WO2009077642A1 - Procédé de criblage - Google Patents

Procédé de criblage Download PDF

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Publication number
WO2009077642A1
WO2009077642A1 PCT/FI2008/050369 FI2008050369W WO2009077642A1 WO 2009077642 A1 WO2009077642 A1 WO 2009077642A1 FI 2008050369 W FI2008050369 W FI 2008050369W WO 2009077642 A1 WO2009077642 A1 WO 2009077642A1
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probe
cdkn2a
cancer
locus
deletion
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PCT/FI2008/050369
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English (en)
Inventor
Sakari Knuutila
Suvi Savola
Piero Picci
Katia Scotlandi
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University Of Helsinki
Istituto Ortopedico Rizzoli
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Priority claimed from PCT/FI2007/050707 external-priority patent/WO2008074924A2/fr
Application filed by University Of Helsinki, Istituto Ortopedico Rizzoli filed Critical University Of Helsinki
Publication of WO2009077642A1 publication Critical patent/WO2009077642A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

Definitions

  • the present invention is related to a method for improving human cancer diagnostics and prognostics, to a probe used in said method and to the method for optimizing clinical trial design for selecting a cancer therapy and to the method for selecting cancer therapy for treatment of cancer.
  • Gene and chromosome abnormalities are often associated with cancer.
  • the deletion of whole chromosomes and the deletion of chromosomal segments or specific regions are common occurrences in cancer.
  • the loss of genetic material results in the loss of the function of tumour suppressor genes.
  • the increase in the genetic material results in the activation of cancer promoting genes, oncogenes.
  • the changes in the number of chromosomes and in the DNA copy number are essential mechanisms in changing the functions of tumour suppressor and oncogenes.
  • the mapping of the entire human genome and the development of microarray technologies have for the first time revealed the genome wide information on gene and chromosome changes related to cancer. Furthermore, the knowledge of known changes in the genome has become more accurate and new cancer related aberrations have been revealed.
  • chromosome 9 The p arm of chromosome 9 is frequently lost or deleted in many human cancers, including the Ewing sarcoma family of tumours (Lopez-Guerrero et al., 2001; Tsuchiya et al., 2000).
  • CDKN2A maps to chromosome band 9p21.3 (location: 21957751-21984490, Ensembl: www.ensembl.org) and encodes two splice site variants, cyclin dependent kinase inhibitors, pl ⁇ 1 ⁇ and pl4 (ARF) Recent studies have provided convincing evidence that these proteins are important cell-cycle regulators in both normal and malignant cells (for review, see Sharpless, 2005).
  • the genes located in said locus encode proteins required for the regulation of cell division and which proteins have the cancer protecting capacity.
  • CDKN2 retinoblastoma
  • pl4 take part in the regulation of retinoblastoma (Rb) and p53 pathways, respectively. Both proteins control the cell-cycle progression from Gl -phase to S-phase.
  • pl6 inhibits CDK4/6 kinases by preventing the interaction between CDK4/6 and cyclin D (Sharpless, 2005). Suppression of CDK4/6 - cyclin Dl complex blocks E2F dependent transcription and leads to pathogenesis in various malignancies (Malumbres and Barbacid, 2001).
  • pl4 regulates p53 stability by inhibiting MDM2 -mediated degradation of p53 via ubiquitination (Sharpless, 2005).
  • CDKN2A is the principal target of the 9p21.3 deletion, although, it is sometimes co -deleted with MTAP or CDKN2B (also known as INK4b, MTS2 or pi 5) or both of these genes (Lopez-Guerrero et al., 2001; Bertin et al., 2003).
  • the CDKN2A locus is frequently deleted in various human neoplasms and the highest deletion frequencies are detected, according to US SEER data, in cancers of the pancreas (85%), esophagus (70%), head and neck (68%), and in melanoma (65%) (Sharpless, 2005 and SEER, 2002).
  • the identification of specific genomic regions associated with cancer is crucial both in developing better means of diagnosis and prognosis.
  • the methods for detecting deletions in patient samples have been developed. The methods have been mainly based on fluorescence in situ hybrization (FISH) or polymerase chain reaction (PCR) . FISH has been most useful for tissue material, especially paraffin embedded samples.
  • Ewing sarcoma is a malignant disease in which cancer cells are found in the bone or soft tissue. Ewing sarcoma usually presents in childhood or early adulthood.
  • the detection of deletion in CDKN2A locus is clinically and prognostically very significant, the deletion being a marker for poor prognosis.
  • the detection of said deletion provides a very useful cancer specific marker for monitoring the efficacy of cancer treatment, progression of disease and for detecting residual disease.
  • the methods include polymerase chain reaction (PCR) based assays, especially competitive PCR, gel electrophoresis of single-strand conformation polymorphisms, direct sequencing and restriction endonuclease digestion.
  • PCR polymerase chain reaction
  • WO 95/25429 discloses detection of mutations in multiple tumour suppressor (MTS) gene in cancer cell lines using PCR.
  • MTS multiple tumour suppressor
  • US 2003/0077582 discloses that an amplification of some genes or an increase in that gene activity or a deletion of the genes or a decrease in gene product activity is a marker for a cancer.
  • the method includes detecting the level of genes in specific cell line samples using Comparative Genomic Hybridization.
  • WO 01/32909 discloses the use of quantitative PCR for detecting mutations in a tumour suppressor gene associated with a leukaemia, especially lymphoblastic leukaemia.
  • US 6,331,390 discloses a diagnostic method for identifying mutations of a pl6 gene using PCR.
  • Fluorescent in situ hybridization is commonly used in the detection of CDKN2A (pl6) deletion in clinical cancer prognostics.
  • Namazie et al. 2002 and 2003 disclose the detection of deletions in pl6 associated with head and neck tumours using FISH.
  • Bleichert et al. 2001 dislcose a method for detecting deletions between pi 5 and pl6 genes using FISH in patients with T-cell acute lymphoblastic leukemia or chronic myeloid leukemia.
  • Dreyling et al. 1997 disclose the generation of FISH probes for mapping deletions in leukemia-derived cell lines. Several commercial kits and custom made probes are accessible for this purpose.
  • An object of the present invention is to provide a novel method for improving human cancer prognostics or diagnostics wherein the method provides an accurate detection of deletions in CDKN2A locus at chromosome region 9p21.3 in a sample of a subject using FISH analysis.
  • An object of the invention is to provide a probe for use in the method, wherein the probe having a nucleic acid sequence of SEQ ID NO:1 is specific for detecting the presence of a deletion in CDKN2A locus at chromosome region 9p21.3
  • the present invention is directed to the use of the method for optimizing clinical trial design for selecting a cancer therapy by detecting deletions in the region covering the
  • the present invention is also directed to the method for selecting a suitable cancer therapy for treatment of cancer by detecting deletions in the region covering the CDKN2A locus at chromosome region 9p21.3.
  • the present invention is also directed to the use of the method for treating malignancies or aberrations caused by deletions in the region covering the CDKN2A locus at chromosome region
  • Malignancies or aberrations include Ewing sarcoma, different types of leukaemia, bone and soft tissue sarcomas, melanoma, glioma, blastoma, mesothelioma and lymphoma, and gastrointestinal, breast, lung, pancreas, liver, prostate, testis, ovarian, head and neck and bladder cancers.
  • Figure 1 depicts IOR/RCH Ewing sarcoma cell line sample array CGH analysis on 9p chromosomal aberrations.
  • Figure IA depicts array CGH profile of chromosome 9 from the high-resolution oligonucleotide array (44K) analysis showing ⁇ 58 kb microdeletion, encompassing
  • CDKN2A/B loci within a gain of 9pl3.3-p22.3.
  • Figure IB depicts Gene view illustrating probespots deleted in 9p21.3 chromosomal area.
  • Figure 1C depicts array CGH profile of chromosome 9 from the ultra high-resolution oligonucleotide array (244K) analysis to confirm microdeletion size and coverage.
  • Figure ID depicts Gene view showing that CDKN2A/B loci are homozygously deleted and adjacent genes MTAP and DMRTAl are gained.
  • Figure 2 depicts tumour samples from patients number 13 and 14 analysed with 44K high- density array CGH.
  • Figure 2A depicts array CGH profile of chromosome 9 in patient sample 13 showing a ⁇ 154kb microdeletion in 9p21.3.
  • Figure 2B depicts that genes affected by microdeletion are CDKN2A/B and MTAP.
  • Figure 2C depicts array CGH profile of chromosome 9 in patient sample 14 showing a ⁇ 41kb microdeletion in 9p21.3.
  • Figure 2D depicts that genes affected by microdeletion are CDKN2A/B .
  • Figure 3 depicts metaphase FISH analysis of IOR/RCH cell line sample with commercial probe (LSI pl6/CEP9 Dual Color probe, Vysis).
  • the figure shows that one CDKN2A locus is gained and both chromosome 9 homologs are present as indicated by three orange signals (CDKN2A containing clone), marked in the picture with dotted arrows, and two green signals (chromosome 9 centrome, 9pl 1-ql 1) marked in the picture with white arrows, respectively (oil immersion, XlOO).
  • the FISH result shown here is in strike contrast with the results from array CGH analysis from the same cell line sample (See Figure 1), which shows that parts of CDKN2A gene and CDKN2B gene are homozygously deleted in the sample.
  • Figures 1 and 3 together show that FISH analysis with commercial CDKN2A probe creates false negative results in this cell line with ⁇ 58 kb microdeletion in CDKN2 locus.
  • Figure 4A depicts genomic location of 9p deletions in Ewing sarcoma patients and cell lines studied. The deletions are arranged by the size of the deletion.
  • Figure 4B depicts schematic representation of the deletions detected on chromosomal band 9p21.3 harboring the genes CDKN2A, CDKN2B and MTAP zoome d to the genomic area 21.7-22.15 Mb on 9p. The smallest overlapping deletion area (12.2 kb) in all cases is indicated. CDKN2A, CDKN2 B and MTAP together with the most commonly used pi 6 FISH probe (Vysis) are featured in the lower part of the figure.
  • CDKN2A which is also referred as CDK4I, INK4a/ARF, MTSI and pl6/lpl4, maps to chromosome band 9p21.3 (location: 21957751-21984490, Ensembl: www.ensembl.org) and encodes two splice site variants, cyclin dependent kinase inhibitors pl6 (INK4a) and pl4 (ARF) .
  • CDKN2 locus includes the genes CDKN2A and CDKN2B. INK4 locus also refers to CDKN2 locus.
  • the deletions of the CDKN2 locus are preferably within the following base pairs:
  • CDKN2B 21992902 - 21999280 (Vega) Locus ID OTTHUMG00000019691 &
  • CDKN2B 21992902 - 21999312 Ensembl Gene ID ENSG00000147883 The locations are based on ENSEMBL (www ⁇ cnscmbLorg) and VEGA (vega.sanger.ac.uk) databases. The locations of the genes vary depending on the database used.
  • Chrosome region 9p21.3 means a designation for the chromosome band (named) 21.3 on the short arm of chromosome number 9.
  • “Poor cancer prognosis” means the likelihood of further development of cancer or malignancy.
  • a deletion in CDKN2A locus at chromosome region 9p21.3 indicates poor cancer prognosis. Poor cancer prognosis also means the likelihood of invasive or metastatic spread of cancer.
  • Improved human cancer diagnostics or prognostics means the methods and means for improved identification of patients who are in the risk of developing cancer of malignancy .
  • Improved human cancer diagnostics or prognostics also means the evaluation of disease progression and prognosis in order to determine the proper course of treatment for an individual patient.
  • “Smallest overlapping region of deletion” means the narrowest chromosomal segment absent in all deletion cases.
  • the smallest overlapping deletion in chromosome 9p is presented in Figure 4B wherein the absence of area of 12.2 kb in all cases is indicated.
  • Microdeletion means a loss of DNA material that is not detectable by conventional cytogenetic methods e .g. metaphase karyotyping or conventional CGH.
  • the detection of microdeletions requires special techniques such as high-resolution chromosome banding or molecular chromosome analysis e.g. with FISH, PCR or by other DNA methods (for example with DNA microarray CGH analysis).
  • nucleic acid or “polynucleic acid” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in all their forms, i.e., single- or double -stranded form.
  • a polynucleotide sequence can be naturally occurring or synthetic or semisynthetic, but is typically prepared by synthetic or semisynthetic means, including PCR.
  • a polynucleotide refers to a molecule comprising a nucleic acid.
  • the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without a signal sequence, the secreted protein coding region, and the genomic sequence with or without the accompanying promoter and transcriptional termination sequences, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as defined.
  • the terms “nucleotide”, “base” and “nucleic acid” are intended to be equivalent.
  • the terms “nucleotide sequence”, “nucleic acid sequence”, “nucleic acid molecule” and “nucleic acid segment” are intended to be equivalent.
  • probe or a “nucleic acid probe”, as used herein, is defined to be a collection of one or more nucleic acid fragments whose hybridization to a sample can be detected.
  • the probe may be unlabeled or labeled as described below so that its binding to the target or sample can be detected. Similarly, depending on context, either the probe, the target, or both can be labeled.
  • the probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products.
  • PCR polymerase chain reaction
  • the probes of the present invention are produced from nucleic acids found in the regions described herein.
  • the probe or genomic nucleic acid sample may be processed in some manner, e.g., by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids.
  • One of skill will recognize that the precise sequence of the particular probes described herein can be modified to a certain degree to produce probes that are "substantially identical" to the disclosed probes, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived (see discussion above). Such modifications are specifically covered by reference to the individual probes described herein.
  • a cell line is a clone of a cell and capable of stable growth in vitro under controlled conditions for several generations.
  • Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumours, most primary cell cultures have limited lifespan.
  • An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene.
  • the "biological sample” refers to a sample of tissue or fluid isolated from a subject or individual. It may be from tumour cells or tissues or fluids, which contain nucleic acids or proteins or polypeptides, polynucleotide, or transcript. Such samples include, but are not limited to, tissue isolated from the subject to be treated and tissues such as biopsy and autopsy samples, or comprise frozen sections taken for histological purposes, archival samples, blood, plasma, serum, spinal fluid, lymph fluid, sputum, stool, tears, mucus, hair, skin, bone marrow, respiratory, intestinal and genitourinary tracts, saliva, milk, tumours, organs and also includes samples of in vivo cell culture constituents. The samples also include explants and primary and/or transformed cell cultures derived from patient tissues.
  • cancer in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumour, but such cells may exist alone within a subject, or may be a non-tumorigenic cancer cell, such as a leukemia cell.
  • Cancers include, but are not limited to Ewing sarcoma, leukaemias, bone and soft tissue sarcomas, melanoma, glioma, blastoma, mesothelioma, lymphoma or gastrointestinal, breast, lung, pancreas, liver, prostate, testis, ovarian, head and neck and bladder cancer.
  • detecting a cancer refers to the ascertainment of the presence or absence of cancer in a subject.
  • Detecting a cancer can also refer to obtaining indirect evidence regarding the likelihood of the presence of cancerous cells in the animal or to the likelihood or predilection to development of a cancer. Detecting a cancer can be accomplished using the methods of this invention alone, or in combination with other methods or in light of other information regarding the state of health of the subject.
  • a “method of treating cancer” refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in an animal, or to alleviate the symptoms of a cancer.
  • “A method of treating cancer” does not necessarily mean that the cancer cells will in fact be eliminated, that the number of cells will in fact be reduced, or that the symptoms of a cancer will in fact be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of an animal, is deemed an overall beneficial course of action.
  • Cancer can be treated by surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibody therapy or combination thereof or other methods. The choice of therapy depends upon the location and grade of the tumour and the stage of the disease, as well as the general state of the patient.
  • a "suitable cancer therapy” in the present invention refers to the treatment for patients with CDKN2A deletion , which is more aggressive than for patients without deletion.
  • New gateways are opened to develop more specific targeted therapy for the loss of function in CDKN2A gene product. It is essential to detect and monitor the deletion status in patients correctly in order to give optional therapy for each patient.
  • Heterozygosity means that an organism is a heterozygote or is heterozygous at a locus or gene when it has different alleles occupying the gene's position in each of the homologous chromosomes. In other words, it describes an individual that has two different alleles for a trait. In diploid organisms, the two different alleles are inherited from the organism's two parents. For example a heterozygous individual would have the allele combination Pp.
  • heterozygosity of deletion means e.g. that only one copy of particular chromosomal segment, gene or part of the gene is present in the sample. Normal sample has both copies of that particular chromosomal segment or gene in genome.
  • Homozygosity means that an organism is referred to as being homozygous at a specific locus when it carries two identical copies of the gene affecting a given trait on the two corresponding homologous chromosomes (e.g., the genotype is PP or pp when P and p refer to different possible alleles of the same gene). Such a cell or such an organism is called a homozygote.
  • a homozygous dominant genotype occurs when a particular locus has two copies of the dominant allele (e.g. PP).
  • a homozygous recessive genotype occurs when a particular locus has two copies of the recessive allele (e.g. pp). Pure -bred or true breeding organisms are homozygous.
  • homozygous individual could have the allele combinations PP or pp. All homozygous alleles are either allozygous or autozygous.
  • homozygosity of deletion means that both copies of particular chromosomal segment, gene or part of the gene are lost, so no copies are present. Recall that normal sample has two copies present.
  • the detection of deletions is carried out with a FISH analysis, wherein a fluorescently labelled nucleic acid probe hybridizes to a complementary nucleotide sequence in a sample.
  • the fluorescent signal is detected with a fluorescence microscope.
  • two CDKN2A signals are observed in both alleles of chromosome 9 in the sample there is no deletion. If the signal is missing (homozygous deletion) or only one signal is observed ( heterozygous deletion) said DNA region has been deleted from the sample.
  • the probe covers the smallest overlapping region (12.2kb) within the CDKN2A gene carrying a deletion, but is not larger than the region.
  • CDKN2A inactivation seems to the principal model in CDKN2A inactivation, at least in Ewing sarcoma (Tsuchiya et al., 2000; Huang et al., 2005).
  • the other less common ways of CDKN2A gene inactivation are a heterozygous deletion combined with methylation on promoter site or with mutation in the coding sequence inactivating the protein produced.
  • negative means that there is no deletion.
  • the term "false negative” refers to a result that appears negative, but fails to reveal a true situation that is positive i.e. there is a deletion.
  • An example of a false negative is : a FISH test designed to detect CDKN2A deletion is negative i.e. indicating that there is no deletion, but the sample has deletion of CDKN2A in reality, e.g. see Figure 3.
  • the present invention is based on the surprising finding that the currently used FISH analyses fail to detect microdeletions in CDKN2A of size less than 190 kb. Therefore the current FISH methods produce false negative results. Thus there is a need for more accurate detection methods for improving human cancer prognostics or diagnostics.
  • the present invention discloses a novel method for improving human cancer prognostics or diagnostics, comprising the steps of providing a probe having the nucleic acid sequence of SEQ ID NO:1 specific for a cyclin dependent kinase 2A (CDKN2A) locus at chromosome region 9p21.3, combining said probe with a biological sample of a subject, detecting by FISH analysis the presence of at least one deletion in CDKN2A locus at chromosome region 9p21.3 on said sample and classifying a subject having a deletion in CDKN2A locus at chromosome region 9p21.3 as indicating poor cancer prognosis.
  • CDKN2A cyclin dependent kinase 2A
  • the detection is carried out with a probe comprising a nucleic acid including a region of nucleotide sequence which hybridizes to CDKN2A gene sequence having the nucleic acid sequence of SEQ ID NO:2 and locating in a genomic location 21957752 - 21985300 according to Vega (Vega Locus ID OTTHUMGOOOOOO 19686) and/or SEQ ID NO:3 locating in a genomic location 21957751
  • the detection is carried out with a probe which is less than 60 kb in length, preferably less than 40 kb, more preferably less than 13 kb, most preferably from 5 to 12,5 kb. In a more preferred embodiment the detection is carried out with a 12.236 kb probe having the nucleic acid sequence of SEQ ID NO: 1 which hybridizes to a nucleic acid sequence or its complementary sequence in a genomic location 21968346 - 21980581 at chromosome region 9p21.3.
  • the sample comprises one or more interphase or mitotic cells.
  • FISH analysis is interphase FISH or metaphase FISH. The method can be also used on mitotic specimens and metaphase chromosome preparations.
  • the probe can be a dual color probe.
  • the cancer or malignancy is selected from Ewing sarcoma, leukaemias, melanoma, glioma, blastoma, mesothelioma, lymphoma or gastrointestinal, breast, lung, pancreas, liver, prostate, testis, ovarian, head and neck or bladder cancer.
  • the cancer or malignancy is Ewing sarcoma.
  • the present invention provides a nucleic acid specific for a CDKN2A locus at chromosome region 9p21.3.
  • the probe comprises preferably a nucleotide sequence which hybridizes to unique sequences of CDKN2A gene. More preferably the probe comprises a nucleic acid including a region of nucleotide sequence which hybridizes to CDKN2A gene sequence having the nucleic acid sequence of SEQ ID NO:2 locating at 21957752 - 21985300 according to Vega (Vega Locus ID OTTHUMG00000019686) and/or SEQ ID NO:3 locating in a genomic location 21957751 - 21984490 according to Ensembl (ENSEMBL Gene ID ENSG00000147889) at chromosome region 9p21.3 on chromosome 9.
  • the probe is less than 60 kb in length, preferably less than 40 kb, more preferably less than 13 kb, most preferably 5 to 12.5 kb. Most preferably the probe is 12.236 kb in length and specific for a genomic location 21968346 - 21980581 at chromosome region 9p21.3.
  • the probe may be a dual color probe.
  • the present invention provides the use of the method or the probe in diagnostics or prognostics of a cancer or malignancy, wherein the cancer of malignancy comprises Ewing sarcoma, leukaemias, melanoma, glioma, blastoma, mesothelioma, lymphoma or gastrointestinal, breast, lung, pancreas, liver, prostate, testis, ovarian, head and neck or bladder cancer.
  • the cancer or malignancy is Ewing sarcoma.
  • the probe of the present invention is used for preparation of a diagnostic tool for detecting or analyzing malignancies or aberrations caused by deletions in the region covering the CDKN2A locus at chromosome region 9p21.3.
  • the probe of the present invention can used for detecting deletions in the subjects suffering from malignancies or aberrations comprising Ewing sarcoma, leukaemias, melanoma, glioma, blastoma, mesothelioma, lymphoma or gastrointestinal, breast, lung, pancreas, liver, prostate, testis, ovarian, head and neck or bladder cancer.
  • the cancer or malignancy is Ewing sarcoma.
  • the present invention also provides a method for optimizing clinical trial design for selecting a cancer therapy, wherein the method comprises providing a probe specific for a cyclin dependent kinase 2 A CDKN2A locus at chromosome region 9p21.3, combining said probe with a biological sample of a subject; and detecting by FISH analysis the presence of at least one deletion in CDKN2A locus at chromosome region 9p21.3 on said sample; and classifying a subject having a deletion in said locus as indicating poor cancer prognosis.
  • the present invention provides a method for selecting a cancer therapy for treatment of cancer, wherein the method comprises the steps of providing a probe specific for a cyclin dependent kinase 2A CDKN2A locus at chromosome region 9p21.3, combining said probe with a biological sample of a subject; and detecting by FISH analysis the presence of at least one deletion in CDKN2A locus at chromosome region 9p21.3 on said sample and classifying a subject having a deletion in said locus as indicating poor cancer prognosis; and applying a suitable cancer therapy.
  • in situ hybridization assays are well known (e.g., Angerer (1987) Meth. Enzymol 152:649). Generally, in situ hybridization comprises the following major steps: 1) fixation of sample to be analyzed; 2) prehybridization treatment of the sample to increase accessibility of target DNA, and to reduce nonspecific binding; 3) hybridization of the nucleic acid probe to the nucleic acid in the biological sample; 4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and 5) detection of the hybridized nucleic acid fragments.
  • the reagent used in each of these steps and the conditions for use vary depending on the particular application.
  • cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein.
  • the targets e.g., cells
  • the targets are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained.
  • the probes are typically labeled, e.g., with radioisotopes or fluorescent labels. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions.
  • the probe in order to detect even the smallest deletions in the patient samples, covers the smallest overlapping region (12.236 kb) within the CDKN2A gene carrying a deletion, but is not larger than the region ( Figure
  • deletion 12.2 kb The smallest overlapping region of deletion 12.2 kb can also be deducted from the Table 1 showing locations of the CDKN2A deletions in cell line and patient samples.
  • the probe can be a dual color probe, wherein the probe for CDKN2A is labeled with different fluorochrome/hapten than the reference probe.
  • Reference probe is designed to hybridize to e.g. alpha satellite sequences specific to chromosome 9.
  • CDKN2A is preferably within the following base pairs:
  • DNA microarray method is not suitable for patient diagnostics due to high costs.
  • DNA array technology is not suitable for detecting clinically significantly residual disease, wherein the sensitivity should be high enough for detecting one cell out of hundreds of cells.
  • PCR based methods are problematic for detecting deletions in tissue samples since method is likely to give false negative results in samples with clonal heterogeneity or normal cell contamination. Consequently, the method should be sensitive, quantitative and cost effective.
  • the present invention provides for the first time the method for detecting the size of the CDKN2 deletions with variability limits and the precise location of the deletion in the genome.
  • the present invention provides a fast and reliable diagnostic method for clinical use suitable for variable patient samples.
  • the probe should not be too small in size, since the use of small probes in patient material creates problems and may lead to false results.
  • the method is also applicable to patient samples, which do not comprise cells of the cell division phase i.e. so called metaphase cells. In other words the method is also suitable for detecting CDKN2 deletions in interphase nuclei in addition to metaphase nuclei.
  • a high -resolution (44K) array comparative genomic hybridization (CGH) on Ewing sarcoma cell lines and patient samples was carried out in order to screen sizes of 9p21.3 deletions.
  • Microarray CGH analysis revealed 9p21.3 deletions, encompassing the CDKN2A locus, in eight Ewing sarcoma cell line samples and in six patient samples (Example 8). In two cell lines and two patient samples the deletion was less than 190 kb in size. In one cell line sample, a microdeletion of ⁇ 58 kb in size was detected in 9p21.3 harboring CDKN2A locus.
  • the present inventors provide a new and more accurate method for detection of deletions in the CDKN2A locus.
  • the cause could be the LSI pl6/CEP9 Dual Color probe, which is much larger (-190 kb) than the deletion (-58 kb) observed in the IOR/RCH cell line.
  • the telomeric and centromeric ends of the LSI pl6 probe can still hybridise to the area around the CDKN2A microdeletion and create false negative results as seen in FISH analysis.
  • FISH probe is designed based on the smallest overlapping region of deletion within CDKN2A carrying a deletion ( Figure 4; Table 1).
  • the FISH probe is preferably less than 60 kb, more preferably less than 40 kb and more preferably less than 13 kb , most preferably from 5 to 12.5 kb.
  • CDKN2A locus Array CGH analysis of ES tissue samples indicates that sharp homozygous microdeletions in the CDKN2A locus might be a common phenomenon in Ewing sarcoma patient samples. Aberrations in the CDKN2A locus have been described not only in Ewing sarcoma but also in other malignancies, including different types of leukaemia, melanoma, glioma/blastoma, mesothelioma and lymphoma, and gastrointestinal, breast, lung, pancreas, liver, prostate, testis, ovarian, head and neck and bladder cancers (Liggett and Sidransky, 1998; Malumbres and Barbacid, 2001).
  • CDKN2A deletion in Ewing sarcomas as well as in other tumours is clinically important for selecting the best possible treatment for the patient (Wei et al., 2000; Lopez-Guerrero et al., 2001).
  • the problem is that these clinically significant aberrations might not be detected when the size of the microdeletion is so small that FISH probes cannot reveal it. Therefore new and more accurate methods to detect CDKN2A deletions are needed in the diagnosis of common cancers.
  • FISH Fluorescence in situ hybridization
  • the labeled DNA is purified, concentrated, resuspended in hybridization buffer and is hybridized onto targeted sequence on sample slides.
  • FISH method enables microscopically detecting small DNA changes in cells.
  • FISH analysis can also be done for interphase cells, wherein the amount of metaphases is not a restricting factor.
  • FISH analysis is suitable for monitoring response for treatment. Furthermore, provided, that enough sample cells are available the method is also suitable for detecting residual disease.
  • small deletions or microdeletions in CDKN2A are detected with fluorescence in situ hybridization (FISH) method (Andreeff et al. 1999).
  • FISH fluorescence in situ hybridization
  • the probe used in the FISH method covers the smallest overlapping region (12.236 kb) within the CDKN2A gene carrying a deletion, but is not larger than the overlapping region ( Figure 4B).
  • the smallest overlapping region of deletion 12.236 kb can also be deducted from the Table 1 showing locations of the CDKN2A deletions in cell line and patient samples. The same data is presented in Figure 4.
  • CDKN2A gene fragment having the nucleic acid sequence of SEQ ID NO:1 is isolated and purified according to the standard methods disclosed e.g. by Lodish et al. 1999, Birnboim et al. 1979, Miesfield 1999 or Sambrook et al. 1989, 2001.
  • the CDKN2A gene fragment, 12 236 bp in length is amplified with long range PCR reaction using suitable high-fidelity DNA polymerase possessing proofreading properties e.g. LongRange PCR Kit (QIAGEN), Long PCR Enzyme Mix (Fermentas), iProofTM High-Fidelity DNA Polymerase Kit (Bio-Rad) or Expand Long Range dNTPack (Roche Applied Science).
  • the primers used for the PCR reaction are designed to contain a suitable restriction enzyme (RE) cleavage sites that allow RE-cleavage and ligation to a plasmid e.g. pUC18/19 (or universal cloning vector) (Fermentas) or pGEM (Promega, USA) containing, an origin of replication, a gene for antibiotic resistance (e.g. for ampicillin, penicillin, kanamycin, tetracycline or chloramphenicol) and a multiple cloning site (MCS) opened with the same restriction enzymes after purification.
  • a suitable restriction enzyme cleavage sites that allow RE-cleavage and ligation to a plasmid e.g. pUC18/19 (or universal cloning vector) (Fermentas) or pGEM (Promega, USA) containing, an origin of replication, a gene for antibiotic resistance (e.g. for ampicillin, penicillin, kanamycin, tetracycl
  • the ligation is done to lambda phage, which can be used to clone large pieces (10-20 kb) of DNA in between the ends of lambda DNA (e.g. Charon 4A Lambda).
  • the plasmid is inserted into bacteria, e.g. Escherichia coli (e.g. K- 12 strain), by transformation using a standard procedure of the manufacturer or the plasmid or cells.
  • the bacteria is then grown in selective conditions on agar plate, exposed to antibiotics such as ampicillin, penicillin, kanamycin, tetracycline or chlorampenicol as examples, harvested and lysed by alkaline lysis procedure to isolate the plasmid containing CDKN2A gene fragment by mini or large scale preparations (Sambrook et al. 2001 ) or using a kit such as GenEluteTM Plasmid Miniprep Kit or GenEluteTM HP Plasmid Kits (Sigma).
  • CDKN2A DNA fragment is cleaved from plasmid vector by the same restriction enzymes as used above, i.e. enzymes such as EcoRl, BamRl, Hindl ⁇ l, Xhol and run on an agarose gel to check the size of the fragment.
  • the fragment can be sequenced, to validate that the fragment contains a correct sequence.
  • Labeling of the probe is done by nick-translation. After labeling, the DNA is purified according to a standard method (Sambrook et al. 1989, 2001), in order to remove the residual free dye.
  • the samples of a patient which can be paraffin embedded tissue or cryosection or a cell preparation, are pretreated according to the sample material with acid (e.g. HCl) and/or protease such as pepsin in order to improve the DNA hybridization by making the tissue and cells more permeable.
  • acid e.g. HCl
  • protease such as pepsin
  • the sample and the labelled probe are heated to the temperature of 70-80 0 C to denaturate the DNA i.e. to make it single-stranded followed by sequential salt buffer treatments at different treatments e.g. with NaOH .
  • the above mentioned treatments are optimized according to the sample material.
  • the samples on slides are dehydrated in increasing alcohol series, e.g. 70%, 80%, 95%, 99% EtOH.
  • the hybridization is carried out by adding the labeled probe in a suitable buffer solution directly on the sample.
  • the hybridization mixture includes formamide, SSC, dextran sulfate, labeled probe, and blocking agents or congruent ingredients, respectively (Sambrook et al. 1989, 2001).
  • the sample is concealed with the cover slide and sealed with rubber glue.
  • a suitable temperature e.g. to 37°C DNA hybridizes i.e. it returns to double-stranded form.
  • the probe is available in excess, it competes with the original complementary strand of the chromosome and hybridizes to the homologous site in the DNA.
  • the hybridization result is improved by fine-tuning the hybridization and denaturation conditions e.g. temperature, salt concentration and pretreatments.
  • the unspecifically bound probe and background is removed from the preparation by washing followed by dehydration in alcohol series. Washing conditions, as well as denaturation and hybridization conditions are optimized according to the specific probe and sample material.
  • DNA is background stained with DAPI (2-(4-amidinophenyl)-lH -indole-6-carboxamidine), which is a fluorescent stain that binds strongly to genomic DNA and allows detection by fluorescence microscopy.
  • DAPI a fluorescent stain that binds strongly to genomic DNA and allows detection by fluorescence microscopy.
  • Other suitable DNA stains used for fluorescence microscopy include Quinacrine and Hoechst 33258 /33342-stains. Detection is carried out directly with a fluorescence microscope, because the fluorescent signal is detected in the nucleus, where the probe is hybridized. When two fluorescently labeled signals are observed in both alleles of the locus there is no CDK2NA deletion in the sample. If the signal is missing in both of the alleles, i.e. in the homozygous deletion or only one signal is observed in either of the alleles, i.e. in the heterozygous deletion, said DNA region has been deleted from the sample.
  • Genomic DNA was digested and labelled according to the manufacturer's instructions, Agilent protocol version 2.0 for 44K arrays and 4.0 for 244K arrays, as previously described by Tyybakinoja et al. (Tyybakinoja et al., 2006). Briefly, for DNA labeling 10 ⁇ g (44K) or 1.5 ⁇ g (224K) of sample and reference DNA were fragmented with AIu I and Rsa I restriction enzymes (Sigma), followed by purification of 44K array samples using QIAprep Spin Miniprep Kit (Qiagen).
  • Fragmented DNA was then quantitated and visualised using standard agarose gel electrophoresis with 1 ⁇ g of DNA for 44K array samples and 20 ng of DNA for 244K samples (Flash GelTM; Cambrex). 1.5 ⁇ g of tumour and reference genomic DNA were labelled via random priming using the BioPrime array labeling kit (Invitrogen) with Cy5-dUTP and Cy3-dUTP (Perkin-Elmer) dyes for 44K array samples or using Agilent Genomic DNA Labeling Kit (Agilent) for 244K array samples.
  • BioPrime array labeling kit Invitrogen
  • Cy5-dUTP and Cy3-dUTP Perkin-Elmer
  • Agilent Genomic DNA Labeling Kit Agilent Genomic DNA Labeling Kit
  • the IOR/RCH cell line obtained from IOR, was cultured at +37 0 C, 5% CO 2 atmosphere in
  • Iscove's Modified Dulbecco's Medium with 10% heat inactivated fetal bovine serum and Pen/Strep (all manufactured by Gibco). The cells were subcultured every 3-4 days by diluting them (1 :4) to fresh culture medium. Cells were prepared according to standard procedures. In brief, colcemid (KaryoMAX; Gibco) was added to concentration of 0.1 ⁇ g/ml in the cell culture suspension when cells were in their exponential growth phase. After 16h colcemid incubation, cells were treated with hypotonic solution (56 mmol/1 KCL) followed by three fixations in fresh 3:1 methanol/acetic acid solution. Nuclear suspension was then dropped on glass slides followed by drying.
  • FISH analysis was performed using a commercial CDKN2A -gene probe (LSI pl6/CEP9 Dual Color probe; Vysis) according to the manufacturer's instructions.
  • the size of 9p21 LSI pl6 spectrum orange probe is -190 kb, covering genomic markers D9S1749, D9S1747, D9S1748 and D9S1752.
  • CEP9 spectrum green probe covers chromosome bands 9pl 1-ql 1.
  • Homozygous or heterozygous deletion of the CDKN2A gene is represented by loss of one or two orange pl6 signal(s), respectively, while retaining two green centromeric signals from chromosome 9.
  • Gain in the CDKN2A gene is represented by gain of orange pl6 signal. At least 20 nuclei were studied per sample to confirm the results and the experiment was performed three times.
  • Pre-designed TaqMan probe and primer sets for target gene were chosen from an on-line catalogue (Applied Biosystems), CDKN2A: Assay ID Hs00233365_ml. Once selected, the sets were factory- loaded into the 384 wells of TaqMan Low Density Arrays (LDAs). Array format was customized on-line with two replicates per target gene. Expression level of target gene was normalized to expression level of GAPDH gene. Samples were analyzed using the 7900HT system with a TaqMan LDA Upgrade (Applied Biosystems), according to the manufacturer's instructions. In short, 100 ng of single-stranded cDNA was combined with water and TaqMan Universal PCR Master Mix, following by loading to the port.
  • LDAs TaqMan Low Density Arrays
  • Thermal cycling conditions were as follows: 50 0 C for 2 min, 94°C for 10 min, 97°C for 30s, and 59.7°C for 1 min.
  • Gene expression values were calculated based on the ⁇ Ct method, where a pool of normal muscle (from three patients) was designated the calibrator, through which all other samples were analyzed. Briefly, ⁇ Ct represents the threshold cycle (Ct) of the target minus that of GAPDH and ⁇ Ct represents the ⁇ Ct of each target minus that of the calibrator.
  • FISH analysis was performed using a commercial probe (LSI pl6/CEP9 Dual Color probe, Vysis). The results from this analysis are shown in Figure 3. Two green signals and three red signals were detected, indicating that the CDKN2A locus was gained. This result was unexpected because the array CGH results of the same sample unambiguously showed that the CDKN2A locus was homozygously deleted.
  • CDKN2A locus can also be inactivated in cancer cells by promoter hypermethylation or by point/frameshift mutations in coding sequence (Liggett and Sidransky, 1998).
  • inactivation events of this kind are rare and homozygous deletion seems to the principal model in CDKN2A inactivation, at least in ES (Tsuchiya et al., 2000; Huang et al., 2005).
  • CDKN2A locus Array CGH analysis of two ES tissue samples (13 and 14) indicates that sharp homozygous microdeletions in the CDKN2A locus might be a common phenomenon in Ewing sarcoma patient samples. Aberrations in the CDKN2A locus have been described not only in Ewing sarcoma but also in other malignancies, including different types of leukaemia, melanoma, glioma/blastoma, mesothelioma and lymphoma, and gastrointestinal, breast, lung, pancreas, liver, prostate-, testis, ovarian-, head and neck and bladder cancers (Liggett and Sidransky, 1998; Malumbres and Barbacid, 2001).
  • CDKN2A deletion in Ewing sarcomas as well as in other tumours might, however, be clinically important for selecting the best possible treatment for the patient (Wei et al., 2000; Lopez-Guerrero et al., 2001).
  • the problem is that these clinically significant aberrations might not be detected when the size of the microdeletion is so small that the commercially available FISH probes cannot reveal it. Therefore new and more accurate methods to detect CDKN2A deletions are needed in the diagnosis of common cancers.
  • FISH fluorescence in situ hybridization
  • CDKN2A gene fragment having the nucleic acid sequence of SEQ ID NO:1 is isolated using restriction enzymes and purified according to a standard method by Sambrook et al. 1989.
  • the CDKN2A gene fragment, 12.236 kb in length, is amplified with long range PCR reaction using high-fidelity DNA polymerase (LongRange PCR Kit (QIAGEN)).
  • the primers used for the PCR reaction are designed to contain restriction enzyme (RE) cleavage sites that allow RE-cleavage and ligation to a plasmid (pUC18, Fermentas), multiple cloning site (MCS) opened with the same RE- enzymes after purification.
  • RE restriction enzyme
  • the plasmid is inserted into bacteria, Escherichia coli K- 12 strain, by transformation according to the method by the manufacturer of the cells. The bacteria is then grown in selective conditions, exposed to antibiotics, harvested and lysed by alkaline lysis procedure to isolate the plasmid containing CDKN2A gene fragment (Sambrook et al. 1989). After plasmid purification, CDKN2A DNA fragment is cleaved from plasmid vector by the same restriction enzymes as used above and run on an agarose gel to check the size of the fragment. The fragment is sequenced to validate that fragment contains a correct sequence.
  • Labeling of the intact CDKN2A fragment is done by nick-translation (Sambrook et al. 1989). After labeling, the DNA is purified, in order to remove the residual free dye (Sambrook et al. 1989). The sample of a patient is pretreated with acid (HCl) and protease (pepsin) in order to improve the DNA hybridization by making the tissue and cells more permeable.
  • HCl acid
  • pepsin protease
  • the sample and a labelled probe are heated to 70-80 0 C to denaturate the DNA i.e. to make it single-stranded followed by sequential salt buffer (NaOH) treatments .
  • NaOH salt buffer
  • the samples on slides are dehydrated in increasing alcohol series (70%, 80%, 95%, 99% EtOH).
  • the hybridization is carried out by adding the labeled probe in a buffer solution containing formamide, SSC, dextran sulfate and blocking agents (Sambrook et al. 1989) directly on the sample.
  • the sample is concealed with the cover slide and sealed with rubber glue. The temperature is decreased to 37°C to hybridization.
  • DAPI 3-(4-amidinophenyl)-lH-indole-6-carboxamidine
  • CDKN2A, CDKN2B, and MTAP gene dosage permits precise characterization of mono- and bi-allelic 9p21 deletions in childhood acute lymphoblastic leukemia. Genes Chromosomes Cancer 2003;l :44-57.
  • INK4a/ARF a multifunctional tumor suppressor locus. Mutat Res 2005;576:22-38.
  • CDK inhibitors positive and negative regulators of Gl -phase progression. Genes Dev 1999;13:1501-12.

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Abstract

La présente invention porte sur un procédé pour améliorer les pronostics et diagnostics du cancer humain, sur une sonde spécifique pour le locus CDKN2A au niveau de la région de chromosome 9p 21.3, utilisée dans ledit procédé, et sur l'utilisation du procédé pour la préparation d'un outil de diagnostic pour analyser les malignités ou aberrations provoquées par des délétions dans les régions spécifiques au niveau du chromosome 9 et pour optimiser la conception d'essais cliniques pour sélectionner une thérapie du cancer, et sur le procédé pour sélectionner une thérapie du cancer pour le traitement du cancer.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
US5801236A (en) * 1994-03-18 1998-09-01 Myriad Genetics, Inc. Probes for MTS1 gene and polynucleotides encoding mutant MTS1 genes
US6689561B1 (en) * 1994-04-14 2004-02-10 The Regents Of The University Of California Tumor suppressor gene and methods for detection of cancer, monitoring of tumor progression and cancer treatment
US6870037B1 (en) * 1995-07-03 2005-03-22 Arch Development Corporation Methylthioadenosine phosphorylase compositions and methods of use in the diagnosis and treatment of proliferative disorders

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US5801236A (en) * 1994-03-18 1998-09-01 Myriad Genetics, Inc. Probes for MTS1 gene and polynucleotides encoding mutant MTS1 genes
US6689561B1 (en) * 1994-04-14 2004-02-10 The Regents Of The University Of California Tumor suppressor gene and methods for detection of cancer, monitoring of tumor progression and cancer treatment
US6870037B1 (en) * 1995-07-03 2005-03-22 Arch Development Corporation Methylthioadenosine phosphorylase compositions and methods of use in the diagnosis and treatment of proliferative disorders

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Title
NAMAZIE, A ET AL.: "Cyclin D1 amplification and p16(MTS1/CDK41) deletion correlate with poor prognosis in head and neck tumors.", LARYNGOSCOPE., vol. 112, no. 3, March 2002 (2002-03-01), pages 472 - 481, XP002725595, DOI: doi:10.1097/00005537-200203000-00013 *
NAMAZIE, A ET AL.: "Fluorescence in situ hybridization for detecting TP16 MTS1/CDK41 gene deletions in squamous cell carcinoma of the head and neck.", CANCER GENETICS AND CYTOGENETICS., vol. 141, no. 1, February 2003 (2003-02-01), pages 49 - 55 *
SAVOLA, S ET AL.: "Microdeletions in 9p21.3 induce false negative results in CDKN2A FISH analysis of Ewing sarcoma", CYTOGENETIC AND GENOME RESEARCH, vol. 119, no. 1-2, 14 December 2007 (2007-12-14), pages 21 - 26, Retrieved from the Internet <URL:10.1159/000109614> [retrieved on 20080611] *
USVASALO, A ET AL.: "CDKN2A deletions in acute lymphoblastic leukemia of adolescents and young adults: an array CGH study.", LEUKEMIA RESEARCH, vol. 32, no. ISS.8, 6 March 2008 (2008-03-06), pages 1228 - 1235, Retrieved from the Internet <URL:10.1016/j.leukres.2008.01.014> [retrieved on 20080611] *

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