n=1088), FISH (n=1209), SNP arrays (SNPA) (n=106), CGH arrays (aCGH) (n=106), dHPLC (n=48) and methylation specific-PCR (MSP) (n=96) we have assessed the mode and frequency of CDKN2A inactivation. Mutation or methylation of CDKN2A was rare occurring in 1 patient each (2% and 1% respectively). In contrast, CDKN2A deletion was highly prevalent. The frequencies of deletion detected by the different methodologies were: CC 166 (15%), FISH 335 (28%), SNPA 17 (16%) and aCGH 35 (33%). The proportion of biallelic deletions also varied: CC 15 (9%), FISH 174 (52%) and aCGH 15 (65%). This variation was directly related to the resolution of each technique with a high degree of concordance across samples investigated by >1 method. Analysis of 50 deletions by aCGH showed that the size of the deletion ranged from 0.03Mb to 39.1Mb with a mean of 14.8Mb. Furthermore, analysis of 15 biallelic deletions demonstrated that they comprised one large deletion (mean size 23.3Mb) and a second much smaller deletion (mean size 1.4Mb). In addition, SNPA revealed copy number neutral LOH in 8 (8%) cases, but only once in association with a CDKN2A mutation. At diagnosis CDKN2A inactivation by any method was noted in 329 (27%) patients which was not different from that observed at relapse [25 (25%)]. However, the frequencies of CDKN2A inactivation and biallelic deletion were significantly greater in T-ALL compared with B cell precursor (BCP) ALL: 135/269 (50%) v 190/918 (21%) (p<0.001) and 83/135 (61%) v 82/190 (43%) (p=0.001), respectively. Within BCP-ALL, older patients (10+ yrs) were more likely to have CDKN2A inactivation compared to younger patients (<10 yrs) whereas the reverse was true in T-ALL: 52/196 (27%) v 138/722 (19%) (p=0.023) and 53/126 (42%) v 82/143 (57%) (p=0.012). Among 46 matched samples CDKN2A inactivation was retained (n=8), lost (n=3) or gained (n=6). The frequency of CDKN2A inactivation was strongly correlated with cytogenetics. Lower frequencies were observed among high hyperdiploid [31/302 (10%) p<0.001] and ETV6-RUNX1 patients [36/236 (15%) p=0.02] with higher frequencies among those with t(9;22) [11/19 (58%) p<0.001], t(1;19) [10/25 (40%) p=0.019] and other abnormalities [92/226 (41%) p<0.001]. In conclusion, we have confirmed the importance of CDKN2A inactivation in childhood ALL and demonstrated that by far the most prevalent method of inactivation is deletion. While it is clear that loss of CDKN2A acts as a cooperating mutation in childhood ALL it is strongly correlated with age, phenotype and genotype. The observation that it is negatively correlated with good risk cytogenetic subgroups may explain why it has been inconsistently associated with a poor outcome. The discovery of copy number neutral LOH at 9p with no evidence of CDKN2A inactivation suggests the presence of another tumour suppressor gene or oncogene in this region.

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Comprehensive Analysis of p16INK4a in Childhood Acute Lymphoblastic Leukemia Reveals Homozygous Deletion, Haploinsufficiency and Acquired Isodisomy at the 9p Locus with Intact p16INK4a.

Blood ◽

10.1182/blood.v108.11.4332.4332 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4332-4332

Author(s):

Sarina Sulong ◽

Julie Irving ◽

Marian Case ◽

Lynne Minto ◽

Nick Bown ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Tumor Suppressor ◽

Tumor Suppressor Gene ◽

Somatic Mutation ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Promoter Hypermethylation ◽

Suppressor Gene ◽

Comprehensive Analysis ◽

Childhood All

Abstract Genetic alterations including chromosomal translocation, promoter hypermethylation, somatic mutation and gene deletion are believed to play a key role in the leukemogenic process in childhood acute lymphoblastic leukemia (ALL). The p16INK4a (CDKN2A/MTS1/p16/INK4a) gene located on chromosome 9p21 is a tumor suppressor gene whose product can block cell division during the G1/S phase of the cell cycle. Inactivation of p16INK4a in ALL can occur by deletion, promoter hypermethylation or somatic mutation. However, published reports are inconsistent in terms of both incidence and route of p16INK4a inactivation suggesting that a detailed analysis of all possible modes of inactivation in a large cohort is essential to clarify the status of this gene in leukemogenesis. In this study, we report the findings of a comprehensive analysis of p16INK4a in 115 DNA samples with childhood ALL (86 cases at presentation and 29 cases at relapse) in which a combination of techniques including, fluorescence in situ hybridization (FISH), mapping arrays, denaturing high performance liquid chromatography (dHPLC) and methylation specific-PCR (MSP) were used to assess the mode of inactivation of this gene. Data from a genome-wide screening in 86 presentation cases and 20 of 29 relapse cases using Affymetrix Mapping 10K and/or 50K single nucleotide polymorphism (SNP) microarray technique showed loss of heterozygosity (LOH) at the p16INK4a locus in 21% (22/106) of cases (14 at presentation and 8 at relapse), 14 (8 at presentation and 6 at relapse) with an associated loss of copy number and 8 (6 at presentation and 2 at relapse) with a normal copy number, indicative of acquired isodisomy (AID). FISH analysis on 19 of the 22 confirmed that those cases with LOH and copy number loss had either p16INK4a homozygous (n=6) or hemizygous (n=6) deletion and those with LOH associated with AID (n=7) retained 2 copies. Mutation and methylation analyses were performed on those cases identified to have one p16INK4a allele or retention of both alleles. Partial methylation of p16INK4a was found in only 1 case. Mutational screening by dHPLC of the coding region revealed a somatic mutation, H83Y, in a subpopulation of leukemic blasts in one patient at relapse. Three common SNPs were identified including A148T in exon 2 and 500C>G and 540 C>T in the 3′ UTR. These data show that mutation and hypermethylation of p16INK4a are rare events in childhood ALL but that homozygous and hemizygous deletion is relatively common. The loss of only one p16INK4a allele in this latter group, without evidence for mutation or hypermethylation of the remaining one suggests that p16INK4a may be haploinsufficient in ALL. The finding that LOH on 9p locus is common but in nearly 40% of these cases is associated with AID with intact p16INK4a, suggests the existence of another tumor suppressor gene or oncogene in this region, which may have importance in leukemogenesis.

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The TP53 tumour suppressor gene in colorectal carcinomas. I. Genetic alterations on chromosome 17

British Journal of Cancer ◽

10.1038/bjc.1993.14 ◽

1993 ◽

Vol 67 (1) ◽

pp. 88-92 ◽

Cited By ~ 19

Author(s):

GI Meling ◽

RA Lothe ◽

A-L Børresen ◽

C Graue ◽

S Hauge ◽

...

Keyword(s):

Suppressor Gene ◽

Genetic Alterations ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Chromosome 17 ◽

Colorectal Carcinomas

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Childhood Acute Lymphoblastic Leukemia: High Genomic Stability from Initial Diagnosis to Early Relapse.

Blood ◽

10.1182/blood.v112.11.1522.1522 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1522-1522

Author(s):

Barbara Meissner ◽

Cornelia Eckert ◽

Anja Moericke ◽

Arend von Stackelberg ◽

Renate Kirschner-Schwabe ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Genomic Stability ◽

Genetic Alterations ◽

Initial Diagnosis ◽

Childhood All ◽

Early Relapse ◽

Resistant Disease ◽

Homozygous Deletions

Abstract Despite considerable improvements regarding treatment outcome about 20% of children with acute lymphoblastic leukemia (ALL) still suffer from recurrent disease. Especially patients with an early relapse occurring before 6 months of treatment cessation have a poor prognosis and, therefore, urgently need therapy optimization. As an initial step, detection of genetic aberrations that are associated with resistant disease and/or early relapse might be of great value as they could serve as prognostic markers to predict disease recurrence and, in addition, may increase our understanding of mechanisms underlying treatment resistance and early relapse. This knowledge may also help to identify new therapeutic strategies including targeted approaches. Surprisingly, almost no information is available on the genetic evolution from initial diagnosis to early relapse. As a first aim in the process of uncovering the pathomechanism of early relapse we wanted to find out if early relapse of childhood ALL reflects primarily resistant disease with little or no additional aberrations at relapse or represents evolution and selection of a new and more resistant clone possibly triggered through chemotherapy. Recently, high throughput technologies (single nucleotide polymorphism [SNP]–Arrays) have been developed to screen the whole genome with high resolution for submicroscopic, genetic alterations by simultaneously analyzing the SNP genotype and determining the copy number state. This prompted us to perform SNP-array analysis encompassing more than 100.000 SNPs in 20 childhood ALL samples collected at initial diagnosis and to compare them to their counterparts obtained at relapse. The analyzed patients belonged to the intermediate or high risk treatment group; 16 exhibited B-precursor and 4 T-precursor ALL. Exclusion criteria were translocations t(12;21), t(9;22) and (4;11) as well as pre-existing conditions (e.g. Down syndrome). At initial diagnosis, the leukemic sample of all patients already displayed a complex karyotype with multiple genetic lesions (average numbers per patient: 5.15 deletions, 1.35 amplifications, and 0.65 copy number neutral LOH (CNN-LOH)). Most alterations remained stable from initial diagnosis to relapse (99 of 103 (96%) initially observed deletions as well as 24 of 27 (89%) amplifications). Four deletions occurring in 3 different patients at initial diagnosis disappeared at relapse. Nevertheless, these patients had additional stable copy number alterations. Newly occurring aberrations at relapse were less frequent (average numbers per patient: 1.8 new deletions, 0.75 new amplifications, and 0.15 new CNN-LOH). In 3 patients genetic alterations at diagnosis and relapse were entirely identical. The most commonly affected chromosome was 9p (75% of samples). Fourteen patients had either heterozygous or homozygous deletions of CDKN2A at initial diagnosis and relapse with 2 patients progressing from heterozygous to homozygous deletions. PAX5 deletions (mainly heterozygous) were detectable in 9 patients at initial diagnosis and 10 patients at relapse. Other sides of recurrent deletions – mainly detected at relapse – included multiple areas of chromosome 7p (p21.3, p15.3, p14.3, p14.3-14.1, p14.1-p13) as well as chromosome 1q (deletion: q42.12-q44, amplification: q25.2-q31.1), chromosome 17p (deletion: p13.3-p11.2) and 17q (amplification: q21.2-q25.1). Potential pathogenetic implications of specific recurrent genetic lesions will be discussed. In conclusion, our results demonstrate a high degree of genomic stability from initial diagnosis to early relapse of childhood ALL. These results suggest that – at least in some patients – early relapse might already be predetermined by a resistant leukemic clone at the time of initial diagnosis.

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Age-dependent copy number variations of TP53 tumour suppressor gene associated with altered phosphorylation status of p53 protein in sporadic schwannomas

Journal of Neuro-Oncology ◽

10.1007/s11060-019-03176-1 ◽

2019 ◽

Vol 143 (3) ◽

pp. 369-379

Author(s):

Hongsai Chen ◽

He Huang ◽

Jingjing Zhao ◽

Zhigang Wang ◽

Mengling Chang ◽

...

Keyword(s):

Copy Number ◽

P53 Protein ◽

Suppressor Gene ◽

Copy Number Variations ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Age Dependent

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CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms19123832 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3832 ◽

Cited By ~ 5

Author(s):

Charles Bailey ◽

Cynthia Metierre ◽

Yue Feng ◽

Kinsha Baidya ◽

Galina Filippova ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Somatic Cells ◽

Suppressor Gene ◽

Genetic Alterations ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Absolute Requirement ◽

The Impact

CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/−) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/− MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/− MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.

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CTCF Expression Is Essential for Somatic Cell Viability and Protection against Cancer

10.20944/preprints201810.0451.v1 ◽

2018 ◽

Author(s):

Charles G. Bailey ◽

Cynthia Metierre ◽

Julie Feng ◽

Kinsha Baidya ◽

Galina N. Filippova ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Somatic Cells ◽

Suppressor Gene ◽

Genetic Alterations ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Tumour Suppressor Genes ◽

Absolute Requirement ◽

The Impact

CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown, functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing and examined the growth and development of heterozygous Ctcf (Ctcf+/-) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/- MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression and decreased survival after apoptotic insult compared to wild type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/- MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.

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Genetic alterations of the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q in human breast carcinomas

Genes Chromosomes and Cancer ◽

10.1002/gcc.2870040203 ◽

1992 ◽

Vol 4 (2) ◽

pp. 113-121 ◽

Cited By ~ 74

Author(s):

Tone I. Andersen ◽

Astrid Gaustad ◽

Anne-Lise Børresen ◽

George W. Farrants ◽

Jahn M. Nesland ◽

...

Keyword(s):

Suppressor Gene ◽

Genetic Alterations ◽

Tumour Suppressor Gene ◽

Tumour Suppressor ◽

Breast Carcinomas ◽

Human Breast ◽

Human Breast Carcinomas

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A comprehensive analysis of the CDKN2A gene in childhood acute lymphoblastic leukemia reveals genomic deletion, copy number neutral loss of heterozygosity, and association with specific cytogenetic subgroups

Blood ◽

10.1182/blood-2008-07-166801 ◽

2009 ◽

Vol 113 (1) ◽

pp. 100-107 ◽

Cited By ~ 120

Author(s):

Sarina Sulong ◽

Anthony V. Moorman ◽

Julie A. E. Irving ◽

Jonathan C. Strefford ◽

Zoe J. Konn ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Loss Of Heterozygosity ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Neutral Loss ◽

Suppressor Gene ◽

Childhood Acute Lymphoblastic Leukemia ◽

Comparative Genomic ◽

Childhood All ◽

Genomic Deletion

Abstract Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.

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