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    • 专利摘要:
    The present invention encompasses compositions and methods for detecting cancer metastasis.
    公开号:NL2007467A
    申请号:NL2007467
    申请日:2011-09-23
    公开日:2012-03-26
    发明作者:Anne Bowcock；James Harbour
    申请人:Univ Washington；
    IPC主号:
    专利说明:

    COMPOSITIONS AND METHODS FOR DETECTING CANCER
    METASTASIS
    FIELD OF THE INVENTION
    The invention encompasses methods for detecting cancer metastasis, as well as biomarkers associated with cancer metastasis.
    BACKGROUND OF THE INVENTION
    Once a primary tumor has metastasized and is clinically detectable by current diagnostic measures, treatment of the tumor becomes more complicated, and generally speaking, survival rates decrease. Consequently, it is advantageous to determine which tumors are more likely to metastasize and to advance the time to detection of metastasis, so that appropriate treatment may be started as soon as possible. Many different types of tumors are capable of metastasizing. Melanomas, in particular, are capable of aggressive metastasis.
    Melanoma is a malignant tumor of melanocytes, and may occur in the eye (uveal melanoma), on the skin, or on mucosal tissues. Uveal melanoma is the most common intraocular malignancy. The incidence of this tumor increases with age and reaches a maximum between the 6th and 7th decade of life. Approximately 50% of patients die of metastases, a proportion that, despite all efforts to improve treatment, has remained constant during the last century. The average life expectancy after diagnosis of metastases is 7 months.
    Around 160,000 new cases of melanoma of the skin are diagnosed worldwide each year, and according to the WHO Report about 48,000 melanoma related deaths occur worldwide per annum, which accounts for 75 percent of all deaths associated with skin cancer. Similar to uveal melanoma, when there is distant metastasis, the cancer is generally considered incurable. The five-year survival rate is less than 10%, with a median survival time of 6 to 12 months. Additionally, specific to uveal melanoma and cutaneous melanoma and generally considered for carcinoma, earlier treatment of malignancies is associated with improved progression-free and overall survival.
    Due to the aggressive nature of these malignancies, there is a need in the art for methods of predicting the risk of metastasis and for earlier detection of metastatic disease, so that treatment may begin as early as possible.
    SUMMARY OF THE INVENTION
    One aspect of the present invention encompasses a method for detecting the presence of a biomarker for metastatic cancer in a subject. The method may encompass a method for determining the risk of metastasis in a subject. Generally speaking, the method comprises analyzing the BAP1 gene nucleotide sequence and/or the BAP1 protein amino acid sequence from a tumor cell in a sample obtained from the subject, and identifying the presence of a mutation in the BAPl gene and/or BAP1 protein sequence. The presence or absence of a mutation is as compared to the gene and/or protein sequence from a non-tumor cell from the same subject. For example, the gene nucleotide and/or protein amino acid sequence from a non-tumor cell may be SEQ ID NOs:3 and 1, respectively. Comparison may also be made between cDNA obtained from mRNA from a tumor cell and cDNA obtained from mRNA from a non-tumor cell, which may have BAP1 nucleotide sequence SEQ ID NO:2. The presence of a mutation, particularly an inactivating mutation as defined elsewhere herein, indicates an increased risk for metastasis in the subject.
    The biomarker may be decreased BAP1 activity in a tumor cell from a subject, as compared to the activity in a non-tumor cell from the same subject. Decreased BAP1 activity may be indicative of an increased risk of metastasis in the subject and/or of the presence of metastatic cancer.
    Another embodiment of the present invention encompasses a method for detecting the presence of metastatic cancer. Generally speaking, the assay comprises analyzing the BAP1 gene nucleotide sequence or the BAP1 protein amino acid sequence in a tumor sample obtained from the subject, and detecting the presence of a mutation in the BAP1 gene or BAP1 protein sequence, as compared to the sequence in a non-tumor sample from the subject, as mentioned above. The presence of the mutation indicates the presence of metastatic melanoma.
    Yet another aspect of the present invention encompasses a metastatic cancer biomarker, which may be detected in a tumor sample obtained from a subject. The biomarker typically comprises a BAP1 nucleotide sequence comprising at least one mutation, as compared to the BAP1 sequence in a non-tumor sample from the subject. The biomarker may also comprise a BAP1 amino acid sequence comprising at least one mutation. Such a biomarker may be detectable, for example, by use of an antibody which specifically recognizes the biomarker and such antibodies are also encompassed by the present invention. The biomarker may be detected by detecting reduced BAP1 activity in a cell from tumor sample from a subject, as compared to the activity in a cell from a non-tumor sample from the same subject.
    Other aspects and iterations of the invention are described more thoroughly below.
    BRIEF DESCRIPTION OF THE FIGURES
    The invention will be described, by way of example only, with reference to Figures 1 -17.
    FIG. 1 depicts a series of panels illustrating that inactivating mutations in BAP1 occur frequently in uveal melanomas. (A) Sanger sequence traces of MM 056 and MM 070 at the sites of the mutations. Location of mutated base in MM 056 and the start of the deletion of MM 070 are indicated (arrows). The non-coding BAP1 strand is shown for MM 070. (B) Map of BAP1 gene and location of BAP l mutations. BAP l contains 17 exons (shaded boxes) that encode a 728 amino acid protein. Introns are not to scale. Mutations are shown below the gene figure as indicated. The UCH domain (aa. 1-188) and UCH37-like domain (ULD) (aa. 635-693) are indicated (12, 13). The critical Q, C, H and D residues of the active site (Gln85, Cys91, Hisl69 and Aspl84) are indicated with asterisks. The catalytic cysteine is indicated with a circle. Also shown are: the NHNY consensus sequence for interaction with HCFC1 (aa. 363-365, exon 11), nuclear localization signals (NLS) at aa. 656-661 (exon 15) and aa. 717-722 (exon 17), the BARD1 binding domain within the region bounded by aa. 182-240 (13), and the BRCA1 binding domain within aa. 598-729 (11). (C) Location of BAP1 gene missense mutations in the UCH domain aligned to the crystal structure of UCH-L3 (21). Three-dimensional structure of UCH-L3 was visualized with MMDB software (22). The small molecule near C91G, H169Q and S172R represents a suicide inhibitor, illustrating the critical location of these mutations for catalytic activity. (D) Conservation of BAP1 in regions containing mutated amino acids. Alignments of segments of BAP I homologs harboring mutated amino acids (missense or in-frame deletions) are shown for the indicated species. Amino acid numbering is on the basis of human BAP1 (SEQ ID NO:l). Positions of mutated amino acids are indicated with asterisks.
    FIG. 2 depicts Sanger sequence trace of one end of the mutated region of the BAP1 gene in tumor sample NB101. The breakpoint at one end of the insertion/deletion is indicated with an arrow. Wild type sequence is indicated below the NB101 sequence. FIG. 3 depicts bar graphs of BAPl mRNA levels. (A) BAPl mRNA levels measured by quantitative RT-PCR in 9 non-metastasizing class 1 UMs and 28 metastasizing class 2 UMs. (B) Relationship between BAPl mRNA levels (measured by quantitative RT-PCR) and type of BAPl mutation in 9 UMs with nonsense mutations, 10 UMs with missense mutations (including small in-frame deletions, splice acceptor, and stop codon read-through mutations), and 4 class 2 UMs in which no BAPl mutations were detected.
    FIG. 4 depicts a series of photographs illustrating that BAPl mutations disrupt BAPl protein expression in human uveal melanoma samples. Immunofluorescence analysis of BAPl protein expression was performed on archival tumor specimens from uveal melanomas of known class and BAPl mutation status, as indicated. All images were captured at 40X and are represented at the same magnification. Scale bar, 10 microns. No BAPl expression is seen in the Class 2 metastasizing UM cells (MM 100, MM071, MM135, MM091) whereas expression is seen in the class 1 non-metastasizing UM cells (MM050, MM085).
    FIG. 5 depicts a series of micrographs illustrating that UM cells depleted of BAPl acquire properties that are typical of metastasizing class 2 tumor cells. Phase contrast photomicrographs of 92.1 uveal melanoma cells transfected with BAPl or control siRNA at the indicated days. Bottom panels show representative examples of class 1 and class 2 uveal melanoma cells obtained from patient biopsy samples (Papanicolaou stain). Scale bars, 10 microns.
    FIG. 6 depicts a gene expression heatmap of the top class 1 versus class 2 discriminating transcripts in 92.1 uveal melanoma cells transfected with control versus BAPl siRNAs.
    FIG. 7 depicts a diagram, a Western blot, and a bar graph showing the effects of BAPl depletion by siRNA. 92.1 cells were transfected with BAPl siRNA and evaluated after five days. (A) BAPl protein levels were efficiently depleted to less than 95% of control levels (see Western blot). Upper panel depicts principal component analysis to show effect of BAPl knockdown on gene expression signature. The small spheres represent the training set of known class 1 (left side) and class 2 (right side) tumors. Large spheres represent the control-transfected (left hand large sphere) and BAPl siRNA transfected (right hand large sphere) cells. Lower panel depicts mRNA levels measured by quantitative RT-PCR of a panel of melanocyte lineage genes, presented as fold change in BAP1 siRNA/control siRNA transfected cells. Results are representative of three independent experiments. (B) mRNA levels of mRNAs of a panel of melanocyte lineage genes measured by quantitative RT-PCR, presented as fold change in BAP1 siRNA/control siRNA transfected cells. (C) RNAi mediated depletion of BAP 1 in 92.1 and Mel290 UM cell lines using two independent siRNAs that target BAP1. Duplicate experiments of each cell line and siRNA are shown.
    FIG. 8 depicts a bar plot, a Western blot and micrographs for characterizing BAP1 stable knockdown cells.
    FIG. 9 depicts Western blots and fluorescence immunohistochemical micrographs showing increased ubiquitination of histone H2A.
    FIG. 10 depicts bar plots showing decreased RNA levels of melanocyte differentiation genes in BAP1 stable knockdown cells.
    FIG. 11 depicts plots showing that transient knockdown of BAP 1 leads to a decrease in cell proliferation.
    FIG. 12 depicts micrographs and a bar plot showing that loss of BAP1 in culture leads to decreased cell motility.
    FIG. 13 depicts images of culture plates and a bar plot showing that loss of BAP1 leads to decreased growth in soft agar.
    FIG. 14. depicts bar plots showing that loss of BAP 1 leads to an increased ability to grow in clonegenic assays.
    FIG. 15. depicts a bar plot showing that loss of BAP 1 leads to increased migration towards a serum attractant.
    FIG. 16 depicts plots showing that loss of BAP 1 in culture leads to decreased tumor growth in the mouse flank.
    FIG. 17 depicts plots showing that loss of BAP 1 in culture leads to decreased tumor growth in the mouse after tail vein injection.
    DETAILED DESCRIPTION OF THE INVENTION
    The present invention provides a method for detection of a metastatic cancer biomarker in a subject, wherein detection of the biomarker comprises identifying a mutation in a BAP1 nucleotide sequence and/or a BAP1 protein sequence in a sample obtained from the subject. The method may comprise determining the risk of metastasis in a subject and/or detecting the presence of a metastasis in a subject. Advantageously, such methods may allow a physician to determine the severity of an oncogenic disease in a subject and to make appropriate, timely, treatment decisions based on this information.
    In one embodiment, the method comprises analyzing the BAPJ nucleotide and/or BAP1 amino acid sequence from a cell in a sample obtained from the subject, and identifying the presence of a mutation in the BAP1 nucleotide sequence and/or the BAP1 amino acid sequence. In this context, “a mutation in the BAP1 nucleotide sequence” refers to a mutation in an exon of BAP1, an intron of BAPl, the promoter of BAP1, the 5’ untranslated region of BAP1, the 3’ untranslated region of BAPl, or any other regulatory region for the BAPl gene, such that the mutation decreases the expression of BAP l mRNA, synthesis of BAP1 protein, or enzymatic activity of BAP1 when compared to the sequence of BAP 1 from a non-tumor cell of the same individual. Nucleotide and amino acid sequence mutations in tumor cells are detected by comparison with the equivalent sequences from non-tumor cells from the same subject and/or by comparison to human wild type sequences SEQ ID NO:3 (genomic nucleotide sequence) and SEQ ID NO:l (amino acid sequence). A mutation may also be identified by comparing cDNA sequences obtained from mRNA in a tumor and non-tumor cell. “Wild type” cDNA may have the sequence SEQ ID NO:2. The presence of the mutation indicates an increased risk for metastasis in the subject. In another embodiment, the method comprises analyzing the level of BAP 1 activity in a tumor cell in a sample obtained from a subject compared with activity in a non-tumor cell from that subject, where a decrease in BAP1 activity indicates an increased risk for metastasis in the subject.
    A method of the invention may comprise analyzing the BAPl nucleotide sequence of a sample obtained as described above. Typically, analyzing the BAP1 nucleotide > sequence may comprise identifying a mutation in the BAP I nucleotide sequence. As detailed above, “a mutation in the BAPl nucleotide sequence” refers to a mutation in an exon of BAPJ, an intron of BAP1, the promoter of BAP1, the 5’ untranslated region of BAP1, the 3’ untranslated region of BAP1, or any other regulatory region for the BAP I gene (e.g. a splice acceptor site), such that the mutation decreases the expression of BAPJ mRNA, synthesis of BAP1 protein, or enzymatic activity of BAP1 when compared to the sequence of BAP 1 from a non-tumor cell of the same individual. Such a mutation may be a point mutation, a deletion mutation, or an insertion mutation. The mutation may be a missense or nonsense sequence. For instance, in one embodiment, the mutation may cause a premature truncation of the BAP1 amino acid sequence. Alternatively, the mutation may affect a conserved amino acid in the ubiquitin carboxy-terminal hydrolase (UCH) domain or the UCH37-like domain (ULD); for example, see Figure IB. Such a mutation may be identified using methods commonly known in the art. For instance, see the Examples. All or a portion of the BAP1 nucleic acid sequence may be sequenced and compared to the wild-type genomic sequence (RefSeq #NM_004656, SEQ ID NO:3) to identify a mutation. Alternatively or additionally, all or a portion of the BAP1 amino acid sequence may be compared to the wild-type amino acid sequence (SEQ ID NO:l) to identify a mutation. Alternatively or additionally, all or a portion of cDNA obtained from BAP1 mRNA may be compared to the cDNA nucleotide sequence SEQ ID NO:2.
    However, with the knowledge of the mutations provided herein, it is a routine matter to design detection means such as primers and/or probes that would be able to detect and/or identify mutated sequences, such as mutated nucleotide sequences which differ from the wild-type SEQ ID NO:3 (or SEQ ID NO:2, if cDNA is being examined). Possible techniques which might be utilized are well-established in the prior art and their use is readily adaptable by the skilled person for the purposes of detecting the BAP1 gene and/or BAP1 protein mutations disclosed herein. For example, amplification techniques may be used, examples including polymerase chain reaction, ligase chain reaction, nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), transcription mediated amplification (TMA), Loop-Mediated Isothermal Amplification (LAMP), Q-beta replicase, Rolling circle amplification, 3 SR, ramification amplification (Zhang et al. (2001) Molecular Diagnosis 6 pl41-150), multiplex ligation-dependent probe amplification (Schouten et al. (2002) Nucl. Ac. Res. 30 e57). Other related techniques for detecting mutations such as SNPs may include restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP) and denaturing high performance liquid chromatography (DHPLC). A summary of many of these techniques can be found in “DNA Amplification: Current technologies and applications” (Eds. Demidov & Broude (2004) Pub. Horizon Bioscience, ISBN:0-9545232-9-6) and other current textbooks.
    A mutation of BAP] may be an inactivating mutation, i.e., expression levels of BAP I mRNA and/or BAP1 protein are reduced and/or BAP1 protein activity is reduced in cells from a tumor sample from a subject, compared to expression level and/or activity in cells from a non-tumor sample from the same subject. BAP1 protein activity may be, for example, ubiquitin carboxy-terminal hydrolase activity. In one embodiment, a mutation of BAP I may be found in exon 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12,13,14,15,16 or 17 of the BAP1 nucleotide sequence. In another embodiment, a mutation of BAP1 may be found in the promoter of BAP1. In yet another embodiment, a mutation of BAP1 may be found in the 5’ untranslated region. In still another embodiment, a mutation of BAP I may be found in the 3’ untranslated region. In a certain embodiment, a mutation of BAP1 may be found in a splice acceptor site.
    In particular embodiments, a mutation may be selected from one or more of: deletion of the nucleotides equivalent to positions 3025-3074 of SEQ ID NO:3; deletion of the nucleotides equivalent to positions 2026-2028 of SEQ ID NO:2; substitution of the nucleotide cytosine with the nucleotide guanine at the position equivalent to position 622 of SEQ ID NO:2; substitution of the nucleotide guanine with the nucleotide adenine at the position equivalent to position 703 of SEQ ID NO:2; substitution of the nucleotide cytosine with the nucleotide thymine at the position equivalent to position 872 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 960-968 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 1083-1093 of SEQ ID NO:2; substitution of the nucleotide adenine with the nucleotide guanine at the position equivalent to position 2130 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 3313-3335 of SEQ ID NO:3; deletion of the nucleotides equivalent to positions 736-751 of SEQ ID NO:2; insertion of the nucleotide adenine between positions equivalent to positions 1318 and 1319 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 468-487 of SEQ ID NO:2 and insertion of the nucleotide adenine; deletion of nucleotide adenine at the position equivalent to position 874 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 726-759 of SEQ ID NO:3; substitution of the nucleotide thymine with the nucleotide ; adenine at the position equivalent to position 2303 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 1829-1833 of SEQ ID NO:2; deletion of nucleotide cytosine at the position equivalent to position 259 of SEQ ID NO:2; substitution of the nucleotide guanine with the nucleotide cytosine at the position equivalent to position 497 of SEQ ID NO:2; substitution of the nucleotide cytosine with the nucleotide guanine at the position equivalent to position 622 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 2112-2120 of SEQ ID NO:2; substitution of the nucleotide thymine with the nucleotide guanine at the position equivalent to position 388 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 2006-2017 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 610-634 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 739-776 of SEQ ID NO:3; substitution of the nucleotide guanine with the nucleotide thymine at the position equivalent to position 7819 of SEQ ID NO:3; substitution of the nucleotide cytosine with the nucleotide guanine at the position equivalent to position 631 of SEQ ID NO:2; deletion of the nucleotides equivalent to positions 2195-2220 of SEQ ID NO:2; substitution of the nucleotide cytosine with the nucleotide thymine at the position equivalent to position 221 of SEQ ID NO :2. As outlined above, nucleotide numbering is by reference to the human wild-type sequences, for example, as represented by SEQ ID NO:3 when comparing genomic DNA or SEQ ID NO:2 when comparing cDNA.
    In a particular embodiment, a mutation may be a truncating mutation in exon 2,3,4, 5, 6, 7, 8, 9,11,13,16 or 17 of BAP1, a missense mutation in exon 5,6, 7 or 16, an in-frame deletion in exon 10,15 or 16, or a termination read-through in exon 17. In another particular embodiment, a BAPl mutation may be a nonsense mutation in a BAP1 protein encoded by the BAP1 nucleotide sequence, selected from Q36X, W196X and Q253X. In yet another particular embodiment, a BAP1 mutation may result in a missense mutation selected from C91G, C91W, G128R, H169Q, S172R or D672G. In still another particular embodiment, an in-frame deletion may be selected from the group E283-S285del, E631-A634del or R666-H669del. Amino acid numbering is by reference to the human wild-type sequences, for example, as represented by SEQ ID NO: 1.
    In some embodiments of the invention, the level of BAP 1 activity in a sample may be analyzed. The “level of BAP1 activity” may refer to the level of expression of BAP1 mRNA, the level of synthesis of BAP 1 protein, or the level of enzymatic activity of BAP 1 in a sample.
    In one embodiment, the level of BAP1 activity may refer to the level of expression of BAP1 mRNA in a sample. Generally speaking, if a sample has a decreased level of expression of BAP1 mRNA, then the subject has an increased risk of metastasis. In certain embodiments, the level of BAP 1 activity is decreased about 50% to about 100% compared to in a non-tumor cell from the same individual. In other embodiments, the level of BAP 1 activity is decreased from about 60% to about 100% compared to in a non-tumor cell from the same individual. In still other embodiments, the level of BAP1 activity is decreased from about 70% to about 95% compared to in a non-tumor cell from the same individual. In certain embodiments, the level of BAP1 activity is decreased about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74,73, 72, 71, 70, 69, 68,67,66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50% compared to in anon-tumor cell from the same individual.
    Determining the level of expression of a nucleic acid sequence, comprises, in part, measuring the level of mRNA expression for a nucleic acid sequence in a tumor sample. Methods of measuring the level of mRNA in a tumor sample for a particular nucleic acid sequence, or several sequences, are known in the art. For instance, in one embodiment, the level of mRNA expression may be determined using a nucleic acid microarray. Methods of using a nucleic acid microarray are well and widely known in the art. In another embodiment, the level of mRNA expression may be determined using PCR. In these embodiments, the mRNA is typically reverse transcribed into cDNA using methods known in the art. The cDNA may, for example, have nucleotide sequence SEQ ID NO:2 when derived from mRNA obtained from a non-tumor cell. Methods of PCR are well and widely known in the art, and may include quantitative PCR, semi-quantitative PCR, multi-plex PCR, or any combination thereof. Other nucleic acid amplification methods are suggested above. In yet another embodiment, the level of mRNA expression may be determined using a TLDA (TaqMan low density array) card manufactured by Applied Biosciences, or a similar assay. The level of mRNA expression may be measured by measuring an entire mRNA transcript for a nucleic acid sequence, or measuring a portion of the mRNA transcript for a nucleic acid sequence. For instance, if a nucleic acid array is utilized to measure the level of mRNA expression, the array may comprise a probe for a portion of the -mRNA of the nucleic acid sequence of interest, or the array may comprise a probe for the full mRNA of the nucleic acid sequence of interest. Similarly, in a PCR reaction, the primers may be designed to amplify the entire cDNA sequence of the nucleic acid sequence of interest, or a portion of the cDNA sequence. One of skill in the art will recognize that there is more than one set of primers that may be used to amplify either the entire cDNA or a portion of the cDNA for a nucleic acid sequence of interest. Methods of designing primers are known in the art.
    Methods of extracting RNA from a tumor sample are known in the art. For instance, see Examples 1 and 2 of PCT/US09/041436, herein incorporated by reference in its entirety.
    The level of expression may or may not be normalized to the level of a control gene. Such a control gene should have a constant expression in a tumor sample, regardless of the risk for metastasis of the tumor. This allows comparisons between assays that are performed on different occasions.
    In another embodiment, the level of BAP1 activity may refer to the level of BAP1 protein synthesis in a sample. Generally speaking, a decreased level of BAP 1 protein synthesis in a sample indicates an increased risk of metastasis in the subject. Methods of measuring the synthesis of BAP 1 are known in the art. For instance, immunofluorescence may be used, as described in the Examples.
    In yet another embodiment, the level of BAP1 activity may refer to the level of BAP1 enzymatic activity in a sample. Generally speaking, a decreased level of BAP1 enzymatic activity indicates an increased risk of metastasis in a subject. BAP1 has ubiquitin carboxy-terminal hydrolase activity. Such activity may be measured using methods well known in the art. See, for instance, Scheuermann JC, et al: Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB, Nature 2010, 465:243-247 (the measurement of histone H2A monoubiquitination); Machida YJ, et al: The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1, J Biol Chem 2009,284:34179-34188 (the measurement of HCFC1 deubiquitination); Russell NS, Wilkinson KD. Deubiquitinating enzyme purification, assay inhibitors, and characterization. Methods Mol Biol 2005;301:207-19 (other strategies for measurement of deubiquitinating enzymatic activity using substrates that can be monitored, such as described in Russell et al.).
    A method of the invention may be conducted on a sample obtained from a subject. Suitable samples comprise one or more tumor cells, either from a primary tumor or a metastasis. In one embodiment, a suitable sample comprises a melanoma cell. In another embodiment, a suitable sample comprises a carcinoma cell. In yet another embodiment, a suitable sample comprises a sarcoma cell. In an exemplary embodiment, a suitable sample comprises a uveal melanoma cell. In some embodiments, a suitable sample may be a circulating tumor cell. Circulating tumor cells may be found in a bodily fluid (e.g. plasma, sputum, urine, etc.) or other excrement (e.g. feces).
    Methods of collecting tumor samples are well known in the art. For instance, a tumor sample may be obtained from a surgically resected tumor. In uveal melanoma, for example, a tumor sample may be obtained from an enucleation procedure. Alternatively, the tumor sample may be obtained from a biopsy. This is advantageous when the tumor is small enough to not require resection. In an exemplary embodiment, the tumor sample may be obtained from a fine needle biopsy, also known as a needle aspiration biopsy (NAB), a fine needle aspiration cytology (FNAC), a fine needle aspiration biopsy (FNAB) or a fine needle aspiration (FNA). A tumor sample may be fresh or otherwise stored so as to reduce nucleic acid degradation. For instance, a tumor sample may be a fresh frozen tumor sample or a formalin-fixed paraffin embedded tumor sample.
    In certain embodiments, the method of the invention may be performed with a tumor sample comprising about five cells or less. In one embodiment, the tumor sample may comprise about 1,2, 3,4, 5,6, 7, 8, 9,10,11,12,13,14,15 or more cells. In another embodiment, the tumor sample may comprise 20,25, 30, 35,40 or more cells.
    A method of the invention may further comprise determining the risk of metastasis. The level of risk is a measure of the probability of a metastasis occurring in a given individual. If a mutation is indentified, as described above, in a sample from a subject, then the subject is at a higher risk (i.e., there is an increased probability) of developing metastases then a subject without a mutation in BAPl and/or BAP1. Alternatively, if the level of BAP1 activity is decreased, as described above, then the subject is at a higher risk of developing metastases then a subject with out a decreased level of BAP1 activity. For instance, the risk may be greater than about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the risk may be greater than about 50%, 55%, 60%, 65%, 70%,> 75%, 80%, 85%, 90%, or 95%. In particular embodiments, the risk may continue to increase over time. For example, the risk may be about 50% at five years after initial cancer diagnosis and 90% for ten years.
    Alternatively, if a mutation in not identified (i.e. the BAP1 and/or BAP1 sequence is wild-type) in a sample from a subject, then the subject is at lower risk of developing metastases. Similarly, if the level of BAP1 activity is not decreased, then the subject is at a lower risk of developing metastasis. For instance, the risk may be less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some embodiments, the risk may be less than about 20%, 15%, 10%, or 5%. In particular embodiments, the risk may be low, but may still increase over time. For example, the risk may be about 5% at five years and 10% at ten years.
    Increased or decreased “risk” or “probability” may be determined, for example, by comparison to the average risk or probability of an individual cancer patient within a defined population developing metastasis. For example, by way of illustration, for a given cancer the overall proportion of patients who are diagnosed with a metastasis within 5 years of initial cancer diagnosis may be 50%. In this theoretical context, an increased risk for an individual will mean that they are more than 50% likely to develop a metastasis within 5 years, whereas a reduced risk will mean that they are less than 50% likely to develop a metastasis. Such comparisons may, in some circumstances, be made within patient populations limited or grouped using other factors such as age, ethnicity, and/or the presence or absence of other risk factors. Some embodiments of the present invention include a method for detecting the presence of a metastasis. In one embodiment, the method generally comprises analyzing the BAP1 gene and/or BAP1 protein sequence in a sample obtained from the subject, and determining the presence of a mutation in the BAP1 and/or BAP1 sequence. The presence of the mutation indicates the presence of a metastasis. As outlined above, the presence of a mutation may be determined by comparison of a sequence from a tumor cell with a sequence from a non-tumor cell from the same subject and/or by comparison to SEQ ID NO:l (wild type amino acid sequence) or SEQ ID NO:3 (wild type genomic DNA sequence). It may also be determined by obtaining cDNA from BAP1 mRNA in the cell and comparing the sequence to SEQ ID NO:2. In another embodiment, the method comprises analyzing the level of BAP 1 activity in a sample obtained from the subject, where a decrease in BAP1 activity indicates the presence of a metastasis in the subject. Suitable samples, methods of analyzing a BAP1 and/or BAP1 sequence, and methods of determining the level of BAP1 activity in a sample are described in section I above.
    In certain embodiments, a method of the invention may be used in conjunction with a method as described in PCT/US09/041436, herein incorporated by reference in its entirety, to determine the risk of metastasis in a subject.
    Yet another aspect of the invention encompasses a tumor metastasis biomarker. In one embodiment, a biomarker of the invention comprises a BAPl and/or BAP1 sequence comprising a mutation, as described above. In another embodiment, a biomarker of the invention comprises a decreased level of BAP1 activity, as described above. This may include a decrease in BAP1 protein expression. Where the bio marker is a BAP1 amino acid sequence comprising a mutation, the presence of the biomarker may be detected by use of an antibody which specifically binds to the biomarker. Such antibodies are encompassed within the scope of the present invention, as well as kits comprising the antibody and methods of use thereof. In each of these embodiments, a tumor may be a melanoma, carcinoma, or sarcoma. In an exemplary embodiment, the tumor is a melanoma. In a further exemplary embodiment, the tumor is a uveal melanoma.
    DEFINITIONS
    As used herein, “carcinoma” refers to a malignant tumor derived from an epithelial cell. Non-limiting examples of carcinoma may include epithelial neoplasms, squamous cell neoplasms, squamous cell carcinoma, basal cell neoplasms, basal cell carcinoma, transitional cell carcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinous and serous neoplasms, ductal, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, and adenocarcinomas such as adenocarcinoma, linitis plastica, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, and grawitz tumor.
    As used herein, “regulatory region” refers to a nucleic acid sequence operably linked to a nucleic acid encoding BAP1 such that the regulatory region modulates the transcription of BAP1 mRNA.
    As used herein, “melanoma” refers to a malignant tumor of a melanocyte. In one embodiment, the melanoma may be a uveal melanoma. In another embodiment, the melanoma may be a cutaneous melanoma. In another embodiment, the melanoma may be a mucosal melanoma.
    As used herein, “sarcoma” refers to a malignant tumor derived from connective tissue. Non limiting examples of a sarcoma may include Askin's Tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, primitive neuroectodermal tumor (PNET), malignant hemangioendothelioma, malignant peripheral nerve sheath tumor (malignant schwannoma), osteosarcoma and soft tissue sarcomas such as alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, desmoid Tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma. Lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma.
    As used herein, “subject” refers to a mammal capable of being afflicted with a carcinoma, melanoma, or sarcoma, and that expresses a homolog to BAP1 (human BAP1 having the amino acid sequence SEQ ID NO:1). In addition to having a substantially similar biological function, a homolog of BAP1 will also typically share substantial sequence similarity with the nucleic acid sequence encoding BAP1 (the human BAP1 gene having the nucleotide sequence SEQ ID NO:3). For example, suitable homo logs preferably share at least about 30% sequence homology, more preferably, about 50%, and even more preferably, are greater than about 75%, 80%, 85%, 90%, or greater than about 95% homologous in sequence. In determining whether a sequence is homologous to BAP1, sequence similarity may be determined by conventional algorithms, which typically allow introduction of a small number of gaps in order to achieve the best fit. In particular, “percent homology” of two polypeptides or two nucleic acid sequences may be determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 87,2264). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (J. Mol. Biol. (1990) 215, 403 ). BLAST nucleotide searches may be performed with the NBLAST program to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. Equally, BLAST protein searches may be performed with the XBLAST program to obtain amino acid sequences that are homologous to a polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul, et al. (Nucleic Acids Res. (1997) 25, 3389). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are employed. See www.ncbi.nlm.nih.gov for more details.
    In an exemplary embodiment, the subject is human. In certain embodiments, the subject may have a carcinoma, sarcoma, or melanoma. In other embodiments, the subject may be suspected of having a carcinoma, sarcoma, or melanoma.
    Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to” and do not exclude other moieties, additives, components or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that may changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
    EXAMPLES
    The following examples illustrate various iterations of the invention.
    Example 1. BAP1 mutations and uveal melanoma metastasis. )
    Uveal melanoma (UM) is the most common primary cancer of the eye and has a strong propensity for fatal metastasis (1). UMs are divided into class 1 (low metastatic risk) and class 2 (high metastatic risk) based on a validated multi-gene clinical prognostic assay included in the TNM classification system (2, 3). However, the genetic basis of metastasis remains unclear. Oncogenic mutations in the Gotq stimulatory subunit GNAQ are common in UM (4), but these mutations occur early in tumorigenesis and are not correlated with molecular class or metastasis (5, 6). On the other hand, class 2 tumors are strongly associated with monosomy 3 (7), suggesting that loss of one copy of chromosome 3 may unmask a mutant gene on the remaining copy which promotes metastasis. However, intensive investigations have previously failed to identify mutations associated with metastasis (1).
    Using exome capture followed by massively parallel sequencing (8, 9), the inventors analyzed two class 2 tumors that were monosomic for chromosome 3 (MM56 and MM70) and matching normal DNA from peripheral blood lymphocytes. Both tumors contained inactivating mutations in BAP1, located at chromosome 3p21.1 (Fig. 1A). MM56 contained a C/G to T/A transition that created a premature termination codon (p.W196X). MM70 contained a deletion of 1 lbp in exon 11, leading to a frameshift and premature termination of the BAP1 protein (p.Q322fsX100). The matched normal DNA samples did not contain these mutations, indicating that they were likely to be somatic in origin. No gene on chromosome 3 other than BAP1 contained deleterious somatic mutations that were present in both tumors (Table 1).
    BAP1 encodes a nuclear ubiquitin carboxy-terminal hydrolase (UCH), one of several classes of deubiquitinating enzymes (10). In addition to the UCH catalytic domain, BAPl contains a UCH37-like domain (ULD) (11), binding domains for BRCA1 and BARD 1, which form a tumor suppressor heterodimeric complex (12), and a binding domain for HCFC1, which interacts with histone-modifying complexes during cell division (11,13,14). BAP1 also interacts with ASXL1 to form the Polycomb group repressive deubiquitinase complex (PR-DUB), which is involved in stem cell pluripotency and other developmental processes (15,16). BAP1 exhibits tumor suppressor activity in cancer cells (10,12), and BAPl mutations have been reported in a small number of breast and lung cancer samples (10,17).
    Table 1: Summary of DNA sequence alterations identified by exome capture and massively parallel sequencing of tumor DNA and matching normal peripheral blood lymphocyte DNA from uveal melanomas MM056 and MM070. (hgl9 refers to the human genomic DNA
    reference sequence used)
    To further investigate BAP1, genomic DNA from 29 additional class 2 UMs, and 26 class 1 UMs were subjected to Sanger re-sequencing of all BAP1 exons. Altogether, BAP I mutations were identified in 26 of 31 (84%) class 2 tumors, including 13 out-of-frame deletions and two nonsense mutation leading to premature protein termination, six missense mutations, four in-frame deletions, and one mutation predicted to produce an abnormally extended BAP1 polypeptide (Fig. 1A-C). Three of the missense mutations affected catalytic residues of the UCH active site (C91 and HI69), two occurred elsewhere in the UCH domain, and one affected the ULD (Fig. 1B-C). All BAPl missense mutations and in-frame deletions affected phylogenetically conserved amino acids (Fig. ID). Only one of 26 class 1 tumors contained a BAPl mutation (NB101). This case may represent a transition state in which the tumor has sustained a BAP1 mutation but has not yet converted to class 2, suggesting that BAPl mutations may precede the emergence of the class 2 signature. Somatic BAP1 mutations were also detected in two of three metastatic tumors. A summary of genetic data on uveal melanoma tumor samples are presented in Tables 2 and 3, to show information from tumors where a BAP1 mutation was identified in tumor DNA. In these tables, “g” before the mutation location indicates the genomic location, whereas “c” indicates the cDNA location.
    Table 2: Summary genetic data on uveal melanoma tumor samples in the study

    Table 3
    One copy of chromosome 3 was missing in all 17 BAP1-mutant class 2 tumors for which cytogenetic data were available, consistent with chromosome 3 loss uncovering recessive BAP1 mutations. Normal DNA from 20 patients with BAPl-rmianX class 2 primary tumors and the two with metastatic tumors was available and did not contain a BAP1 mutation, indicating that the mutations were somatic in origin. However, one germline mutation (p.E402fsX2; c,1318-1319insA) was detected in the patient with the class 1 tumor NB101 (Table 2), and this case was particularly interesting. Resequencing of this tumor revealed a deletion of a segment of exon 6 of BAP1, including its splice acceptor. This mutation is predicted to result in a premature truncation of the encoded protein (Table 2). However, the wildtype allele was present at levels similar to the mutant allele, indicating that it was disomic for chromosome 3 (Fig. 2). Hence, this case may represent a transition state in which the tumor is still class 1 but has sustained a BAP I mutation. This might suggest that the BAP1 mutations precede loss of chromosome 3 and the emergence of the class 2 signature during tumor progression. Thus, germline alterations in BAP1 can predispose to UM. Another germline (blood) mutation in exon 13 (g.chr3:52437465insT; pE566X; c,1695-1696insT leading to premature protein termination) FUM1-01 and FUM-02 was also detected.
    GNAQ mutation status was available in 15 cases. GNAQ mutations were present in 4/9 BAP1 mutant tumors and 3/6 BAP1 wildtype tumors, indicating that there was no correlation between GNAQ and BAP1 mutation status.
    UM usually metastasizes to the liver, where it is difficult to obtain specimens for research. However, we were able to obtain sufficient DNA from three UM liver metastases for analysis. BAPl mutations were detected in two of the three metastatic tumors, supporting the hypothesis that cells mutant for BAP1 are indeed the ones responsible for metastasis (Table 2). NB071M contained a nonsense mutation (Q36X), and MM152M contained an out-of-frame deletion (p.E693fsX13). Both mutations are predicted to cause premature protein truncation. Primary tumor DNA on either case was unavailable.
    Quantitative RT-PCR showed that BAPl mRNA levels were significantly lower in class 2 tumors compared to class 1 tumors (P<0.0001) (Fig. 3A). Truncating mutations were associated with significantly lower mRNA levels than missense mutations (P=0.001) (Fig. 3B), consistent with nonsense mediated mRNA decay in the former group. Class 2 tumors in which BAPJ mutations were not identified expressed very low levels of BAP1 mRNA (Fig. 3B).
    To determine whether the low BAP1 mRNA levels in class 2 tumors without detectable BAP I mutations may be explained by DNA methylation, the inventors performed a preliminary analysis of DNA methylation of BAP1. This did not reveal a convincing difference between class 1 and class 2 tumors. However, analysis of the BAP I promoter was limited by an unusually complex CpG island that will require further work to resolve. Thus, a role for methylation in class 2 tumors in which BAPJ mutations were not found cannot be ruled out. However, with almost 85% of class 2 tumors harboring mutations, methylation is not expected to be a major mechanism of BAPJ inactivation. An alternative explanation is that these tumors may contain very large deletions of the BAPJ locus not detectable by the sequencing method used. Immunofluorescence revealed abundant nuclear BAP1 protein in two class 1 tumors but virtually none in four BAPJ mutant class 2 tumors (Fig. 4). This was expected for the two tumors with mutations expected to cause premature protein terminations (MM 091 and MM 100), but it was surprising for the two tumors with missense mutations (MM 071 and MM 135) and suggests that these mutations lead to protein instability. RNAi-mediated knock down of BAP 1 in 92.1 UM cells, which did not harbor a detectable BAPJ mutation, recapitulated many characteristics of the de-differentiated class 2 UM phenotype (18). Cells transfected with control siRNA exhibited typical melanocytic morphology, including dendritic projections and cytoplasmic melanosomes (Fig. 5), whereas cells transfected with BAP1 siRNA lost these features, developed a rounded epithelioid morphology and grew as multicellular non-adherent spheroids, strikingly similar to the features of class 2 clinical biopsy samples (Fig. 5). Microarray gene expression profiling of 92.1 UM cells transfected with control versus BAP1 siRNA showed that most of the top genes that discriminate between class 1 and class 2 tumors shifted in the class 2 direction in BAP1 depleted cells compared to control cells (Fig. 6). Similarly, depletion of BAP1 shifted the gene expression profile of the multi- gene clinical prognostic assay towards the class 2 signature (Fig. 7 A). BAP1 depletion caused a reduction in mRNA levels of neural crest migration genes (ROBO1), melanocyte differentiation genes (CTNNB1, EDNRB and SOX10) and other genes that are down-regulated in class 2 tumors (LMCD1 and LTA4H) (18). In contrast, BAP1 depletion caused an increase in mRNA levels of CDH1 and the proto oncogene KIT, which are highly expressed in class 2 tumors (19). Similarly, mRNA transcripts of KIT, MITF and PAX3, whose protein products are associated with proliferation of pre-terminally differentiated melanocytes and have oncogenic effects when overactive in melanoma (20-22), were significantly up-regulated by BAP1 depletion (Fig. 7B). Similar results were seen in other UM cell lines and with an independent BAP1 siRNA (Figure 7C).
    GNAQ mutations occur early in UM and are not sufficient for malignant transformation (4), but they may create a dependency of the tumor cells on constitutive GNAQ activity. In contrast, BAP1 mutations occur later in UM progression and coincide with the onset of metastatic behavior. Thus, simultaneous targeting of both genetic alterations might have synergistic therapeutic effects. One potential strategy to counteract the effects of BAP1 mutation would be to inhibit the RING1 ubiquinating activity that normally opposes the deubiquinating activity of BAP1 (16). The present findings strongly implicate mutational inactivation of BAP 1 as a key event in the acquisition of metastatic competence in UM, and they dramatically expand the role of BAP1 and other deubiquitinating enzymes as potential therapeutic targets in cancer.
    Materials and Methods for Example 1.
    Patient materials:
    Acquisition of patient material (matched tumor and normal samples) has been described elsewhere (25) (Table 4). This study was approved by the Human Studies Committee at Washington University (St. Louis, MO), and informed consent was obtained from each subject. Tumor tissue was obtained immediately after eye removal, snap frozen, and prepared for RNA and DNA analysis. UM metastases were collected from liver biopsies at the time of metastatic diagnosis. All samples were histopathologically verified. Genomic DNA from tumors was prepared using the Wizard Genomic DNA Purification kit (Promega, Madison, WI). DNA from blood was isolated using the Quick Gene DNA whole blood kit S (Fugifilm, Tokyo, Japan). RNA was isolated using the PicoPure kit (including the optional DNase step). All RNA samples were converted to cDNA using the High Capacity cDNA Reverse Transcription kit from Applied Biosystems (Applied Biosystems Inc., Foster City, CA) following the manufacturer’s protocol.
    Table 4: Summary of clinical and pathologic data on uveal melanoma patients in the study
    Cell Culture: 92.1 (generous gift from Dr. Martine Jager) and Mel290 (generous gift of Dr. Bruce Ksander) human UM cells were grown in RPM1-1640 (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics. Transfections were performed with HiPerFect (Qiagen, Valencia, CA) and Silencer® Select BAP1 (sl5820 and sl5822) or Control #1 siRNA (Ambion, Austin, TX). Knockdown of BAP 1 protein levels was confirmed by western blot with antibodies that recognize the BAP1 protein (Santa Cruz, Santa Cruz, CA) and alpha-tubulin (Sigma-Aldrich, St. Louis, MO). Cell morphology data were collected by digital imaging of phase contrasted cells at 200X magnification. After five days, transfected cells were harvested for RNA and protein analyses.
    RNA and protein analysis:
    All RNA samples were converted to cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems Inc., Foster City, CA) and then pre-amplified for 14 cycles with pooled probes and TaqMan Pre-Amp Master Mix following manufacturer’s protocol. Expression of mRNA for individual genes was quantified using the 7900HT Real-Time PCR System with either custom-made primers and iQ SYBRGreen SuperMix (Bio-Rad Laboratories Inc, Hercules, CA) for CTNNB1, EDNRB, KIT, SOX 10 and UBC (endogenous control) or TaqMan® Gene Expression Assays and Gene Expression Master Mix (Applied Biosystems Inc., Foster City, CA) for BAP1, CDH1, LTA4H, LMCD1 and ROBOl. The 15-gene prognostic assay for assignment of tumors to class 1 or class 2 was performed as described elsewhere (26). Staining with BAP1 antibody (201C, the generous gift of Dr. Richard Baer) was performed on 4pm sections obtained from paraffin-embedded tissue blocks. Statistical significance was assessed using Student’s t-test with Medcalc software version 10.4.0.0.
    Exome capture and DNA sequencing: gDNA libraries were prepared using the Illumina Pair-End Gènomic DNA Sample Prep Kit (Cat # PE-102-1001) according to the manufacturer’s instructions. Each paired-end library was enriched for exomic sequence using the Roche-Nimblegen SeqCap EZ Exome kit (Cat # 5977215001). The captured genomic DNA fragments were sequenced with the Illumine Genome Analyzer II (GAIIx) for 76-cycles (one lane per sample).
    Sequence analysis:
    Illumina Solexa 76 cycle paired-end sequencing data was received as compressed raw reads exported from the Illumina software pipeline (>1.3). Raw reads were parsed into FASTQ format, and the original raw reads were archived. The FASTQ files were aligned to the hgl9 version of the human reference sequence using bowtie. The Bowtie software (vO.12.3) was compiled with g++ (v4.3.3) using the additional compilation switches “-03 —mtune=amdfamlO” with pthreads enabled. Mapped reads were directed to a SAM format file for downstream analysis, and unmapped reads were exported to a separate file.
    Variant bases were extracted with the samtools software (vO. 1.7) with the additional samtools.pl VarFilter switch “-D 1000”, and only positions with at least 8 reads and a SNP quality score of at least 20 were considered for further analysis. Filtered variants were stored in a relational database table (MySQL v5.0.75). Known SNPs (dbSNP130 on hgl9; exact location known, single base changes) and variants found in 8 HapMap samples (27) were filtered from our variant lists using database queries.
    Candidate variants in coding sequence of genes mapping to chromosome 3 were identified and manually annotated for amino acid changes. The 30 base pairs around coding variants were used to query genomic sequence (hgl9) to determine if the sequence mapped to multiple genomic locations. Regions with multiple identical mappings were removed. This included the removal of sequences mapping to pseudogenes.
    HapMap Variants: FASTQ files for 8 HapMap individual’s exomes (NA19240, NA19129, NA18956, NA 18555, NA 18517, NA18507, NA12878, NA 12156) were downloaded from the NCBI Short Read Archive (3) (accession SRP000910). All reads for each individual were aligned to hgl9 (see above). Multiple sequencing runs were merged into one. SAM formatted file. Variants were extracted with samtools (see above) and stored in a relational database table.
    Sequence Validation:
    Oligonucleotide primers were designed from intronic sequences to amplify all coding sequence of BAPl with PCR (Table 5). Genomic DNA of tumor and blood from the same patient were subjected to PCR amplification with routine approaches. Sanger DNA sequencing was performed with routine methods to validate variants found with NextGen sequencing, and to query all tumor and matched normal samples for all coding sequences of BAP1.
    Table 5 Sequencing primers
    DNA m ethylation analysis:
    Following bisulfite treatment and amplification of genomic DNA from region chr3:52,442,270-52,442,651 (hgl9) with bisulfite specific primers, methylation of this region was evaluated with Sequenom's MassARRAY Epityper technology in the inventors’ core facility (hg.wustl.edu/gtcore/methylation.html). Controls for 0% and 100% methylation were also included. Nine class 1 tumors and ten class 2 tumors were analyzed.
    Molecular classification:
    Gene expression data from custom TaqMan Low-Density Arrays were used to determine tumor class assignment, as previously described (26). Briefly, molecular class assignments were made by entering the 12 ACt values of each sample into the machine learning algorithm GIST 2.3 Support Vector Machine (SVM) (bioinformatics.ubc.ca/svm). SVM was trained using a set of 28 well-characterized uveal melanomas of known molecular class and clinical outcome. SVM creates a hyperplane between the training sample groups (here, class 1 and class 2), then places unknown samples on one or the other side of the hyperplane based upon their gene .; expression profiles. Confidence is measured by discriminant score, which is inversely proportional to the proximity of the sample to the hyperplane.
    Loss of heterozygosity for chromosome 3 was determined using 35 SNPs with minor allele frequencies >0.4 at approximate intervals of 6 megabases across the euchromatic regions of chromosome 3 using the MassARRAY system (Sequenom Inc, San Diego, CA), as previously described (25).
    Microarray gene expression profiling
    Expression data, received as flat files exported from the Illumina software, were analyzed in R (v2.10.1) using Bioconductor packages (Biobase v2.6.1). Non-normalized data were imported into the R environment using the beadarray package (vl.14.0). Expression values were quantile normalized and log2 transformed using limma (v3.2.3). Each of three independent siRNA knockdown experiments as well as each of three siRNA control experiments was treated as biological replicates. Linear models were fitted to the expression values and expression differences calculated using a contrast comparing the difference in knockdown / control experiments. For each gene log2 fold change, average expression, and moderated t-statistics were calculated for the defined contrast using the “ebayes” function of the limma package. Nominal p-values were corrected for multiple comparisons using the Benjamini and Hochberg false discovery rate method. Heatmaps were generated using the heatmap function of the R base stats package. Quantile normalized data were filtered down to 29 known discriminating genes plus BAP1. Heatmap colors were generated using the maPalette function of the marray Bioconductor package (vl .24.0), specifying green as low, red as high, and black as mid color values with 20 colors in the palette.
    Example 2. Indirect methods for detecting BAP1 loss.
    BAP1 loss leads to biochemical changes in the cell, such as histone H2A ubiquitination, that may be easier to detect and monitor than direct BAP1 activity. BAP1 stable knockdown cells were produced using lentiviral vectors expressing a short hairpin RNA (shRNA) against BAP1 (Fig. 8). Both transient and stable knockdown of BAP 1 lead to increased ubiquitination of histone H2A (Fig. 9). Thus, the measurement of histone H2A ubiquitination levels could be used as a surrogate indicator of BAP 1 loss.
    Stable knockdown of BAP 1 also leads to a decrease in the RNA levels of melanocyte differentiation genes (Fig. 10). Transient knockdown of BAP1 leads to a decrease in proliferation (Fig. 11) as measured using a BrdU assay. In addition, loss of BAP 1 in culture leads to decreased cell motility (Fig. 12) and a decreased growth in soft agar (Fig. 13). On the other hand, loss of BAP 1 leads to an increased ability to grow in clonegenic assays (Fig. 14) and increased migration towards a serum attractant (Fig. 15).
    Example 3. Loss of BAP1 and tumor behavior in mouse.
    Uveal melanoma cells stably knocked down for BAP1 using lentiviral expression of shRNA against BAP1 were implanted into mouse flank. Cells deficient for BAP1 grew less rapidly in the mouse flank compared to control cells infected with lentiviral vector expression shRNA against GFP (Fig. 16). After injection into the tail vein of mice, knockdown BAP1 cells exhibited decreased tumor growth (Fig. 17). These findings, coupled with the cell culture experiments above, indicate that the major effect of BAP1 loss in uveal melanoma is not increased proliferation, migration, motility or tumorigenicity upon flank injection.
    Example 4. BAP1 mutations in cutaneous melanoma.
    BAP1 mutations may also be analyzed in cutaneous melanoma tumors as described in the examples and materials and methods above. Cutaneous melanoma tumors analyzed may be atypical moles (Dysplastic Nevus), basal cell carcinomas, blue nevi, cherry hemangiomas, dermatofibromas, halo nevi, keloid and hypertrophic scars, keratoacanthomas, lentigos, metastatic carcinomas of the skin, nevi of ota and ito, melanocytic nevi, seborrheic keratosis, spitz nevi, squamous cell carcinomas, and vitiligos.
    Cutaneous melanoma samples and matching normal DNA from peripheral tissue may be analyzed for inactivating mutations in BAP1 using exome capture followed by massively parallel sequencing. Sanger re-sequencing of all BAP1 exons may also be used to further investigate BAP1 mutations. Normal DNA from patients with cutaneous melanoma may be analyzed to determine if BAP 1 mutations are somatic or germline in origin. Germline alterations in BAP I may predispose to cutaneous melanoma.
    Mutation status of other genes may also be analyzed in the cutaneous melanoma samples. For example, GNAQ, BRAF, KIT or NRAS mutation status may be determined, and compared to the results obtained for uveal melanoma samples described above.
    BAP] mRNA levels may be analyzed using quantitative RT-PCR. If BAP 1 mRNA levels are lower in cutaneous melanoma samples than in normal samples, DNA methylation of the BAP l locus may be analyzed to determine if the lower mRNA levels may be explained by DNA methylation. BAP1 protein levels in various tumor and normal samples may also be analyzed using immunofluorescence.
    BAP1 may be knocked down in cell culture using RNAi. BAP1 mRNA and protein expression levels, cell morphology, and gene expression profiling using microarrays may be used to characterize cell cultures after knock down of BAP1 expression.
    Example 5. BAP1 mutations in the germline.
    BAP I mutations may be detected in germline DNA as a means of detection of affected family members in hereditary syndromes. Germline DNA may be any normal patient DNA such as DNA extracted from peripheral blood lymphocytes or buccal swabs. Standard Sanger sequencing may be used as described in Example 1 above.
    Example 6. BAP1 as a marker of circulating tumors.
    BAP1 mutations may be detected in peripheral blood as a marker of circulating tumor cells. This may be performed using targeted capture and deep sequencing of BAP I in blood samples from patients. Targeted capture may be used in combination with NexGen sequencing to provide a very powerful approach for rapidly sequencing genomic regions of interest. The Agilent SureSelect enrichment system is one such method that allows enrichment for genomic regions from a sample of total human genomic DNA. The Agilent system also supports multiplexing of samples in the sequencing reaction, reducing the overall cost of the procedure.
    A l-2Mb genomic region harboring BAP1 may be captured. This may allow detection of deletions of several exons or the entire gene, as well as the smaller mutations identified in the examples above. Targeted capture with Agilent’s SureSelect system starts with querying their eArray web site for a region of interest. This is designed to identify an overlapping set of oligonucleotides (120 mers) over a particular region, but without regions containing repeat (which confound the selection procedure). Agilent synthesizes biotinylated cRNA oligonucleotides and provides them in solution (the probe). l-3mg of genomic DNA (the driver) may then be sheared to -200 bp, end-repaired, A-tailed and ligated to adaptors for Illumina paired-end sequencing.
    Libraries may be amplified for 6-8 cycles to produce at least 500 ng of product. The product may be hybridized to the oligonucleotide baits to enrich for targeted regions then the resultant hybrids may be captured onto streptavidin-labeled magnetic beads. This may be followed by washing and digestion of the RNA bait. Resultant selection products may be subjected to PCR for 12-14 cycles. At this stage, unique oligonucleotide identifiers may be incorporated into the selected DNAs and their concentrations are determined. These are then adjusted it to a final concentration of 15 pM for sequencing. In this way multiple samples may be loaded onto one flow cell lane on the Sequencer. Currently, 12 samples may be run in a single lane of an Illumina HiSeq2000. Illumina and Nimblegen are also developing similar technologies that could be used for targeted capture. This technology was originally developed by Dr. Michael Lovett (8), and instead of oligonucleotides, bacterial artificial chromosomes (BACs) were used as probes. Hence, there are a variety of ways of identifying the genomic target of interest.
    Sequences obtained from targeted capture may be analyzed in a similar manner to those obtained from exome-capture and as described elsewhere.
    This may potentially be used for (1) non-invasive determination of patients with class 2 high risk uveal melanomas, (2) assessment of circulating tumor burden for uveal, cutaneous or other BAP1 mutant cancer, and (3) to monitor response to therapy.
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    权利要求:
    Claims (50)
    [1]
    A method for detecting a metastatic cancer biomarker in a subject, wherein detecting the biomarker comprises identifying a mutation in a BAP1-n ucl cotide cell in a tumor sample cell obtained from the subject.
    [2]
    The method of claim 1, which compares the genomic DNA sequence of a cell of the tumor sample with the genomic DNA sequence of a cell of a non-tumor sample of the same person and identifying a difference on at least one site in the sequence of the tumor sample compared to the same site in the sequence of the non-tumor sample.
    [3]
    The method of claim 1 or 2, comparing the genomic DNA sequence of a cell of the tumor sample with the sequence SEQ ID NO: 3 and identifying a difference at least one location in the sequence of the tumor sample compared to the equivalent site in SEQ ID NO: 3.
    [4]
    The method of claim 1, which comprises obtaining cDNA from a BAP1 mRNA from a tumor sample cell, comparing the cDNA sequence with a non-tumor sample cell from a cell of a non-tumor sample and identifying a difference at least one location in the sequence of the tumor sample compared to the equivalent location in the sequence of the non-tumor sample.
    [5]
    The method according to claim 1 or 4, which comprises obtaining cDNA from a BAP1 mRNA from a cell of the tumor sample, comparing the cDNA sequence with the sequence SEQ ID NO: 2 and identifying a difference on at least one site in the tumor sample sequence compared to the equivalent site in SEQ ID NO: 2.
    [6]
    The method of any one of the preceding claims, wherein the mutation is an inactivation mutation.
    [7]
    A method according to any one of the preceding claims, which identifying at least one mutation in one or more of the exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 and / or 17 of the δ / fPi nucleotide sequence.
    [8]
    The method of claim 7, comprising identifying one or more mutations selected from: a. A truncation mutation in exon 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 16 or 17 from BAP1; b. a miss mutation in exon 5, 6, 7 or 16; c. an in-frame deletion in exon 10, 15 or 16; or d. a termination translecture in exon 17.
    [9]
    The method of claim 7, wherein the truncation mutation causes a nonsense mutation in a BAP1 protein encoded by the BAPI nucleotide sequence selected from Q36X, W196X and Q253X.
    [10]
    The method of claim 7, wherein the miss-mutation results in a substitution in a BAPI protein encoded by the BAPI nucleotide sequence selected from C91G, C91W, G128R, H169Q, S172R or D672G.
    [11]
    The method of claim 7, wherein the in-frame deletion results in a deletion in a BAPI protein encoded by the BAPI nucleotide sequence selected from E283-S285del, E631-A634del or R666-H669del.
    [12]
    The method of any one of the preceding claims for determining the risk of melanoma metastasis in the subject, wherein the presence of the biomarker indicates an increased risk of metastasis in the subject.
    [13]
    The method of any one of claims 1 to 11 for detecting the presence of metastatic melanoma in the subject, wherein the presence of the mutation indicates the presence of metastatic melanoma.
    [14]
    A method according to claim 12 or 13, wherein the melanoma is uvaleal melanoma.
    [15]
    The method of any one of claims 12-14, wherein the sample comprises a tumor sample.
    [16]
    The method of claim 15, wherein the sample can be obtained from a primary tumor or from a circulating tumor cell.
    [17]
    The method of claim 16, wherein the circulating tumor cell can be obtained from a body fluid.
    [18]
    A method according to any one of the preceding claims, which comprises the use of a nucleic acid amplification reaction to amplify a region of the 2 -PF nucleic acid sequence comprising a mutation.
    [19]
    The method of claim 18, wherein the amplification reaction is a polymerase chain reaction (PCR).
    [20]
    The method of claim 18, wherein a mutation present in the nucleotide sequence of the amplified region of BAP1 differs from an equivalent region of SEQ ID NO: 3.
    [21]
    A method for determining the risk of melanoma metastasis and / or the presence of metastatic melanoma in a subject, the method comprising: a. Analyzing the degree of BAP1 activity in a cell of a tumor sample obtained from the person, and b. determining whether the rate of BAP1 activity has decreased compared to the activity in a cell of a non-tumor sample obtained from the subject, with a decreased rate of BAP1 activity indicating an increased risk of metastasis in the subject and / or indicates the presence of a metastatic melanoma in the person.
    [22]
    The method of claim 21, wherein the melanoma is uvaleal melanoma.
    [23]
    The method of claim 21 or 22, wherein the sample comprises a tumor sample.
    [24]
    The method of claim 23, wherein the sample can be obtained from a primary tumor or from a circulating tumor cell.
    [25]
    The method of claim 24, wherein the circulating tumor cell can be obtained from a body fluid.
    [26]
    A detection means for detecting the presence of a metastatic cancer biomarker in a subject, comprising a means for detecting a mutation in the BA PI-nucleotide sequence in a cell of a tumor sample obtained from the subject.
    [27]
    The detection means of claim 26, wherein the mutation detecting means comprises means for detecting a difference between the genome ΌΝΑ-ΒΑΡΙ sequence of a tumor sample cell and the genomic DNA-5 / 1PI- sequence of a cell from a non-tumor sample from the same person.
    [28]
    The detection means according to claim 26, wherein the detection of a mutation comprises means for detecting a difference between the genomic DN A-BAPI sequence of a tumor sample cell and the sequence SEQ ID NO: 3.
    [29]
    The detection means according to claim 26, wherein the detection of a mutation comprises means for detecting a difference between the sequence of a cDNA obtained from a 2 P / mRNA from a tumor sample cell and the sequence of a cDNA obtained from a Z4P / mRNA from a cell from a non-tumor sample from the same person.
    [30]
    The detection means according to claim 26, wherein the detection of a mutation comprises means for detecting a difference between the sequence of a cDNA obtained from a Z P / mRNA of a tumor sample cell and the sequence of SEQ ID NO: 2.
    [31]
    The detection means of any one of claims 26-30, wherein the mutation is in one or more of the exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 and / or 17 of the Z4P7 nucleotide sequence.
    [32]
    The detection means of claim 31, wherein the mutation is selected from: a. A truncation mutation in exon 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 16 or 17 of the BAP1 gene ; b. a miss mutation in exon 5, 6, 7 or 16; c. an in-frame deletion in exon 10, 15 or 16; or d. a termination translecture in exon 17.
    [33]
    The detection means of claim 32, wherein the truncation mutation causes a nonsense mutation in a BAP1 protein encoded by the BAPI nuclide sequence selected from Q36X, W196X and Q253X.
    [34]
    The detecting agent of claim 32, wherein the miss-mutation results in a substitution in a BAPI protein encoded by the BAPI-n ucl cotide sequence selected from C91G, C91W, G128R, H169Q, S172R or D672G.
    [35]
    The detection means according to claim 32, wherein the in-frame deletion results in a deletion in a BAPI protein encoded by the 7 4P / nucleotide sequence selected from E283-S285del, E631-A634del or R666-H669del.
    [36]
    The detection means of any one of claims 26-35, which comprises a system for performing a polymerase chain reaction on the sample.
    [37]
    The detecting agent of claim 37, wherein the polymerase chain reaction amplifies a region of the 5 P / nucleotide sequence comprising a mutation.
    [38]
    Use of a detection means according to any of claims 26-37 in a method according to any of claims 1-25.
    [39]
    39. A metastatic melanoma biomarker comprising a BAP1 nucleotide sequence comprising at least one mutation as compared to SEQ ID NO: 3.
    [40]
    The biomarker of claim 39, which is a biomarker for the risk or presence of a metastatic melanoma in a subject, wherein the mutation is contained in a β4P7 nucleotide sequence in a tumor sample obtained from the subject.
    [41]
    The biomarker of claim 39 or 40, wherein the mutation is in one or more of the exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16 and / or 17 of the BAP1 nucleotide sequence.
    [42]
    The biomarker of claim 41, wherein the mutation is selected from: a. A truncation mutation in exon 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 16 or 17 of the BAP1 gene ; b. a miss mutation in exon 5, 6, 7 or 16; c. an in-frame deletion in exon 10, 15 or 16; or d. a termination translecture in exon 17.
    [43]
    The biomarker of claim 42, wherein the truncation mutation causes a nonsense mutation in a BAP1 protein encoded by the 774P / nucleotide sequence selected from Q36X, W196X and Q253X.
    [44]
    The biomarker of claim 42, wherein the miss-mutation results in a substitution in a BAP1 protein encoded by the BAPI nucleotide sequence selected from C91G, C91W, G128R, H169Q, S172R or D672G.
    [45]
    The biomarker of claim 42, wherein the in-frame deletion results in a deletion in a BAPI protein encoded by the βAP / nucleotide sequence selected from E283-S285del, E631-A634del or R666-H669del.
    [46]
    46. A metastatic melanoma biomarker comprising a BAP1 amino acid sequence comprising at least one mutation as compared to SEQ ID NO: 1.
    [47]
    The metastatic melanoma biomarker according to claim 46, which is encoded by a Z AP / nucleotide protocol comprising at least one mutation compared to SEQ ID NO: 2 and / or SEQ ID NO: 3.
    [48]
    An antibody that specifically recognizes a biomarker according to claim 46 or 47.
    [49]
    A kit comprising a detecting agent according to any of claims 26-37 and / or a biomarker according to any of claims 39-47 and / or an antibody according to claim 48.
    [50]
    Use of an antibody according to claim 48 or a kit according to claim 49 in a method according to any one of claims 21-25.
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    同族专利:
    公开号 | 公开日
    GB2484003B|2013-04-24|
    US20160362750A1|2016-12-15|
    US9441277B2|2016-09-13|
    GB2497444A|2013-06-12|
    GB201116471D0|2011-11-09|
    US9133523B2|2015-09-15|
    GB201302520D0|2013-03-27|
    US20160053330A1|2016-02-25|
    CA2810039A1|2012-03-29|
    US20120077682A1|2012-03-29|
    GB2484003A|2012-03-28|
    WO2012040614A1|2012-03-29|
    NL2007467C2|2012-04-24|
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    US38569610P| true| 2010-09-23|2010-09-23|
    US38569610|2010-09-23|
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