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WO2012038744A2 - Détection de mutations - Google Patents

Détection de mutations Download PDF

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Publication number
WO2012038744A2
WO2012038744A2 PCT/GB2011/051774 GB2011051774W WO2012038744A2 WO 2012038744 A2 WO2012038744 A2 WO 2012038744A2 GB 2011051774 W GB2011051774 W GB 2011051774W WO 2012038744 A2 WO2012038744 A2 WO 2012038744A2
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Prior art keywords
clear cell
mutation
pbrml
gene
cell
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WO2012038744A3 (fr
Inventor
Andrew Futreal
Michael Stratton
Bin Tean Teh
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Genome Research Ltd
Van Andel Research Institute
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Genome Research Ltd
Van Andel Research Institute
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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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the field of oncology, in particular to molecular markers associated with renal and other types of cancer.
  • ccRCC clear cell carcinoma
  • the present invention provides a method for detecting a mutation associated with renal cancer in a subject, comprising screening a test sample derived from the subject for the presence of one or more mutations in a PBRM1 (polybromo-1) gene or a product thereof.
  • PBRM1 polybromo-1
  • the mutation is a truncation mutation.
  • the mutation comprises a PBRM1 mutation as defined in Table 2, 4 or 5 herein.
  • the test sample comprises a renal tissue sample which is suspected to be cancerous or at risk of cancer, and presence of the mutation is indicative of renal cancer or an increased risk of renal cancer in the subject.
  • the method further comprises screening a control sample derived from a normal tissue of the subject for the presence of the mutation, wherein presence of the mutation in the test sample and absence of the mutation in the control sample is indicative of a somatic mutation associated with renal cancer or an increased risk of renal cancer in the subject.
  • the method comprises obtaining nucleic acids from the sample, and detecting one or more mutations in a polypeptide-encoding nucleic acid sequence of the PBRMl gene.
  • the method comprises screening the sample with a ligand which binds selectively to a mutant polypeptide product of the PBRMl gene.
  • the cancer is clear cell renal carcinoma.
  • the method further comprises screening the sample for one or more mutations in a VHL and/or SETD2 gene or a product thereof.
  • the present invention provides an isolated nucleic acid encoding at least a portion of a PBRMl gene product, wherein the nucleic acid comprises a PBRMl mutation as defined in Table 2, 4 or 5 herein.
  • the present invention provides an isolated nucleic acid which is complementary to, or hybridises specifically to, a mutant nucleic acid as defined above. In a further aspect, the present invention provides an isolated nucleic acid primer which directs specific amplification of a mutant nucleic acid as defined above.
  • the present invention provides an isolated polypeptide comprising at least a portion of a product of a PBRMl gene, wherein the polypeptide comprises a PBRMl mutation as defined in Table 2, 4 or 5 herein.
  • the present invention provides a ligand which binds selectively to a mutant polypeptide as defined above.
  • the ligand is an antibody.
  • PBRMl somatic mutations Representation of PBRMl transcript with boxes BR1-BR6, BAH1-2 and HMG indicating the positions of the bromodomains 1-6, bromo-adjacent homology domains and high-mobility group domain, respectively. Relative positions of mutations are indicated by symbols. Stars - nonsense, dots - missense, red triangles - frameshift deletions, black triangles - frameshift insertions and green triangles - in-frame deletions. Splice-site mutations are not depicted.
  • the somatic set is significantly different from the null set (p-value 0.01). They have a higher negative mean score and are thus predicted to be more deleterious on average.
  • Figure 3
  • Pbrml is frequently mutated in a mouse model of pancreatic cancer.
  • a conditional allele of K- Ras G12D and Pdxl-Cre were combined with a conditional Sleeping Beauty transposase driver and the T20nc tg transposon donor allele 29 .
  • Expression of Cre results in expression of K- Ras GI2D and transposon mobilization within the epithelial compartment of the pancreas.
  • Knockdown f PBRMl expression in RCC cell lines (A) Verification of PBRM 1 knockdown by quantitative PCR in renal cancer cell lines. (B)Silencing PBRMl increased the proliferation of ACHN and 786-0 with wild type PBRMl, but not A704 with a homozygous PBRMl trancating mutation. Data represent means of triplicate experiments with standard deviation, p ⁇ 0.0 ⁇ . (C) Knockdown of PBRMl enhanced colony formation in SN12C cells. Data represent means of triplicate experiments with standard deviation, jcO.Ol. (D) Knockdown of PBRMl enhanced cell migration in 786-0, SN12C and TK10 cells.
  • E Gene sets that are most significantly deregulated following PBRMl knockdown in three RCC cell lines using curated gene sets obtained from MSigDB (http://www.broadinstitute.org/gsea/msigdb/) and additional curated gene sets obtained from the PGSEA package (see Example for details).
  • Figure 5 shows the nucleotide sequence of the human PBRMl gene (NM_018313.4, SEQ ID NO:l).
  • Figure 6 shows the amino acid sequence of the human BAF180 protein (NP_060783.3, SEQ ID NO:2).
  • the present invention relates to detecting mutations in a polybromo-1 ⁇ PBRMl) gene or a product thereof.
  • the nucleotide sequence of the human PBRMl gene is given in NCBI database accession no. NM_018313.4, and is shown in SEQ ID NO:l (Fig. 5).
  • the amino acid sequence of the human BAF180 (BRG1 -associated factor 180) protein, which is a product of the human PBRMl gene, is given in NCBI database accession no. NPJ360783.3 and is shown in SEQ ID NO:2 (Fig. 6).
  • Embodiments of the present inventions may involve detecting mutations in any of the above sequences.
  • PBRMl gene or a product thereof preferably includes nucleic acids or polypeptides which have at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:l or SEQ ID NO:2, preferably over at least 20, 50, 100, 500 or 1000 residues of the sequence or over the entire length of the sequence.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage (%) homology (i.e. sequence identity) between two or more sequences.
  • Percentage homology can be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • a sequence comparison method may be used which is designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
  • the method may assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system.
  • % homology can be measured in terms of identity
  • the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • the present method may comprise detecting one or more mutations in a PBRMl gene or a product thereof. Mutations include addition, deletion or substitution of one or more nucleotides or amino acid residues, e.g. in the sequence of SEQ ID NO: 1 or 2. In a preferred embodiment, the mutation results in a truncation of the expressed PBRMl gene product (BAF180 protein), i.e. the mutation is a truncating mutation.
  • the mutation in PBRMl is a naturally occurring mutation, i.e. the mutation has not been intentionally induced in cell s or tissue by the application of carcinogens or other tumorigenic factors.
  • the mutations identified herein accurately reflect natural tumorigenesis in human tissues in vivo and are suitable for diagnostic use.
  • the mutation is a somatic mutation.
  • somatic it is meant a mutation which is not transmitted through the germ line of an organism, i.e. the mutation occurs in somatic tissues of the organism.
  • a somatic mutation is one which is determined to be somatic though normal/tumour paired sample analysis.
  • somatic mutations can be identified as mutations found in a sample derived from a suspected cancer tissue of the subject, but not found in normal tissue from the same subject.
  • the method may comprise identifying any mutation in a PBRMl gene or a product thereof, including any of those disclosed herein (e.g. a mutation in a PBRMl gene or a PBRMl gene product as disclosed in Table 2, 4 or 5 herein). All amino acid and nucleotide numbering used herein starts from amino acid +1 of the in PBRMl gene product (BAF180 protein) or the first ATG of the nucleotide sequence encoding it.
  • the present invention relates to isolated nucleic acids and polypeptides comprising a PBRMl mutation as disclosed herein, e.g. as defined in Table 2, 4 or 5.
  • isolated nucleic acid and polypeptides may comprise fragments, variants or homologues of a PBRMl sequence (e.g. SEQ ID NO: l or 2).
  • PBRMl sequence e.g. SEQ ID NO: l or 2
  • such isolated nucleic acids and polypeptides show at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: l or SEQ ID NO:2, preferably over at least 20, 50, 100, 500 or 1000 residues of the sequence or over the entire length of the sequence.
  • a portion or fragment of a polypeptide in accordance with the invention is a polypeptide fragment which encompasses the mutant amino acid(s) described in accordance with the invention.
  • the fragment can be any length up to the full length of PBRMl (BAF180) polypeptide; it thus encompasses PBRMl (BAF180) polypeptides which have been truncated by a few amino acids, as well as shorter fragments.
  • the polypeptide comprises at least 10, at least 20, at least 50, at least 100, at least 500 or at least 1000 amino acids of SEQ ID NO:2, provides that the polypeptide comprises a mutation as defined herein.
  • fragments may be between about 5 and about 1580 amino acids in length, e.g.
  • fragments may be useful for immunisation of animals to raise antibodies.
  • fragments of polypeptides according to the invention advantageously comprise at least one antigenic determinant (epitope) characteristic of mutant PBRMl (BAF180) as described herein. Whether a particular polypeptide fragment retains such antigenic properties can readily be determined by routine methods known in the art. Peptides composed of as few as six amino acid residues ore often found to evoke an immune response.
  • a "nucleic acid" of the present invention may be, for example, a nucleic acid which encodes a mutant human PBRMl (BAF180) polypeptide as described above.
  • the nucleic acid may comprise at least 10, at least 20, at least 50, at least 100, at least 500 or at least 1000 nucleotides of SEQ ID NO: l , provided that the nucleic acid comprises a mutation as described herein.
  • the present invention also provides polynucleotides complementary to a mutant PBRMl (BAF180)-encoding nucleic acid, as well as polynucleotides which hybridise specifically to a mutant PBRMl (BAF180)-encoding nucleic acid.
  • hybridise specifically it is typically meant that the polynucleotide is capable of hybridising to the mutant nucleic acid (i.e. a nucleic acid sequence comprising a PBRMl mutation as defined herein) but not to a non-mutant nucleic acid (e.g. a nucleic acid sequence comprising the sequence of SEQ ID NO:l or a fragment thereof) under the same conditions.
  • the polynucleotide may hybridise specifically to a nucleic acid mutation as defined in Table 2, 4 or 5.
  • the invention provides polynucleotides capable of hybridising, under stringent hybridisation conditions, to a mutant PBRMl nucleic acid, or the complement thereof.
  • Stringent hybridisation conditions refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulphate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.l SSC at about 65°C.
  • nucleic acids as referred to herein, are generally natural nucleic acids found in nature, the term can include within its scope modified, artificial nucleic acids having modified backbones or bases, as are known in the art.
  • an "isolated" polypeptide or nucleic acid refers to material removed from its original environment (for example, the natural environment in which it occurs in nature), and thus is altered by the hand of man from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • the methods of the present invention may comprise detecting mutations in PBRM1 at the gene or protein level. Any known methods for detecting specific nucleic acid or polypeptide sequences may be used. Thus the methods of the invention can be based on detection of mutations in genomic DNA, as well as transcripts and proteins. In some embodiments mutations in genomic DNA may be confirmed by analysis of transcripts and/or polypeptides, in order to ensure that the detected mutation is indeed expressed in the subject.
  • the screening method of the present invention is performed on a test sample.
  • the sample may be any type of sample derived from the subject, provided that it permits the identification of PBRM1 mutations in the subject.
  • the sample comprises DNA derived from the subject, e.g. genomic or cDNA from the subject.
  • the sample may comprise a tissue or cellular sample or a purified DNA preparation from the subject.
  • the test sample may comprise a protein sample derived from the subject. Methods for the isolation of genomic DNA or protein, or the preparation of cDNA from tissue samples are well-known in the art.
  • the test sample comprises a tissue sample from the kidney, e.g. a renal tissue sample which is suspected to be cancerous or at risk of cancer.
  • nucleic acids e.g. in genomic or cDNA
  • the present invention provides nucleic acids (e.g. oligonucleotide primers and probes) which selectively hybridize to a mutant PBRMl polynucleotide sequence.
  • the nucleic acid may be an oligonucleotide primer or probe which comprises a region which is complementary to the mutant sequence.
  • a skilled person can easily design appropriate primers or probes based on a knowledge of the mutant sequence.
  • mutant nucleic acid sequences may be detected directly in a DNA sample derived from a subject.
  • the method involves first enriching the sample for PBRMl gene sequences.
  • the method may involve a step of selectively amplifying a PBRMl gene locus using appropriate primers, followed by a further amplification step using mutation-specific primers or probes.
  • the products of the locus-specific amplification step are purified in a DNA purification step before the mutation- specific detection step.
  • the present invention provides nucleic acid primers which direct specific amplification of mutant PBRMl sequences.
  • the primer is capable of amplifying the mutant nucleic acid (i.e. a nucleic acid sequence comprising a PBRMl mutation as defined herein) but not a non-mutant nucleic acid (e.g. a nucleic acid sequence comprising the sequence of SEQ ID NO:l or a fragment thereof) under the same conditions.
  • the primer may hybridize specifically to the mutant nucleic acid.
  • the primer is typically extended (e.g. using known methods such as PCR) only when annealed to the mutant nucleotide sequence (template).
  • the primer comprises a sequence complementary to a mutant PBRMl sequence.
  • the primer binds the complementary mutant PBRMl sequence and produces an amplified product
  • the PCR product can be detected by standard techniques. If only a non-mutant sequence is present, no PCR product is detected.
  • the nature of the primer is not particularly limited, provided that it is capable of specifically hybridising to a mutant PBRMl sequence.
  • the length of the primer is preferably 5 to 50 nucleotides, more preferably 10 to 50 nucleotides, more preferably 15 to 30 nucleotides, e.g. 17 to 23 nucleotides.
  • Suitable primers may be designed according to standard techniques known to those skilled in the art for selecting primers for polymerase reactions, such as for amplification of DNA by the polymerase chain reaction (PCR).
  • the primer may comprise, or be specifically complementary to, at least 10, at least 15 or at least 20 nucleotides of a mutant PBRM1 sequence, wherein the mutant PBRM1 sequence comprises a mutant of sequence of SEQ ID NO:l as defined in Table 2, 4 or 5 herein.
  • an aqueous solution of the primer is added to a DNA sample.
  • Hybridisation conditions are then selected so that the primer hybridises selectively to the mutant sequence, according to criteria well known to those skilled in the art.
  • An appropriate temperature and salt content for hybridisation needs to be selected according to the length of the oligonucleotide primer and its G-C content, amongst other things (see Old & Primrose (1994), Principles of Gene Manipulation, Blackwell Science and Maniatis et al. (1992), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • the hybridisation temperature should be close to the melting temperature (T m ) of the primer.
  • Tm is defined as the temperature at which the primer and its target are 50% dissociated.
  • the hybridisation temperature should be within 2°C of T m .
  • the method utilizes a set of primer pairs designed for specifically detecting individual PBRM1 mutations.
  • the PCR reactions accomplished with these primers produce well defined DNA fragments of different length if their respective mutant sequences are present in the sample.
  • Control primers for use as an internal standard e.g. for amplifying the human globin or human growth hormone gene
  • the PCR reaction products may be detected on an electrophoretic gel, e.g. an agarose gel by dyeing the double stranded DNA with ethidium bromide and exposure to ultraviolet light. The gel may be documented by photography and interpreted. According to some embodiments of the present invention, only one primer in a pair is specific for the mutant PBRM1 sequence.
  • Oligonucleotide probes may be used in combination with well-known hybridisation assays to detect mutant PBRM1 nucleic acid sequences.
  • the oligonucleotide probe may be attached to a solid support.
  • a plurality of oligonucleotide probes may be attached to a solid support in the form of an array, e.g. a DNA micro-array.
  • Oligonucleotide arrays may be prepared, for example, by in situ combinatorial oligonucleotide synthesis or by conventional synthesis followed by on-chip immobilization of the oligonucleotide onto the solid support.
  • the solid support may be, for example, a glass slide.
  • different probes may be attached to individual beads or microspheres, e.g. LuminexTM microspheres.
  • Oligonucleotide probes suitable for immobilization on an array may be of any suitable length provided that they are specific for a mutant PBRM1 gene sequence. Typically oligonucleotide probes may be 10 to 50, 15 to 30, 17 to 23 or about 20 nucleotides in length. In one embodiment the oligonucleotide probe may comprise, or be specifically complementary to, at least 10, at least 15 or at least 20 nucleotides of a mutant PBRM1 sequence, wherein the mutant PBRM1 sequence comprises a mutant of sequence of SEQ ID NO:l as defined in Table 2, 4 or 5 herein. Preferably an array comprises at least 10, 30, 50, 100, 200, 500, 1000, 10,000 or 100,000 different oligonucleotide probes. A single array may detect a plurality of PBRM1 mutant sequences and/or other nucleic acids, e.g. cancer-related mutations in other genes.
  • nucleic acid sequences may be detected by techniques based on mobility shift in amplified nucleic acid fragments. Chen et al, Anal Biochem 1996 Jul 15;239(l):61-9, describe the detection of single-base mutations by a competitive mobility shift assay. Moreover, assays based on the technique of Marcelino et al, BioTechniques 26(6): 1134-1148 (June 1999) are available commercially.
  • capillary heteroduplex analysis may be used to detect the presence of mutations based on mobility shift of duplex nucleic acids in capillary systems as a result of the presence of mismatches.
  • Amplification reactions are nucleic acid reactions which result in specific amplification of target nucleic acids over non-target nucleic acids.
  • the polymerase chain reaction (PCR) is a well known amplification reaction.
  • Many amplification methods rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self-sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned.
  • the amplification according to the invention is an exponential amplification, as exhibited by for example the polymerase chain reaction.
  • amplification methods have been described in the literature, for example, general reviews of these methods in Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10: 1, 54-55 (1990).
  • amplification methods can be used in the methods of the invention, and include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridisation, Qbeta bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridisation.
  • Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
  • PCR is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202.
  • PCR consists of repeated cycles of DNA polymerase generated primer extension reactions.
  • the target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridised. These oligonucleotides become primers for use with DNA polymerase.
  • the DNA is copied by primer extension to make a second copy of both strands. By repeating the cycle of heat denaturation, primer hybridisation and extension, the target DNA can be amplified a million fold or more in about two to four hours.
  • PCR is a molecular biology tool, which must be used in conjunction with a detection technique to determine the results of amplification.
  • An advantage of PCR is that it increases sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in approximately 4 hours.
  • PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., (1994), Gynaecologic Oncology, 52: 247- 252).
  • Self-sustained sequence replication is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874). Enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation. RNase H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplifications of 106 to 109 have been achieved in one hour at 42 °C.
  • Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridise to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated.
  • RNA replicase for the bacteriophage Qp which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6: 1197.
  • the target DNA is hybridised to a primer including a T7 promoter and a QP 5' sequence region.
  • reverse transcriptase generates a cDNA connecting the primer to its 5' end in the process.
  • the resulting heteroduplex is heat denatured.
  • a second primer containing a QP 3' sequence region is used to initiate a second round of cDNA synthesis.
  • T7 RNA polymerase then transcribes the double- stranded DNA into new RNA, which mimics the QP. After extensive washing to remove any unhybridised probe, the new RNA is eluted from the target and replicated by QP replicase. The latter reaction creates 107 fold amplification in approximately 20 minutes. Alternative amplification technology can be exploited in the present invention.
  • rolling circle amplification (Lizardi et al, (1998) Nat Genet 19:225) is an amplification technology available commercially (RCATTM) which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions.
  • RCATTM rolling circle amplification technology available commercially
  • RCAT 12 amplification occurs via DNA strand displacement and hyperbranching to generate 10 or more copies of each circle in 1 hour. If a single primer is used, RCAT generates in a few minutes a linear chain of thousands of tandemly linked DNA copies of a target covalently linked to that target.
  • SDA strand displacement amplification
  • SDA comprises both a target generation phase and an exponential amplification phase.
  • target generation double-stranded DNA is heat denatured creating two single-stranded copies.
  • a series of specially manufactured primers combine with DNA polymerase (amplification primers for copying the base sequence and bumper primers for displacing the newly created strands) to form altered targets capable of exponential amplification.
  • the exponential amplification process begins with altered targets (single-stranded partial DNA strands with restricted enzyme recognition sites) from the target generation phase.
  • An amplification primer is bound to each strand at its complementary DNA sequence.
  • DNA polymerase then uses the primer to identify a location to extend the primer from its 3' end, using the altered target as a template for adding individual nucleotides.
  • the extended primer thus forms a double-stranded DNA segment containing a complete restriction enzyme recognition site at each end.
  • a restriction enzyme is then bound to the double stranded DNA segment at its recognition site.
  • the restriction enzyme dissociates from the recognition site after having cleaved only one strand of the double-sided segment, forming a nick.
  • DNA polymerase recognises the nick and extends the strand from the site, displacing the previously created strand.
  • the recognition site is thus repeatedly nicked and restored by the restriction enzyme and DNA polymerase with continuous displacement of DNA strands containing the target segment.
  • SSCP Single Stranded Conformational Polymorphism
  • SCCP detection is based on the aberrant migration of single stranded mutated DNA compared to reference DNA during electrophoresis. Mutation produces conformational change in single stranded DNA, resulting in mobility shift. Fluorescent SCCP uses fluorescent-labelled primers to aid detection. Reference and mutant DNA are thus amplified using fluorescent labelled primers. The amplified DNA is denatured and snap-cooled to produce single stranded DNA molecules, which are examined by non-denaturing gel electrophoresis.
  • SSCP Single Stranded Conformational Polymorphism
  • Chemical mismatch cleavage is based on the recognition and cleavage of DNA mismatched base pairs by a combination of hydroxylamine, osmium tetroxide and piperidine.
  • CMC Chemical mismatch cleavage
  • both reference DNA and mutant DNA are amplified with fluorescent labelled primers.
  • the amplicons are hybridised and then subjected to cleavage using osmium tetroxide, which binds to an mismatched T base, or hydroxylamine, which binds to mismatched C base, followed by Piperidine which cleaves at the site of a modified base. Cleaved fragments are then detected by electrophoresis.
  • RFLPs restriction fragment polymorphisms
  • SNPs single nucleotide polymorphisms
  • PIRA-PCR primer-induced restriction analysis PGR
  • Primers for PIRA-PCR which introduce suitable restriction sites can be designed by computational analysis, for example as described in Xiaiyi et ah, (2001) Bioinformatics 17:838-839.
  • the present invention provides for the detection of PBRMl mutations at the RNA level.
  • Typical assay formats utilising ribonucleic acid hybridisation include nuclear run-on assays, RT-PCR and RNase protection assays (Melton et al, Nuc. Acids Res. 12:7035). Methods for detection which can be employed include radioactive labels, enzyme labels, chemiluminescent labels, fluorescent labels and other suitable labels.
  • a polypeptide encoded by a mutant PBRMl gene is detected, e.g. the method involves detecting a mutant BAF180 protein. Proteins can be detected by protein gel assay, antibody binding assay, or other detection methods known in the art.
  • mutant PBRMl (BAF180) polypeptides can be detected by differential mobility on protein gels, or by other size analysis techniques such as mass spectrometry, in which the presence of mutant amino acids can be determined according to molecular weight. Peptides derived from mutant polypeptides, in particular, as susceptible to differentiation by size analysis.
  • the detection means is sequence-specific, such that a particular point mutation can accurately be identified in the mutant polypeptide.
  • polypeptide or RNA molecules can be developed which specifically recognise mutant PBRMl (BAF180) polypeptides in vivo or in vitro.
  • RNA aptamers can be produced by SELEX.
  • SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. It is described, for example, in U.S. patents 5654151, 5503978, 5567588 and 5270163, as well as PCT publication WO 96/38579, each of which is specifically incorporated herein by reference.
  • the SELEX method involves selection of nucleic acid aptamers, single-stranded nucleic acids capable of binding to a desired target, from a library of oligonucleotides.
  • the SELEX method includes steps of contacting the library with the target under conditions favourable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched library of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
  • SELEX is based on the principle that within a nucleic acid library containing a large number of possible sequences and structures there is a wide range of binding affinities for a given target.
  • a nucleic acid library comprising, for example a 20 nucleotide randomised segment can have 4 20 structural possibilities. Those which have the higher affinity constants for the target are considered to be most likely to bind.
  • the process of partitioning, dissociation and amplification generates a second nucleic acid library, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favour the best ligands until the resulting library is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands.
  • Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle.
  • the iterative selection/amplification method is sensitive enough to allow isolation of a single sequence variant in a library containing at least 10 14 sequences. The method could, in principle, be used to sample as many as about 10 different nucleic acid species.
  • the nucleic acids of the library preferably include a randomised sequence portion as well as conserved sequences necessary for efficient amplification. Nucleic acid sequence variants can be produced in a number of ways including synthesis of randomised nucleic acid sequences and size selection from randomly cleaved cellular nucleic acids.
  • variable sequence portion can contain fully or partially random sequence; it can also contain subportions of conserved sequence incorporated with randomised sequence. Sequence variation in test nucleic acids can be introduced or increased by mutagenesis before or during the selection/amplification iterations and by specific modification of cloned aptamers.
  • PBRM1 (BAF180) polypeptides or peptides derived therefrom can be used to generate antibodies for use in the present invention.
  • the PBRM1 (BAF180) peptides used preferably comprise an epitope which is specific for a mutant PBRM1 (BAF180) polypeptide in accordance with the invention.
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 and, most preferably, between about 15 to about 30 amino acids.
  • Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues in length.
  • Antibodies can be generated using antigenic epitopes of PBRM1 (BAF180) polypeptides according to the invention by immunising animals, such as rabbits or mice, with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections can be needed, for instance, at intervals of about two weeks, to provide a useful titre of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titre of anti-peptide antibodies in serum from an immunised animal can be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • the PBRMl (BAF180) polypeptides disclosed herein, and immunogenic and/or antigenic epitope fragments thereof can be fused to other polypeptide sequences.
  • the polypeptides can be fused with immunoglobulin domains.
  • Chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins have been shown to possess advantageous properties in vivo (see, for example, EP 0394827; Traunecker et al., (1988) Nature, 331 : 84-86).
  • Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (such as insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, for example, WO 96/22024 and WO 99/04813).
  • mutant polypeptides disclosed herein can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • HA hemagglutinin protein
  • the invention provides immunoglobulins (e.g. antibodies) which specifically recognise PBRM1 (BAF180) mutants as described herein.
  • An "immunoglobulin” is one of a family of polypeptides which retain the immunoglobulin fold characteristic of immunoglobulin (antibody) molecules, which contains two ⁇ sheets and, usually, a conserved disulphide bond.
  • Members of the immunoglobulin superfamily are involved in many aspects of cellular and non-cellular interactions in vivo, including widespread roles in the immune system (for example, antibodies, T-cell receptor molecules and the like), involvement in cell adhesion (for example the ICAM molecules) and intracellular signalling (for example, receptor molecules, such as the PDGF receptor).
  • the present invention is preferably applicable to antibodies, which are capable of binding to target antigens with high specificity.
  • Antibodies can be whole antibodies, or antigen-binding fragments thereof.
  • the invention includes fragments such as Fv and Fab, as well as Fab' and F(ab') 2 , and antibody variants such as scFv, single domain antibodies, Dab antibodies and other antigen-binding antibody-based molecules.
  • Antibodies as described herein are especially indicated for diagnostic applications. Accordingly, they can be altered antibodies comprising an effector protein such as a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in vivo. Such labels can be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient.
  • chimeric antibodies can be constructed in order to decrease the iminunogenicity thereof in diagnostic or therapeutic applications.
  • immunogenicity can be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [EP 0 239 400; Riechmann, L. et al., Nature, 332, 323-327, 1988; Verhoeyen M. et al, Science, 239, 1534-1536, 1988; Kettleborough, C. A. et al, Protein Engng., 4, 773-783, 1991; Maeda, H.
  • Antibodies as described herein can be produced in cell culture. Recombinant DNA technology can be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system optionally secretes the antibody product, although antibody products can be isolated from non-secreting cells. Therefore, the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e.g. E.
  • coli an insect cell or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said antibody protein, and isolating said protein.
  • Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. foetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like.
  • suitable culture media which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium
  • a mammalian serum e.g. foetal calf serum
  • trace elements and growth sustaining supplements e.g. feeder cells
  • feeder cells such as normal mouse peritoneal exudate cells, sple
  • Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
  • suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
  • In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies.
  • Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in
  • the desired antibodies can also be obtained by multiplying mammalian cells in vivo.
  • hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours.
  • the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection.
  • pristane tetramethyl-pentadecane
  • hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
  • ascitic fluid is taken from the animals.
  • the cell culture supernatants are screened for the desired antibodies, preferentially by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • an enzyme immunoassay e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • the immunoglobulins in the culture supernatants or in the ascitic fluid can be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like.
  • the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity chromatography with the target antigen, or with Protein-A.
  • customary chromatography methods for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity chromatography with the target antigen, or with Protein-A.
  • the invention further concerns hybridoma cells secreting the monoclonal antibodies of the invention.
  • the preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of the desired specificity and can be activated from deep-frozen cultures by thawing and reclo ing.
  • the invention in a preferred embodiment, relates to the production of anti mutant PBRMl (BAF180) antibodies.
  • the invention also concerns a process for the preparation of a hybridoma cell line secreting monoclonal antibodies according to the invention, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with a one or more mutant PBRMl (BAF180) polypeptides or antigenic fragments thereof, or an antigenic carrier containing a mutant PBRMl (BAF180) polypeptide; antibody-producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected.
  • spleen cells of Balb/c mice immunised with mutant PBRMl are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Agl4, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.
  • a process for the preparation of a hybridoma cell line characterised in that Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally between 1 and lOC ⁇ g mutant PBRMl (BAF180) and a suitable adjuvant, such as Freund's adjuvant, several times, e.g.
  • spleen cells from the immunised mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol.
  • a fusion promoter preferably polyethylene glycol.
  • the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30 % to about 50 % polyethylene glycol of a molecular weight around 4000.
  • the invention also concerns recombinant nucleic acids comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to mutant PBRM1 (BAF180) as described hereinbefore.
  • PBRM1 mutant PBRM1
  • Such DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.
  • DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to mutant PBRM1 can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof.
  • a mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids.
  • said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody.
  • Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s).
  • Such a mutant sequence is also a degenerated sequence.
  • Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded.
  • Such degenerated sequences can be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.
  • mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.
  • the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.
  • the invention therefore also concerns recombinant nucleic acids comprising an insert coding for a heavy chain murine variable domain of an anti mutant PBRM1 (BAF180) antibody fused to a human constant domain ⁇ , for example ⁇ , ⁇ 2, ⁇ 3 or ⁇ 4, preferably ⁇ or ⁇ 4.
  • the invention concerns recombinant DNAs comprising an insert coding for a light chain murine variable domain of an anti mutant PBRMl (BAF180) antibody directed to mutant PBRM1 (BAF180) fused to a human constant domain ⁇ or ⁇ , preferably ⁇ .
  • the invention pertains to recombinant DNAs coding for a recombinant polypeptide wherein the heavy chain variable domain and the light chain variable domain are linked by way of a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a cleavage site and/or a peptide spacer and/or an effector molecule.
  • Antibodies and antibody fragments according to the invention are useful in diagnosis. Accordingly, the invention provides a composition for diagnosis comprising an antibody according to the invention.
  • the antibody is preferably provided together with means for detecting the antibody, which can be enzymatic, fluorescent, radioisotopic or other means.
  • the antibody and the detection means can be provided for simultaneous, simultaneous separate or sequential use, in a diagnostic kit intended for diagnosis.
  • the antibodies of the invention can be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA, sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
  • Such assays are routine in the art (see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2,1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e. g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2,1% Trasylol
  • protein phosphatase and/or protease inhibitors e. g., EDTA, PMSF, aprotin
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.
  • a membrane such as nitrocellulose, PVDF or nylon
  • blocking the membrane in blocking solution e. g., PBS with 3% BSA or non-fat milk
  • washing the membrane in washing buffer e. g., PBS-Tween 20
  • exposing the membrane to a primary antibody the antibody of interest
  • a secondary antibody which recognises the primary antibody, e. g., an antihuman antibody
  • conjugated to an enzymatic substrate e.
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtitre plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound can be added following the addition of the antigen of interest to the coated well.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labelled antigen (e. g., 3 H or I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labelled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis.
  • Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest conjugated to a labelled compound (e. g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
  • a labelled compound e. g., 3 H or 125 I
  • mutant PBRM1 (BAF180) nucleic acids and polypeptides can be employed, in the context of the present invention, to diagnose the presence or predisposition to cellular transformation and cancer.
  • "Cancer” is used herein to refer to neoplastic growth arising from cellular transformation to a neoplastic phenotype. Such cellular transformation often involves genetic mutation.
  • transformation involves genetic mutation by alteration of one or more PBRM1 (BAF180) genes as described herein.
  • the cancer is renal cancer, e.g. clear cell renal carcinoma.
  • the cancer may be a cancer as defined in Table 5.
  • the cancer may be small-cell lung cancer, gall bladder cancer, squamous-cell lung cancer or pancreatic cancer, e.g. pancreatic adenocarcinoma.
  • Screening for mutant PBRM1 sequences according to the present invention may be combined with detection of additional markers known to be associated with cancer, particularly renal cancer (e.g. clear cell renal carcinoma).
  • the method comprises further screening for one or more mutations in a VHL, UTX, JARID1C, ARID 1 A, ARID5B and/or SETD2 gene or a product thereof, preferably in a VHL and/or SETD2 gene or a product thereof.
  • the present invention provides further cancer-associated genes and cancer- associated mutations, i.e. other than in PBRMl.
  • the invention provides further cancer-associated genes and specific mutations therein as defined in Table 2 (somatic mutations).
  • Methods for detecting such mutations, as well as isolated nucleic acids, polypeptides, ligands etc. useful in such methods, are also provided by analogy to the methods and products described herein with respect to PBRMl.
  • Nucleotide and amino acid sequences of, e.g. the human VHL, UTX, JARID1C, ARID 1 A, ARID5B and SETD2 genes and their protein products are available from publicly-accessible databases, e.g. at http://www.ncbi.nlm.nih.gov/ and http://www.ensembl.org as shown below:
  • VHL von Hippel-Lindau ENST00000256474 NM_000551.2 NPJ
  • VHL von Hippel-Lindau ENST00000256474 NM_000551.2 NPJ
  • ARID 1 A AT rich interactive ENST00000457599 NM_006015.4 NP_006006.3 domain 1A
  • ARID5B AT rich interactive ENST00000279873 NMJ32199.2 NPJ 15575.1 domain 5B
  • EXAMPLE Exome sequencing identifies frequent mutation of the SWI/SNF complex gene, PBRMl, in renal carcinoma
  • the custom design included additional exonic regions over those present in CCDS and comprised a total of 288,654 unique exons from 46,275 transcripts of 20,921 Ensembl protein-coding genes, 33,621 transcripts of 13,772 manually annotated protein-coding genes, and 1635 miRNA genes. Baits for both exomes were provided in a single tube solution format.
  • Genomic DNA ⁇ g was fragmented by Adaptive Focused Acoustics on a Covaris El 20 (Covaris Inc, Woburn, MA, USA) for 90 sec with a duty cycle of 20%, intensity of 5 and cycles per burst of 200.
  • the fragmented DNA was purified using a Qiaquick PGR purification column (Qiagen, 28104) and quantified on a Bioanalyser using the Agilent DNA 1000 kit (Agilent, 5067-1504).
  • the resulting DNA ranged in size from ⁇ 100-400bp, with a modal fragment size of ⁇ 250bp.
  • Genomic libraries were prepared using the Illumina Paired End Sample Prep Kit kit following the manufacturer's instructions (Illumina, San Diego CA, USA).
  • Adapter-ligated DNA was purified using AMPure beads (Agencourt Biosciences Corporation, Beverly, MA, USA) following the manufacturer's protocol, and eluted in 40 ⁇ 1 of nuclease-free water. The prepared library was used directly in the subsequent enrichment procedure without prior size-selection or PGR amplification. Exon enrichment
  • the genomic library (500ng) was mixed with 7.5 ⁇ £ human C 0 tl DNA, lyophilized in a speedvac for 30 min at 45°C and rehydrated in 3.4 ⁇ 1 of nuclease-free water. Enrichment of the genomic DNA was performed using the Agilent SureSelect kit with minor modifications to the manufacturer's protocol. Briefly, the genomic DNA library (3.4 ⁇ 1) was combined with 2.5 ⁇ 1 of Block reagent 1, 2.5 ⁇ 1 of Block reagent 2 and 0.6 ⁇ 1 of Block reagent 3 and transferred to a well of a microtitre plate. The sample was denatured by incubating the plate on a thermocycler at 95°C for 5 min then snap-cooled on ice.
  • a hybridization mix was prepared comprising 25 ⁇ of Hyb reagent 1, ⁇ ⁇ of Hyb reagent 2, ⁇ of Hyb reagent 3 and 13 ⁇ 1 of Hyb reagent 4.
  • a 13 ⁇ 1 aliqout of this mastermix was added to the denatured DNA, and the sample incubated at 95°C for 5 min, then 65°C for 5 min.
  • the baits were prepared by combining 5 ⁇ 1 of SureSelect capture library with ⁇ ⁇ of nuclease free water and ⁇ of RNAse block, and the plate incubated at 65°C for 3 rnin.
  • the pre- warmed DNA (22 ⁇ ) was transferred to the pre- warmed bait mix and the solution incubated for 24h at 65°C. Following hybridization, the captured DNA was isolated using streptavidin-coated magnetic Dynabeads, (Invitrogen, 653.05) and washed following the standard Agilent SureSelect protocol. The isolated DNA was purified using a Qiagen MinElute purification column, eluted in 15 ⁇ 1 of elution buffer and PCR-amplified for 14 cycles as previously described a .
  • Mapping of paired-end read data to the human genome was done using BWA b .
  • An average of 5 gigabases of uniquely mapping and 3.7 gigabases of uniquely mapping reads on target were obtained per sample, with an average of 74% of all reads mapping on target.
  • Sixty-percent of target bases had 20X or greater coverage and 50 percent had 40X or greater coverage.
  • CaVEMan (Cancer Variants through Expectation Maximisation), a bespoke Java application using a simple expectation maximisation algorithm implementation 0 was used to call single nucleotide substitutions. Through comparison of reads from both tumour and normal with the reference genome, CaVEMan calculates a probability for each possible genotype per base (given tumour and normal copy number). In order to provide more accurate estimates of sequence error rates within the algorithm, thus aid identification of true variants, variables such as base quality, read position, lane, and read orientation are incorporated into the calculations. Once CaVEMan was run, several post processing filters were applied in order to further increase the specificity of somatic mutation calls.
  • At least 1 mutant allele in a tumour read must fall in the middle third of the read, unless the tumour read depth is less than 10, when a mutant allele the first third is acceptable.
  • Insertion/Deletion variant calling A modified version of Pindel d was used to call insertions and deletions. By modifying the input file generation process we were able to increase sensitivity and increase confidence in events detected by BWA which was used as the initial mapping tool.
  • the accepted approach for generating input for Pindel is to provide all read pairs where one end is unmapped and the other is confidently mapped to the genome, an anchor read. We found that by including readpairs where both ends map to the genome but allowing for one of the pair to have mismatches, insertions or deletions we could greatly increase coverage over smaller events (in some cases both ends are used as an anchor, creating two input records). The majority of these small events are detected by the BWA mapping algorithm, however, this increases confidence that the events are worth investigating.
  • a second modification to the input generation was included to help identify small events close to large scale deletions or repetitive regions. In regions such as these we would not be able to capture any of the smaller events that can be detected within a single end of a read that is confidently mapped but with some form of mismatch, insertion or deletion. In these cases we generated an artificial anchor co-ordinate so that Pindel can attempt a realignment of these reads. Software that can generate input files of this form can be obtained by contacting the authors.
  • tumour BAM depth > wildtype BAM depth
  • Tumour BAM must have ⁇ 8% BWA reference calls vs BWA variant calls.
  • PBRM1 The coding exons of PBRM1 were sequenced via PCR-based capillary sequencing as previously described. Data were analysed semi-automated mutation detection followed by visual inspection of sequencing traces as previously described 6 . The primer sequences for PBRM1 amplification and sequencing are given in Table 7. Missense mutation analyses
  • PBRM1 contains three kinds of functional domains: six copies of the Bromo domain (Pfam entry PF00439), two copies of the BAH domain (PF01426) and one copy of the HMG-box domain (PF00505).
  • Pfam entry PF00439 six copies of the Bromo domain
  • PF01426 two copies of the BAH domain
  • PF00505 one copy of the HMG-box domain
  • PF00505 HMG-box domain
  • N is the total number of residues in the column.
  • the above construct of the observed distribution uses pseudo-counts' 1 ' 1 proportional to po to account for non-observed residues in the finite sample.
  • the two extreme cases are columns that are highly conserved - where the most prevalent letter receives a large positive score and all others large negative ones - and columns that are highly variable and close to neutral - where all letters receive scores close to zero.
  • For similar conservation based scoring schemes for disease related variation see e.g. the recent review" and in the context of cancer mutations 1 '' 1 .
  • we can now record the score difference between the final and the initial residue we can now record the score difference between the final and the initial residue :
  • PBRMl or scrambled control siRNAs were transfected into renal cell lines using Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer's conditions.
  • Primers used for amplification were: PBRMl -F (5 '-GTGTGATGAACCAAGGAGTGGC- 3 '); PBRMl -R (5 '-GATATGGAGGTGGTGCCTGCTG-3 '); ⁇ -actin-F (5 '- GATCAGCAAGCAGGAGTATGACG-3 ') and ⁇ -actin-R (5 '-
  • PBRMl siR A- and scramble siRNA-transfected cells were determined using the colorimetric 3-(4,5-dimethyltMazol-2yl)-5-(3-carboxyixiethoxyphenyl)-(4-sulfophenyl)-2H-tetrazoluim assay according to the manufacturer's protocol (MTS; Promega, WI). The assay was performed in triplicate.
  • SN12C cells were cultured in a two-layer agar system to prevent their attachment to the plastic surface. After transfection, cells (4 ⁇ 10 4 ) were trypsinized to single-cell suspensions, resuspended in 0.4% agar (Sigma, LA), and added to a preset 1% bottom agar layer in six- well plates. The top agar cell layers were covered with culture medium. Cells were incubated in 5% C0 2 at 37°C for 14 days, and colonies were counted under x2.5 object. Experiments were performed in triplicate.
  • Captured material was sequenced using 76 basepair paired-end reads on the Illumina GAIIx platform. After read alignment, variant calling was performed using a naive Bayesian classifier algorithm for substitutions and a split-read mapping approach (PinDel 7 with substantial cancer-aware output filtering) for insertion/deletions (See Materials and Methods for details). These algorithms aim to identify somatically acquired coding and splice-site variants (i.e. present in the tumour but not in the matching normal), and all mutations reported here were confirmed by PCR-based capillary sequencing. 156 somatic mutations were identified, of which 92 were missense, 9 nonsense, 1 canonical splice site, 1 stop codon read-through, 11 frameshift mutations and 42 synonymous. Some of the mutations identified are shown in Table 2.
  • PBRMl maps to chromosome 3p21 and encodes the BAF180 protein, the chromatin targeting subunit of the PBAF SWI/SNF chromatin remodelling complex 8 .
  • the gene is comprised of 6 bromodomains involved in binding acetylated lysine residues on histone tails, 2 bromo-adjacent homology domains important in protein-protein interaction and an HMG DNA binding domain 5 .
  • PBAF complex-mediated chromatin remodelling is implicated in replication, transcription, DNA repair and control of cell proliferation/differentiation 5 ' 8 .
  • the SMARCB1 and BRG1 components of this complex have inactivating mutations in rhabdoid tumours 9 ' 10 and BRG1 mutations have been reported in multiple tumour types 11 .
  • PBRMl mutations included three frame-shifting insertions and a nonsense mutation; all judged to be homozygous from SNP array and mutant allele read count data.
  • PBRMl was not included in our previous PCR-based sequencing screen 2 and was the only gene, apart from VHL, with recurrent truncating mutations in the seven cases screened.
  • PBRMl was next sequenced in a further 257 RCC cases, including 36 cases of papillary, chromophobe and other non-ccRCC cancers. Truncating mutations were identified in a remarkable 85/257 (33%) (Figure 1) of cases, all diagnosed as ccRCC (for full data see Tables 3 and 4). PBRMl mutations were all found in the context of chromosome 3p loss of heterozygosity (38/38) where SNP array data was available (http://www.sanger.ac.uk/cgi- bin/genetics/CGP/cghviewer/CghHome.cgi) .
  • missense mutations having functional impact was assessed using a scoring system calibrated with protein domain alignments from Pfam 13 (see Materials and Methods). Three missense mutations (p.T232P, p.A597D and p.H1204P) could be scored with these alignments. This set of mutations was predicted to be deleterious, having a significantly lower mean score than a typical null set of in silico generated random missense mutations falling onto the scorable parts of the gene (p-value 0.01 Fi gure 2).
  • PBRMl Transcriptional profiling before and after PBRMl knockdown was performed using gene expression microarrays.
  • Gene set enrichment analysis following PBRMl knockdown showed that PBRMl activity regulates pathways associated with chromosomal instability and cellular proliferation ( Figure 4E, Table 6).
  • Xia et al. 14 reported that PBRMl was a critical transcriptional regulator of p21/CDKNl A in breast cancer cell lines.
  • the PBAF complex has been shown to localise at kinetochores during mitosis 20 and SMARCB1 has been implicated in spindle checkpoint control 21 , which would support the loss of PBRMl giving rise to a chromosomal instability/spindle checkpoint expression phenotype.
  • the SWI/SNF complex has been implicated in the normal cellular response to hypoxia, with impairment of the complex rendering cells resistant to hypoxia-induced cell cycle arrest 26 , which would be consistent with selection for frequent loss of PBRMl in ccRCC.
  • Multiple cancers have apparently concomitant VHL, PBRMl and SETD2 mutations, with all three genes mapping to chromosome 3p, suggesting that the mutations are non-redundant functionally.
  • all 9 cases with a SETD2 mutation have a mutation in either PBRM1 or VHL, with 7 of 9 cases having mutations in all three genes.
  • ARID1A encoding the BAF250A subunit of the SWI/SNF complex was found to have two heterozygous missense mutations - p.R1020K,c.3059G>A and p.L1872P,c.5615T>C. Both cases (PD2126, PD2127) have a PBRM1 truncating mutation. Loss of ARID1A expression
  • PD2127 was also found to have a heterozygous truncating mutation in ARID5B, related to ARID1A and recently- implicated in childhood acute lymphoblastic leukaemia susceptibility 28 .
  • PBRMl like the majority of the other non-VHL mutated cancer genes identified in ccRCC, is involved in chromatin regulation - again at least in part at the level of histone H3 modification and recognition. That this adult epithelial cancer develops with little demonstrable involvement of the more commonly seen mutational activation of canonical growth signalling pathways speaks to its unique biology.
  • HCC2998 colon Adenocarcinoma Hetero- 3 52643768 G A ENST00000337303 C.21280T P.R710* NO
  • Biliary tract c.3489_349
  • HCC2998 colon Adenocarcinoma Hetero- 3 52582255 A C ENST00000337303 T>G P.? INT
  • OVCAR-5 Ovary Carcinoma Hetero- 3 52682407 C T ENST00000337303 c.766G>A P.A256T MIS
  • HCC2998 colon Adenocarcinoma Hetero- 3 52637543 T G ENST00000337303 C.2773A>C P.K925Q MIS
  • HCE-T sinus carcinoma Hetero- 3 52613207 TCT ENST00000337303 8delAGA 133>D
  • IDX_TSA_UP_CLUSTER3 0.0001 0.0022 0.0001
  • ADIP_DIFF_CLUSTER4 0.0038 0.0083 0.0016
  • HSA00240_PYRIMIDINE_1 ETABOLISM 0.0015 0.0038 0.0189
  • IGF_VS_PDGF_DN 0.0016 0.0267 0.0295
  • stCE03- 616895 AAACAAGGAAGTCCAGGGC AAAAAGTG G AG ATG C CTTG C stCE03- 616896 TTGGAAGCGGGATTTGGA GGCACACGTTGTCCAGGAT stCE03- 616897 TTTGTCTGCAGGTTATATTTCACT G I N C AAG C AG G ACTTTGTGT AG stCE03- 616898 CCCTCTAGATCTGAGTTGCCTG ATCCTTCTTGCTCGTTCCAA stCE03- 616899 CCCAAATGTGAC I I I I GCTGA AAGAGATTTTCAATTTTGTCTTCCTC stCE03- 616900 AAGTATC I I I I I CATGTGTTTAATGGG AAAAAG C AC AAAT AC CT AC CG A stCE03- 616901 CCATATGGACAACAGGTGAGC AAACATGCAAAGAAACTCCAAAC stCE03- 616902 GAAATGTGCCTGGAAATATTCTG TTGAAATA
  • Versteege I. et al. Truncating mutations of hSNF5/INIl in aggressive paediatric cancer. Nature 394, 203-206 (1998).
  • Sekine, I. et al. The 3p21 candidate tumor suppressor gene BAF180 is normally expressed in human lung cancer. 24, 2735-2738 (2005). 16. Bignell, G.R. et al. Signatures of mutation and selection in the cancer genome. Nature

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Selon un de ses aspects, l'invention concerne une méthode de détection d'une mutation associée au cancer rénal chez un sujet, comprenant le criblage d'un échantillon d'essai dérivé du sujet pour rechercher la présence d'une ou plusieurs mutations dans un gène PBRMA ou un produit de celui-ci.
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WO2018132287A1 (fr) * 2017-01-11 2018-07-19 Dana-Farber Cancer Institute, Inc. Biomarqueurs pbrm1 prédictifs de la réponse de point de contrôle anti-immunitaire
WO2019178611A1 (fr) * 2018-03-16 2019-09-19 Thomas Jefferson University Anticorps pbrm1 pour détecter une réponse à l'immunothérapie
US11773449B2 (en) 2017-09-01 2023-10-03 The Hospital For Sick Children Profiling and treatment of hypermutant cancer

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2018132287A1 (fr) * 2017-01-11 2018-07-19 Dana-Farber Cancer Institute, Inc. Biomarqueurs pbrm1 prédictifs de la réponse de point de contrôle anti-immunitaire
US11377696B2 (en) 2017-01-11 2022-07-05 Dana-Farber Cancer Institute, Inc. PBRM1 biomarkers predictive of anti-immune checkpoint response
US11773449B2 (en) 2017-09-01 2023-10-03 The Hospital For Sick Children Profiling and treatment of hypermutant cancer
WO2019178611A1 (fr) * 2018-03-16 2019-09-19 Thomas Jefferson University Anticorps pbrm1 pour détecter une réponse à l'immunothérapie

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