# Patent

## Title
Compositions and methods for detecting cancer metastasis

## Abstract

The present invention encompasses compositions and methods for detecting cancer metastasis.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the priority of U.S. Ser. No. 13/243,572, filed Sep. 23, 2011, which claims the priority of U.S. provisional application No. 61/385,696, filed Sep. 23, 2010, each of which is hereby incorporated by reference in its entirety.


GOVERNMENTAL RIGHTS
This invention was made with government support under R01 CA125970 awarded by the National Cancer Institute, and under P30 EY02687c and AR007279-31A1 awarded by the National Institutes of Health. The government has certain rights in the invention.


REFERENCE TO SEQUENCE LISTING
A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f).
FIELD OF THE INVENTION
The invention encompasses compositions and methods for detecting 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 determining the risk of metastasis in a subject. Generally speaking, the method comprises collecting a sample from a subject, analyzing the BAP1 nucleotide and/or BAP1 amino acid sequence from a cell in the sample, and identifying the presence of a mutation in the BAP1 nucleotide and/or BAP1 amino acid sequence. The presence of the mutation indicates an increased risk for metastasis in the subject.
Another aspect of the invention encompasses a method for determining the risk of metastasis in a subject, where the method comprises determining the level of BAP1 activity in a sample from a subject, wherein in decreased BAP1 activity indicates an increased risk for metastasis in the subject.
Still another aspect of the present invention encompasses a method for detecting the presence of metastatic cancer. Generally speaking, the method comprises collecting a sample from a subject, analyzing the BAP1 nucleotide and/or BAP1 amino acid sequence in the sample, and determining the presence of a mutation in the BAP1 nucleotide and/or BAP1 amino acid sequence. The presence of the mutation indicates the presence of metastatic melanoma.
Yet another aspect of the present invention encompasses a method for detecting the presence of metastatic cancer, the method comprising determining the level of BAP1 activity in a sample from a subject, wherein decreased BAP1 activity indicates the presence of metastatic cancer in the subject.
Still yet another 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 BAP1 nucleotide sequence 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.
Certain aspects of the present invention encompass 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 nucleotide sequence 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.
Several aspects 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 application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.
FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D illustrate that inactivating mutations in BAP1 occur frequently in uveal melanomas. (FIG. 1A) 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. (SEQ ID NOs: 44-47) (FIG. 1B) Map of BAP1 gene and location of BAP1 mutations. BAP1 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, His169 and Asp184) 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). (FIG. 1C) 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 C91W, H169Q and S172R represents a suicide inhibitor, illustrating the critical location of these mutations for catalytic activity. (FIG. 1D) Conservation of BAP1 in regions containing mutated amino acids. Alignments of segments of BAP1 homologs harboring mutated amino acids (missense or in-frame deletions) are shown for the indicated species. (SEQ ID NO: 48-60) Amino acid numbering is on the basis of human BAP1 (SEQ ID NO: 1). Positions of mutated amino acids are indicated with asterisks.
FIG. 2 depicts Sanger sequence trace of one end of the mutated region of NB101. The breakpoint at one end of the insertion/deletion is indicated with an arrow. Wild type sequence is indicated below the NB101 sequence. (SEQ ID NO: 61-62)
FIG. 3A and FIG. 3B depict bar graphs of BAP1 mRNA levels. (FIG. 3A) is a graph of BAP1 mRNA levels measured by quantitative RT-PCR in 9 non-metastasizing class 1 UMs and 28 metastasizing class 2 Ums, and (FIG. 3B) is a graph showing the relationship between BAP1 mRNA levels (measured by quantitative RT-PCR) and type of BAP1 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 BAP1 mutations were detected.
FIG. 4 depicts a series of photographs illustrating that BAP1 mutations disrupt BAP1 protein expression in human uveal melanoma samples. Immunofluorescence analysis of BAP1 protein expression was performed on archival tumor specimens from uveal melanomas of known class and BAP1 mutation status, as indicated. All images were captured at 40× and are represented at the same magnification. Scale bar, 10 microns. No BAP1 expression is seen in the Class 2 metastasizing UM cells (MM100, 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 BAP1 acquire properties that are typical of metastasizing class 2 tumor cells. Phase contrast photomicrographs of 92.1 uveal melanoma cells transfected with BAP1 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 BAP1 siRNAs.
FIG. 7A, FIG. 7B and FIG. 7C depict a diagram, a Western blot, and a bar graph, respectively, showing the effects of BAP1 depletion by siRNA. 92.1 cells transfected with BAP1 siRNA and evaluated after five days. (FIG. 7A) BAP1 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 BAP1 knockdown on gene expression signature. The small spheres represent the training set of known class 1 (blue) and class 2 (red) tumors. Large spheres represent the control-transfected (gray) and BAP1 siRNA transfected (red) 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. (FIG. 7B) 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. (FIG. 7C) RNAi mediated depletion of BAP1 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. 8A, FIG. 8B, FIG. 8C and FIG. 8D depict a bar plot (FIG. 8A), a Western blot (FIG. 8B), and two micrographs, respectively, showing BAP1 levels in shGFP (FIG. 8C) and shBAP1 (FIG. 8D) cells.
FIG. 9A and FIG. 9B depict Western blots showing increased ubiquitination of histone H2A in siBAP1 (FIG. 9A) and shBAP1 (FIG. 9B) cells compared to controls. FIG. 9C and FIG. 9D depict fluorescence immunohistochemical micrographs showing increased ubiquitination of histone H2A in shBAP1 (FIG. 9D) cells compared to control cells (FIG. 9C).
FIG. 10A and FIG. 10B depict bar plots from two experiments showing decreased RNA levels of melanocyte differentiation genes in BAP1 stable knockdown cells.
FIG. 11A, FIG. 11B and FIG. 11C depict plots showing that transient knockdown of BAP1 using siRNA (FIG. 11A) leads to a decrease in cell proliferation. Transient knockdown of BAP1 using shRNA did not alter cell proliferation (FIG. 11B and FIG. 11C).
FIG. 12A and FIG. 12B depict micrographs (FIG. 12A) and a bar plot (FIG. 12B) showing that loss of BAP1 in culture leads to decreased cell motility.
FIG. 13A, FIG. 13B and FIG. 13C depict images of shGFP (FIG. 13A) or shBAP1 (FIG. 13B) culture plates and a bar plot (FIG. 13C) showing that loss of BAP1 leads to decreased growth in soft agar.
FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14 D depict bar plots from four experiments showing that loss of BAP1 leads to an increased ability to grow in clonegenic assays.
FIG. 15 depicts a bar plot showing that loss of BAP1 leads to increased migration towards a serum attractant.
FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E and FIG. 16F depict plots showing that loss of BAP1 in culture leads to decreased tumor growth in the mouse flank. FIG. 16A and FIG. 16D depict a decrease in weight of the tumor with loss of BAP1. FIG. 16B and FIG. 16E depict a decrease in volume of the tumor with loss of BAP1. FIG. 16C and FIG. 16F depict a decrease of BAP1 RNA expression in the presence of BAP1 shRNA.
FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D depict plots showing that loss of BAP1 in culture leads to decreased tumor growth in the mouse after tail vein injection.
FIG. 18 depicts an illustration of a family with germline BAP1 mutations.
FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E and FIG. 19F depict the genomic sequence of BAP1. Exons are bolded, and select mutations are highlighted (see Table 2).



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for determining the risk of tumor metastasis in a subject. Additionally, the invention provides a method for detecting the presence of a tumor metastasis in a subject. The invention further 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, identifying a mutation in a BAP1 protein sequence, or identifying a decrease in BAP1 activity in a sample obtained from the 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.
I. Method for Determining the Risk of Tumor Metastasis
One aspect of the present invention encompasses a method for determining the risk of tumor metastasis in a subject. In one embodiment, the method comprises collecting a sample from a subject, analyzing the BAP1 nucleotide and/or BAP1 amino acid sequence from a cell in the sample, 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 BAP1, the promoter of BAP1, the 5′ untranslated region of BAP1, the 3′ untranslated region of BAP1, or any other regulatory region for the BAP1 gene, such that the mutation decreases the expression of BAP1 mRNA, synthesis of BAP1 protein, or enzymatic activity of BAP1 when compared to the sequence of BAP1 from a non-tumor cell of the same individual. The presence of such a BAP1 mutation indicates an increased risk for metastasis in the subject. 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: 1 (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.
In another embodiment, the method comprises collecting a sample from a subject, and analyzing the level of BAP1 activity in the sample, where a decrease in BAP1 activity indicates an increased risk for metastasis in the subject.
Each of these embodiments are discussed in more detail below.
(a) Analyzing the BAP1 Sequence to Determine Risk of Tumor Metastasis
One embodiment comprises analyzing the BAP1 nucleotide sequence and/or BAP1 amino acid sequence of a sample collected from a subject as described in section (c) below. Typically, analyzing the BAP1 nucleotide sequence may comprise identifying a mutation in the BAP1 nucleotide sequence. As detailed above, “a mutation in the BAP1 sequence,” refers to a mutation in an exon of BAP1, 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 BAP1 gene (e.g. a splice acceptor site), such that the mutation decreases the expression of BAP1 mRNA, synthesis of BAP1 protein, or enzymatic activity of BAP1 when compared to the sequence of BAP1 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 mutation. For instance, in one embodiment, the mutation may cause a premature truncation of BAP1. Alternatively, the mutation may affect a conserved amino acid in the ubiquitin carboxy-terminal hydrolase (UCH) domain or the UCH37-like domain (ULD) (for instance, see FIG. 1B). Such a mutation may be identified using methods commonly known in the art. For instance, see the Examples. Generally speaking, all or a portion of the BAP1 nucleic acid sequence may be sequenced and compared to the wild-type genomic sequence (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: 1) 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 (RefSeq #NM_004656; 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), or mutated nucleotide sequences which differ from the BAP1 nucleotide sequence from a non-tumor cell of the subject. 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. Non-limiting examples of amplification techniques may include 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, 3SR, ramification amplification (Zhang et al. (2001) Molecular Diagnosis 6 p141-150), 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 BAP1 may be an inactivating mutation, i.e., expression levels of BAP1 mRNA and/or synthesis of 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 synthesis 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 BAP1 may be found in exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 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 BAP1 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 BAP1 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 be a missense mutation selected from 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.
(b) Analyzing the Level of BAP1 Activity
In other embodiments of the invention, the level of BAP1 activity in a sample is analyzed. The “level of BAP1 activity” may refer to the level of expression of BAP1 mRNA, the level of synthesis of BAP1 protein, or the level of enzymatic activity of BAP1 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 BAP1 activity is decreased about 50% to about 100% compared to a non-tumor cell from the same individual. In other embodiments, the level of BAP1 activity is decreased from about 60% to about 100% compared to 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 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 a non-tumor cell from the same individual.
Determining the level of expression of a BAP1 nucleic acid sequence, comprises, in part, measuring the level of BAP1 mRNA expression 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 techniques and 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 BAP1 synthesis in a sample indicates an increased risk of metastasis in the subject. Methods of measuring the synthesis of BAP1 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 J C, 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 Y J, 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 N S, Wilkinson K D. 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.).
(c) Collecting a Sample from a Subject
A method of the invention comprises, in part, collecting a sample 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 another exemplary embodiment, a suitable sample comprises a cutaneous 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.
(d) Determining the Risk of Metastasis
A method of the invention further comprises 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 identified, as described in section (a) 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 a BAP1 nucleotide sequence and/or BAP1 amino acid sequence. Alternatively, if the level of BAP1 activity is decreased, as described in section (b) 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 nucleotide and corresponding amino acid 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.
(e) Combination of Methods
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.
II. Method for Detecting a Metastasis
Another aspect of the present invention is a method for detecting the presence of a metastasis. In one embodiment, the method generally comprises collecting a sample from a subject, analyzing the BAP1 nucleotide and/or BAP1 amino acid sequence in the sample, and determining the presence of a mutation in the 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: 1 (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 collecting a sample from a subject, and analyzing the level of BAP1 activity in the sample, where a decrease in BAP1 activity indicates the presence of a metastasis in the subject. Suitable samples, methods of analyzing a BAP1 nucleotide sequence and/or BAP1 amino acid sequence, and methods of determining the level of BAP1 activity in a sample are described in section I above.
III. Biomarker for Metastasis
Yet another aspect of the invention encompasses a biomarker for tumor metastasis. In one embodiment, a biomarker of the invention comprises a mutation in a BAP1 nucleotide sequence and/or BAP1 amino acid sequence, as described in section I(a) above. In another embodiment, a biomarker of the invention comprises a decreased level of BAP1 activity, as described in section I(b) above. This may include a decrease in BAP1 protein synthesis. Where the biomarker 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 the above 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. In yet another exemplary embodiment, the tumor is a cutaneous 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, “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, “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, “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. 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 of BAP1. For example, suitable homologs preferably share at least 30% sequence homology, more preferably, 50%, and even more preferably, are greater than about 75% 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 87, 2264 (1993)]. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (J. Mol. Biol. 215, 403 (1990)). 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. 25, 3389 (1997)). 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.
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 Gαq 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.
Using exome capture followed by massively parallel sequencing (8, 9), we 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 11 bp 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).







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





















Chr 3

Indel

Ref-
Ref.



location

End

erence
Amino
New


(hg19)
Tumor
(hg19)
Gene
Codon
Acid
Codon





20,216,514
MM56

SGOL1
GGA
G
GTA


 


43,641,970
MM70

AN010
CTA
L
CTG


 


44,488,382
MM56

ZNF445
AGC
S
AGA


 


44,636,130
MM70

ZNF660
CTT
L
ATT


 


45,715,863
MM56

LIMD1
TCC
S
TAC


 


46,008,495
MM70

FYCO1
CAG
Q
CAT


 


48,457,135
MM56

PLXNB1
TGT
C
TGC


 


48,630,022
MM56

COL7A1
GTG
V
GTT


 


49,690,418
MM56

BSN
CCT
P
CCA


 


49,699,662
MM56

BSN
TGG
W
GGG


 


52,439,264
 MM70*
52,439,274
BAP1
deletion




 


52,439,264
 MM70*

BAP1
CAC
H
CAG


 


52,439,266
 MM70*

BAP1
CAC
H
AAC


 


52,440,916
MM56

BAP1
TGG
W
TGA


 


52,814,305
MM56

ITIH1
GAG
E
GAT


 


56,650,054
MM70

CCDC66
TCT
S
CCT


 


123,695,755
MM70

ROPN1
CGG
R
TGG


 


135,825,122
MM70

PPP2R3A
ACG
T
ATG


 


172,351,305
MM56

NCEH1
ACT
T
AAT


 


180,327,975
MM70

TTC14
GGA
G
GAA


 


183,041,104
MM56

MCF2L2
GGC
G
GGA


 


194,062,926
MM56

CPN2
ACC
T
AAC


 


194,408,437
MM56

FAM43A
GAG
E
GAT


 


195,306,227
MM70

APOD
AAT
N
GAT




















Present on


Chr 3
New
Ref


Sanger


location
Amino
base
Con-
Read
sequence


(hg19)
Acid
(hg19)
sensus
Depth
validation2





20,216,514
V
C
M
16
No


 


43,641,970
L
T
Y
77
Not done


 


44,488,382
R
G
K
14
Not done


 


44,636,130
I
C
M
 9
No


 


45,715,863
Y
C
M
53
No


 


46,008,495
H
C
M
19
Not done


 


48,457,135
C
A
R
11
Not done


 


48,630,022
V
C
M
 9
Not done


 


49,690,418
P
T
W
11
No


 


49,699,662
G
T
K
21
No


 


52,439,264

CCCC
deleted
19
Yes




ATCC







CAC







(SEQ ID







NO: 5)





 


52,439,264
Q
G
Q
15
Yes


 


52,439,266
N
G
N
13
Yes


 


52,440,916
X
C
X
27
Yes


 


52,814,305
D
G
K
15
No


 


56,650,054
P
T
Y
13
No


 


123,695,755
W
G
R
39
Yes







(germline)


 


135,825,122
M
C
Y
33
Yes







(somatic)


 


172,351,305
N
G
K
20
No


 


180,327,975
E
G
K
22
No


 


183,041,104
G
G
K
25
Not done


 


194,062,926
N
G
K
24
No


 


194,408,437
D
G
K
 9
No


 


195,306,227
D
T
Y
21
Not done





1In the case of Insertion/Deletions (InDels) that were detected with Novoalign, column 1 defnes the start and column 3 defines the end. The BAP1 mutations reported in the current manuscript are asterisked and were incorrectly detected as base substitutions in the case of the InDel in MM70, in addition to being correctly detected as an InDel with Novoalign. This is why several substitutions are reported in MM70 for this gene, although they correspond to a single mutational event. We detected 20 additional putative somatic mutations in genes on chromosome 3. The predicted codon and amino acid changes for the appropriate strand are indicated where applicable, along with the base in the hg19 reference sequence and the base change reported as a consensus using IUPAC nomenclature. Reference bases and reference base changes are reported for the plus strand. Depth refers to the read depth of the altered base in the tumor sample. Sanger resequencing was performed to validate each variant detected in the tumor but not the germline.


2In the case of one mutation residing in ROPN1 the mutation was confirmed in the tumor and was also seen in the blood. This had been missed with exome capture. In the case of one mutation residing in PPP2R3A in tumor MM070 the mutation was confirmed to be a somatic alteration.






BAP1 encodes a nuclear ubiquitin carboxy-terminal hydrolase (UCH), one of several classes of deubiquitinating enzymes (10). In addition to the UCH catalytic domain, BAP1 contains a UCH37-like domain (ULD) (11), binding domains for BRCA1 and BARD1, 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 BAP1 mutations have been reported in a small number of breast and lung cancer samples (10, 17).
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, BAP1 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 H169), two occurred elsewhere in the UCH domain, and one affected the ULD (FIG. 1B-C). All BAP1 missense mutations and in-frame deletions affected phylogenetically conserved amino acids (FIG. 10). Only one of 26 class 1 tumors contained a BAP1 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 BAP1 mutations may precede the emergence of the class 2 signature. Somatic BAP1 mutations were also detected in two of three metastatic tumors. The summary of genetic data on uveal melanoma tumor samples are presented in Tables 2 and 3.







TABLE 2





Summary genetic data on uveal melanoma tumor samples in the


study





















Source


BAP1




of
Gene
Loss
mutation
BAP1


Tumor
tumor
expression
of
In normal
Mutation


Number
analyzed
class
Chr 3
DNA
In Tumor





MM 010
Primary
Class 1
No
No
No


 


MM 016
Primary
Class 1
No
No
No


 


MM 018
Primary
Class 1
No
No
No


 


MM 050
Primary
Class 1
No
No
No


 


MM 074
Primary
Class 1
No
No
No


 


MM 086
Primary
Class 1
No
No
No


 


MM 089
Primary
Class 1
No
No
No


 


MM 092
Primary
Class 1
No
No
No


 


MM 101
Primary
Class 1
No
No
No


 


MM 109
Primary
Class 1
No
No
No


 


MM 113
Primary
Class 1
No
No
No


 


MM 122
Primary
Class 1
Yes
No
No


 


NB 092
Primary
Class 1
Yes
No
No


 


NB 096
Primary
Class 1
No
No
No


 


NB 099
Primary
Class 1
No
No
No


 


NB 101
Primary
Class 1
No
No
Yes


 


NB 102
Primary
Class 1
No
No
No


 


NB 104
Primary
Class 1
Yes
No
No


 


NB 107
Primary
Class 1
No
No
No


 


NB 108
Primary
Class 1
No
No
No


 


NB 109
Primary
Class 1
No
No
No


 


NB 112
Primary
Class 1
No
No
No


 


NB 113
Primary
Class 1
No
No
No


 


NB 116
Primary
Class 1
Yes
No
No


 


NB 119
Primary
Class 1
No
No
No


 


NB 126
Primary
Class 1
No
No
No


 


MM 046
Primary
Class 2
Yes
No
Yes


 












MM 054
Primary
Class 2
Yes
No
Yes


 


MM 055
Primary
Class 2
Yes
No
Yes


 


MM 056
Primary
Class 2
Yes
No
Yes


 


MM 060
Primary
Class 2
Yes
No
Yes


 


MM 066
Primary
Class 2
Yes
No
Yes


 


MM 070
Primary
Class 2
Yes
No
Yes


 


MM 071
Primary
Class 2
Yes
No
Yes


 


MM 080
Primary
Class 2
Yes
No
No


 


MM 081
Primary
Class 2
Yes
No
Yes


 


MM 083
Primary
Class 2
Yes
No
Yes


 


MM 087
Primary
Class 2
Yes
Yes
Yes


 


MM 090
Primary
Class 2
Yes
No
Yes


 


MM 091
Primary
Class 2
Yes
No
Yes


 


MM 100
Primary
Class 2
Yes
No
Yes


 


MM 103
Primary
Class 2
Yes
No
Yes


 


MM 110
Primary
Class 2
Yes
NO
Yes


 


MM 120
Primary
Class 2
Yes
No
Yes


 


MM 121
Primary
Class 2
Yes
No
Yes


 


MM 125
Primary
Class 2
Yes
No
Yes


 


MM 127
Primary
Class 2
No
No
No


 


MM 128
Primary
Class 2
Yes
No
Yes


 


MM 133
Primary
Class 2
No
No
No


 


MM 134
Primary
Class 2
No
No
No


 


MM 135
Primary
Class 2
Yes
No
Yes


 


NB 185
Primary
Class 2
No
No
Yes


 


NB 191
Primary
Class 2
No
No
Yes


 


NB 195
Primary
Class 2
No
No
Yes


 


NB 199
Primary
Class 2
No
No
Yes


 


NB 200
Primary
Class 2
No
No
Yes


 


NB 214
Primary
Class 2
No
No
No


 


MM 152M
Metastasis
NO
NO
No
Yes


 


NB 07M
Metastasis
Class 2
Yes
No
Yes


 


PV L8
Metastasis
No
No
No
No
















BAP1





Mutation in
Exon

Predicted


Tumor
cDNA of
With
Protein
effect on


Number
gDNA (hg19)
mutation
change
protein





MM 010






 


MM 016






 


MM 018






 


MM 050






 


MM 074






 


MM 086






 


MM 089






 


MM 092






 


MM 101






 


MM 109






 


MM 113






 


MM 122






 


NB 092






 


NB 096






 


NB 099






 


NB 101
g chr3.52,441,485-
 6
Unknown
Loss of 



52,441,436delTCCCCGT


splice



AGAGCAAAGGATATGC


acceptor of



GATTGGCAATGCCCCG


exon 6 and



GAGTTGGCAA


potential



(SEQ ID NO: 4)


cryptic 






splice






leading to






out of frame






peptide and






premature






termination


 


NB 102






 


NB 104






 


NB 107






 


NB 108






 


NB 109






 


NB 112






 


NB 113






 


NB 116






 


NB 119






 


NB 126






 


MM 046
C 2026-2028delGTG
15
P
Deletion of





K637_C63
K637 and





8delinsN
C638 and






substitution






of N


 


MM 054
G chr3 52,441,434-
 6
Unknown
Los of 



52,441,483del


splice






acceptor of






exon 6 and






potential






cryptic






splice 






leading to






out of frame






peptide and






premature






termination


 


MM 055
c 622C > G
 7
pH169Q
UCH active






site mutated


 


MM 056
c 703G > A
 8
pW196X
Premature






Termination


 


MM 060
c 872C > T
 9
pQ253X
Premature






Termination


 


MM 066
c 960-
10
P. E283-
In-frame



968delCTGAGGAGT

S285del
deletion






between 






BARD1 and






HCFC1






binding






domains


 


MM 070
c 1083-
11
P
Premature



1093delCCCCatCCCAC

Q322fsx100
Termination



(SEQ ID NO: 5)





 


MM 071
c 2130A > G
16
Pd72G
AA change






in ULD






domain


 


MM 080






 


MM 081
g chr3.52441197-
7
unknown
Loss of



52441174delTGACCATG


splice



GTAGGCACCATGAGC


acceptor of



(SEQ ID NO: 6)


exon 7






and potential






cryptic 






splice 






leading to 






out of frame






peptide and






premature






termination


 


MM 083
c 736-
8
pR207fsX32
Premature



751delCGGGTCATCATG


termination



GAG






(SEQ ID NO: 7)





 


MM 087
c 1318-1319insA
12
pE402fsX2
Premature






termination


 


MM 090
c 468-487delinsA
5
p.F118X
Premature






termination


 


MM 091
c 874delG
9
pQ253fs
Premature






termination


 


MM 100
g chr3 52,443,784-
2
Unknown
Loss of



42,443,750del


splice



CCCCTCCTCTTGTCGC


acceptor of



CCCACCCAGGCCTCTT


exon 2



CAC


and potential



(SEQ ID NO: 8)


cryptic 






splice 






leading to 






out of frame






peptide and






premature






termination


 


MM 103
c 2303T > A
17
pTer729R
Read through






termination






codon


 


MM 110
c 1829-1833delCCCCT
13
ps571fsX25
Premature






termination


 


MM 120
C 259delC
4
pF48fsX22
Premature






termination


 


MM 121
c 497G > C
6
pG128R
Missense


 


MM 125
c 622C > G
7
pH169Q*
UCH active






site mutated


 


MM 127






 


MM128
c 2112-2120del9
16
R666-
RRTH



GAAGGACCC

H669
deletion in





delinsN
ULD domain


 


MM 133






 


MM 134






 


MM 135
c 388T > G
5
p.C91W
UCH active






site mutated






(active site)


 


NB 185
c 2006-2017
15
p E631-
Internal



delGAGCTGCTGGCA

A634del
in-frame



(SEQ ID NO: 9)


deletion in






ULD domain


 


NB 191
C 610-634
7
P
Premature



delGGAGGCGTTCCACT

M166fsX12
termination



TTGTCAGCTAT






(SEQ ID NO: 10)





 


NB 195
g chr3 52,443,771-
2
Unknown
Loss of



52,443,734


splice



delCGCCCCACCCAGGC


acceptor of



CTCTTCACCCTGCTCG


exon 2



TGGAAGAT


and potential



(SEQ ID NO: 11)


cryptic 






splice 






leading to 






out of frame






peptide and






premature






termination


 


NB 199
chr3:52,436,691G > T
16
Unknown
Unknown,



Splice acceptor AG 


likely



to AT


premature






termination


 


NB 200
c 631C > G
7
p S172R
Missense


 


NB 214






 


MM 152M
C 2195-2220
17
p
Premature



delCAGAACCATCTCCG

E693fsX13
termination



TGCGGCGGCGCCA






(SEQ ID NO: 12)





 


NB 07M
c 221C > T
3
Q36*
Premature






termination


 


PV L8






















TABLE 3











BAP1








muta-
BAP1




Source of


tion
muta-
Mutation


Tumor
tumor
GEP

normal
tion
in gDNA


Number
analyzed
class
LOH3
DNA
tumor
(hg19)





MM 133
Primary/fresh
2
?
NA
No
NA



frozen







 


MM 134
Primary/fresh
2
?
NA
No
NA



frozen







 


MM 137
Primary/fresh
2
?
No
Yes
g.chr3:52443889-



frozen




52443927delATTC








ATCTTCCCGCGG








GGCGGCCCCTC








AGCGCCATGTCC








(SEQ ID NO: 13)


 


MM 138
Primary/fresh
2
?
NA
No
NA



frozen







 


MM 144
Primary/fresh
2
?
No
Yes
c.265delC;



frozen




g.chr3:52442595








delC


 


MM 150
Primary/fresh
2
?
NA
No
NA



frozen







 


MM 151A
Primary/fresh
2
?
No
Yes
g.chr3:52440925-



frozen




52440918delAGG








GCCCT


 


Mouse


?
NA
Yes
g.chr3:52440925-


204





52440918delAGG


(MM151





GCCCT


A met)








 


MM 161
Primary/fresh
2
?
?
Yes
c.1013-1014delAG;



frozen




g.chr3:52439814-








52439813delAG


 


MM 162
Primary/fresh
2
?
?
Yes
g.chr3:52437431



frozen




G > C and








chr3:52437433








delA 


 


OP-11-
Primary/paraf

?
?
Yes
g.chr3:52442086-


953
fin embedded




52442106delGGTA


(Emory)





TCAGCTGTGAAA








CCAAG








(SEQ ID NO: 14)


 


MM 131T
Primary/fresh
 1b
?
NA
No
NA



frozen







 


MM 159T
Primary/fresh
2
?
NA
No
NA



frozen

















Predicted



Tumor
BAP1
Mutant
Protein
Predicted effect


Number
cDNA
exon
change
on protein





MM 133

NA
NA



 


MM 134

NA
NA



 


 


MM 137
c.82-
1
premature
deletes first two



121del

truncation
 aa (MN) and 33 bp






from 5′UTR






(ATTCATCTTCCCGCG






GGGCGGCCCCTCAG






CGCCATGTCC)






(SEQ ID NO: 13)


 


MM 138

NA
NA



 


MM 144
c.265delC
4
premature






truncation






(p.F50LfsX






22)



 


MM 150

NA
NA



 


MM 151A

8
Deletion of
delete AG splice 





exon 6
donor of exon 8 






and then deletion 






of 6 bp in exon 6-






leaves 48 bp. Might






be exon skipping.


 


Mouse

8
Deletion 



204


of exon 6



(MM151






A met)






 


MM 161

10
premature
Premature 





truncation
termination


 


MM 162

13
premature
Splice mutation,





truncation 
deletion of A


 


OP-11-

10
premature



953


truncation



(Emory)






 


MM 131T






 


MM 159T

NA
NA





NA: Not applicable






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 BAP1-mutant 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, we detected one germline mutation (p.E402fsX2; c.1318-1319insA) in the patient with the class 1 tumor NB101 (Table 2), and this case was particularly interesting. Re-sequencing 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 wild-type 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 BAP1 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.
Other germline (blood) mutations in exon 13 (g.chr3:52437465insT; pE566X; c.1695-1696insT leading to premature protein termination) in FUM1-01 and FUM-02 were also detected (see FIG. 18).
GNAQ mutation status was available in 15 cases. GNAQ mutations were present in 4/9 BAP1 mutant tumors and 3/6 BAP1 wild-type 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. BAP1 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 BAP1 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 BAP1 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 BAP1 mutations may be explained by DNA methylation, we 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 BAP1 promoter was limited by an unusually complex CpG island that will require further work to resolve. Thus, we cannot rule out a role for methylation in class 2 tumors in which BAP1 mutations were not found. However, with almost 85% of class 2 tumors harboring mutations, we do not expect that methylation will be a major mechanism of BAP1 inactivation. An alternative explanation is that these tumors may contain very large deletions of the BAP1 locus or other mutations not detectable by our sequencing method.
Immunofluorescence revealed abundant nuclear BAP1 protein in two class 1 tumors but virtually none in four BAP1 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 BAP1 in 92.1 UM cells, which did not harbor a detectable BAP1 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. 7A). 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 (FIG. 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 BAP1 (16). Our findings strongly implicate mutational inactivation of BAP1 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, Wis.). 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, Calif.) following the manufacturer's protocol.







TABLE 4







Summary of clinical and pathologic data on uveal melanoma patients in the study



















Age at


Tumor








Source
primary

Tumor
thickness

Pathologic



of
tumor

diameter
of
Ciliary
cell type of

Mons


Tumor
tumor
diag-

of primary
primary
body
primary
Treatment of
follow-


Number
analyzed
nosis
Gender
tumor
tumor
involvement
tumor
primary tumor
up
Metastasis




















MM 010
Primary
41
Male
17
9.9
No
Mixed
Enucleation
131.2
Yes


MM 016
Primary
24
Female
24
12.6
Yes
Spindle
Enucleation
87.8
Yes


MM 018
Primary
55
Male
12
9.2
No
Epithelioid
Enucleation
67.4
No


MM 050
Primary
50
Female
19
8.9
Yes
Epithelioid
Enucleation
71.4
No


MM 074
Primary
77
Male
N/A
22.0
N/A
Mixed
Enucleation
24.4
No


MM 086
Primary
47
Male
14
14.0
No
Spindle
Enucleation
17.7
No


MM 089
Primary
74
Male
18
8.1
Yes
Spindle
Enucleation
8.1
Yes


MM 092
Primary
61
Male
20
13.4
Yes
Epithelioid
Enucleation
8.0
No


MM 101
Primary
66
Female
15
6.4
No
Spindle
Enucleation
10.8
No


MM 109
Primary
56
Male
15
12.7
Yes
Spindle
Enucleation
8.8
No


MM 113
Primary
54
Male
14
11.0
No
Mixed
Enucleation
8.0
No


MM 122
Primary
52
Male
N/A
N/A
Yes
Epithelioid
Enucleation
1.0
No


NB 092
Primary
53
Male
11
4.4
No
Mixed
Brachytherapy
25.2
No


NB 096
Primary
55
Female
15
3.8
No
Spindle
Brachylerapy
24.1
No


NB 099
Primary
76
Male
N/A
N/A
No
Other
Biopsy
1.0
No


NB 101
Primary
57
Female
14
2.6
Yes
Other
Brachytherapy
23.2
No


NB 102
Primary
83
Female
13
2.4
No
Epithelioid
Brachytherapy
27.5
No


NB 104
Primary
62
Male
13
5.5
No
Epithelioid
Brachytherapy
24.1
No


NB 107
Primary
58
Male
15
10.0
Yes
Epithelioid
Brachytherapy
19.9
No


NB 108
Primary
66
Female
10
2.6
No
Other
Brachytherapy
17.8
No


NB 109
Primary
76
Male
18
8.4
Yes
Spindle
Brachytherapy
9.1
No


NB 112
Primary
53
Male
12
6.1
No
Other
Brachytherapy
21.4
No


NB 113
Primary
70
Male
14
3.2
Yes
Spindle
Brachytherapy
13.2
No


NB 116
Primary
85
Female
17
7.1
Yes
Spindle
Brachytherapy
13.4
No


NB 119
Primary
69
Female
16
5.9
Yes
Spindle
Brachytherapy
21.8
No


NB 126
Primary
34
Female
17
8.1
No
Spindle
Brachyterhapy
22.4
No


MM 046
Primary
69
Female
22
9.0
Yes
Epithelioid
Enucleation
32.6
Yes


MM 054
Primary
80
Female
15
6.7
No
Mixed
Enucleation
34.6
Yes


MM 055
Primary
82
Female
19
8.6
Yes
Epithelioid
Enucleation
81.3
Yes


MM 056
Primary
63
Male
18
11.7
Ye
Epithelioid
Enucleation
16.3
No


MM 060
Primary
67
Male
14
9.5
Yes
Epithelioid
Enucleation
37.0
Yes


MM 066
Primary
47
Male
22
9.2
Yes
Mixed
Enucleation
52.5
No


MM 070
Primary
62
Male
24
15.6
Yes
Epithelioid
Enucleation
31.5
Yes


MM 071
Primary
63
Female
N/A
12.5
N/A
Spindle
Enucleation
46.3
No


MM 080
Primary
37
Male
N/A
11.3
Yes
Epithelioid
Enucleation
31.5
Yes


MM 081
Primary
65
Male
18
11.3
Yes
Epithelioid
Enucleation
28.2
Yes


MM 083
Primary
43
Male
 5
3.7
Yes
Epithelioid
Enucleation
51.1
Yes


MM 087
Primary
53
Female
16
5.8
No
Epithelioid
Enucleation
17.5
Yes


MM 090
Primary
72
Female
19
14.0
Yes
Mixed
Enucleation
27.5
No


MM 091
Primary
64
Male
17
10.2
Yes
Mixed
Enucleation
26.4
Yes


MM 100
Primary
68
Male
18
12.3
Yes
Epithelioid
Enucleation
16.2
No


MM 103
Primary
63
Male
15
12.7
Yes
Mixed
Enucleation
33.4
Yes


MM 110
Primary
48
Female
15
8.0
No
Epithelioid
Enucleation
37.4
No


MM 120
Primary
68
Female
20
10.4
Yes
Spindle
Enucleation
26.7
Yes


MM 121
Primary
52
Female
17
5.8
Yes
Spindle
Enucleation
32.3
Yes


MM 125
Primary
79
Female
18
3.9
No
Mixed
Enucleation
7.
Yes


MM 127
Primary
78
Male
N/A
N/A
N/A
Epithelioid
Enucleation
20.0
No


MM 128
Primary
69
Female
 8
2.4
No
Mixed
Enucleation
21.1
Yes


MM 133
Primary
54
Female
20
15.0
No
Epitheliod
Enucleation
4.5
No


MM 134
Primary
57
Female
19
9.1
Yes
Mixed
Enucleation
6.5
No


MM 135
Primary
36
Female
20
NA
Yes
Mixed
Enucleation
7.1
No


NB 185
Primary
85
Male
15
6.9
Yes
Epithelioid
Brachytherapy
3.8
No


NB 191
Primary
60
Female
 9
2.7
No
Spindle
Brachytehrapy
3.4
No


NB 195
Primary
74
Male
18
9.0
Yes
Mixed
Biopsy
3.1
No


NB 199
Primary
71
Female
13
8.7
Yes
Mixed
Brachytherapy
1.8
No


NB 200
Primary
61
Femlae
18
4.4
Yes
Spindle
Brachytherapy
6.9
No


NB 214
Primary
86
Male
15
4.5
No
Epithelioid
Brachytherapy
3.4
No


MM
Metastasis
68
Male
19
15.3
N/A
Epitheliod
Unknown
4.2
Yes


152M


NB 071M
Metastasis
51
Male
18
8.6
Yes
Epitheliod
Brachytherapy
36.5
Yes


PVLB
Metastasis
43
Female
18
9.0
N/A
Epithelioid
Unknown
64.4
Yes









Cell Culture:

92.1 (generous gift from Dr. Martine Jager) and Me1290 (generous gift of Dr. Bruce Ksander) human UM cells were grown in RPMI-1640 (Lonza, Walkersville, Md.) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.) and antibiotics. Transfections were performed with HiPerFect (Qiagen, Valencia, Calif.) and Silencer® Select BAP1 (s15820 and s15822) or Control #1 siRNA (Ambion, Austin, Tex.). Knockdown of BAP1 protein levels was confirmed by western blot with antibodies that recognize the BAP1 protein (Santa Cruz, Santa Cruz, Calif.) and alpha-tubulin (Sigma-Aldrich, St. Louis, Mo.). Cell morphology data were collected by digital imaging of phase contrasted cells at 200× 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, Calif.) 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, Calif.) for CTNNB1, EDNRB, KIT, SOX10 and UBC (endogenous control) or TaqMan® Gene Expression Assays and Gene Expression Master Mix (Applied Biosystems Inc., Foster City, Calif.) for BAP1, CDH1, LTA4H, LMCD1 and ROBO1. 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 4 μm 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 Genomic 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 Illumina 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 hg19 version of the human reference sequence using bowtie. The Bowtie software (v0.12.3) was compiled with g++ (v4.3.3) using the additional compilation switches “—O3—mtune=amdfam10” 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 (v0.1.7) 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 hg19; 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 (hg19) 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, NA18555, NA18517, NA18507, NA12878, NA12156) were downloaded from the NCBI Short Read Archive (3) (accession SRP000910). All reads for each individual were aligned to hg19 (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 BAP1 with the 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. Oligonucleotide primer sequences are available upon request.







TABLE 5







Sequencing primers














PCR






product



Primers
Seq.
Exon
size
Location (hg19)





BAP1-e1-3-F2
SEQ ID NO. 15:
1
566 bp
chr3:52443441 +



AGGCTGCTGCTTTCTGTGAG


3052444006


 


BAP1-e1-3-R2
SEQ ID NO. 16:
1





CGTTGTCTGTGTGTGGGAC





 


BAP1-e4-F
SEQ ID NO. 17:
4
261 bp
chr3:52442418 -



ATGCTGATTGTCTTCTCCCC


52442678


 


BAP1-e4-R
SEQ ID NO. 18:
4





CTCCATTTCCACTTCCCAAG





 


BAP1-e5-F
SEQ ID NO. 19:
5
255 bp
chr3:52441894 -



CTTGGGGCTTGCAGTGAG


52442148


 


BAP1-e5-R
SEQ ID NO. 20:
5





ATGTGGTAGCATTCCCAGTG





 


BAP1E8L
SEQ ID NO. 21:
8
250 bp
chr3:52440750 +



GGCCTTGCAATTTACAAATCA


52440999


 


BAP1E8R
SEQ ID NO. 22:
8





TGTCTTCCTTCCCACTCCTG





 


BAP1-e9-F
SEQ ID NO. 23:
9
256 bp
chr3:52440207 -



GGATATCTGCCTCAACCTGATG


52440462


 


BAP1-e9-R
SEQ ID NO. 24:
9





GAAGGGAGGAGGAATGCAG





 


BAP1-e10-F
SEQ ID NO. 25:
10
287 bp
chr3:52439727 -



TTCCTTTAGGTCCTCAGCCC


52440013


 


BAP1-e10-nest
SEQ ID NO. 26:
10




This is a nested
CTGAGGTCCACAAGAGGTCC





primer used for






sequencing






 


BAP1-e10-R
SEQ ID NO. 27:
10





CAGACATTAGCGGGTGGC





 


BAP1E11L
SEQ ID NO. 28:
11
227 bp
chr3:52439107 +



AAGGGTGCTCCCAGCTTAC


52439333


 


BAP1E11R
SEQ ID NO. 29:
11





CCTGTGTTCTTGCCCTGTCT





 


BAP1-e12-F
SEQ ID NO. 30:
12
270 bp
chr3:52438402 -



GCTGTGAGTGTCTAGGCTCAG


52438671


 


BAP1-e12-R
SEQ ID NO. 31:
12





AGACTGAGATATTCAGGATGGG





 


BAP1-e14-F
SEQ ID NO. 32:
14
275 bp
chr3:52437098 -



CCAAGTGACCACAAAGTGTCC


52437372


 


BAP1-e14-R
SEQ ID NO. 33:
14





AGCTCAGGCCTTACCCTCTG





 


BAP1-e17-F2
SEQ ID NO. 34:
17
496 bp
chr3:52436103 +



CTGAGCACTATGGGGCTGAT


52436598


 


BAP1-e17-R2
SEQ ID NO. 35:
17





TCTTAACTGGAATGCCCTGC





 


BAP1-e13A-F2
SEQ ID NO. 36:
13A
567 bp
chr3:52437269 +



CTGCCTTGGATTGGTCTGAT


52437835


 


BAP1-e13A-R2
SEQ ID NO. 37:
13A





CAACACCATCAACGTCTTGG





 


BAP1-e13B-F2
SEQ ID NO. 38:
13B
595 bp
chr3:52437489 +



TGATGACAGGACCCAGATCA


52438083


 


BAP1-e13B-R2
SEQ ID NO. 39:
13B





GCTGTCAGAACTTGATGCCA





 


BAP1-e15-16-F
SEQ ID NO. 40:
15-16
409 bp
chr3:52436552 -



CTAGCTGCCTATTGCTCGTG


52436960


 


BAP1-e15-16-R
SEQ ID NO. 41:
15-16





GAGGGGAGCTGAAGGACAC





 


BAP1-e6-7-F
SEQ ID NO. 42:
6-7
412 bp
chr3:52441134 -



TTTGCCTTCCACCCATAGTC


52441545


 


BAP1-e6-7-R
SEQ ID NO. 43:
6-7





AGCTCCCTAGGAGGTAGGC









DNA Methylation Analysis:

Following bisulfite treatment and amplification of genomic DNA from region chr3:52,442,270-52,442,651 (hg19) with bisulfite specific primers, methylation of this region was evaluated with Sequenom's MassARRAY Epityper technology in our 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 ΔCtvalues 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, Calif.), 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 (v1.14.0). Expression values were quantile normalized and log 2 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 log 2 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 (v1.24.0), 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 BAP1 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 BAP1 loss.
Stable knockdown of BAP1 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 BAP1 in culture leads to decreased cell motility (FIG. 12) and a decreased growth in soft agar (FIG. 13). On the other hand, loss of BAP1 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 BAP1 mutations are somatic or germline in origin. Germline alterations in BAP1 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.
BAP1 mRNA levels may be analyzed using quantitative RT-PCR. If BAP1 mRNA levels are lower in cutaneous melanoma samples than in normal samples, DNA methylation of the BAP1 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
BAP1 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.
For instance, FIG. 18 illustrates a family with stomach cancer, bone cancer, breast cancer, bladder cancer, uveal melanoma, and cutaneous melanoma. The individuals labeled FUM1-01, FUM1-02, FUM1-03, and FUM1-04 were positive for germline BAP1 mutations. These data support the conclusion that germline BAP1 mutations may be used to detect affected family members in hereditary cancers and/or syndromes.
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 BAP1 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 1-2 Mb 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). 1-3 mg 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 (Bashiardes et al. 2005), 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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## Claims

1. A method for determining the risk of melanoma metastasis in a subject, the method comprising:
(a) analyzing BAP1 nucleic acid from a cell in a sample obtained from a subject,
(b) detecting the presence of a truncating mutation in the BAP1 nucleic acid, wherein the mutation is selected from the group consisting of:
i. a nonsense mutation selected from the group consisting of Q36X, W196X and Q253X of BAP1;
ii. an insertion or deletion mutation in exon 2, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 17 of BAP1; and
iii. a splice acceptor mutation in exon 16 of BAP1; and

(c) identifying the subject as having an increased risk for metastasis when a mutation is detected.

2. The method of claim 1, wherein the melanoma is uveal melanoma.

3. The method of claim 1, wherein the sample is a tumor sample.

4. The method of claim 3, wherein the sample is collected from a primary tumor or from a circulating tumor cell.

5. The method of claim 4, wherein the circulating tumor cell is collected from a bodily fluid.

6. A method for prognosing melanoma in a subject, the method comprising:
(a) analyzing BAP1 nucleic acid from a cell in a sample obtained from a subject,
(b) detecting the presence of a truncating mutation in the BAP1 nucleic acid, wherein the mutation is selected from the group consisting of:
i. a nonsense mutation selected from the group consisting of Q36X, W196X and Q253X of BAP1; and
ii. an insertion or deletion mutation in exon 2, 4, 5, 6, 7, 8 or 9 of BAP1; and

(c) identifying the subject as having poor prognosis when a mutation is detected.

7. The method of claim 6, wherein the melanoma is uveal melanoma.

8. The method of claim 6, wherein the sample is a tumor sample.

9. The method of claim 8, wherein the sample is collected from a primary tumor or from a circulating tumor cell.

10. The method of claim 9, wherein the circulating tumor cell is collected from a bodily fluid.