Patent Description:
In general, a comparison of sequences present at the same locus on each chromosome (each autosomal chromosome for males) of a chromosome pair can reveal whether that particular locus is homozygous or heterozygous within the genome of a cell. Polymorphic loci within the human genome are generally heterozygous within an individual since that individual typically receives one copy from the biological father and one copy from the biological mother. In some cases, a polymorphic locus or a string of polymorphic loci within an individual are homozygous as a result in inheriting identical copies from both biological parents. In other cases, homozygosity results from a loss of heterozygosity (LOH) from the germline. <NPL>) describe an integrated analysis of genotype, loss of heterozygosity (LOH), and copy number for DNA derived from FFPE tissues using oligonucleotide micro-arrays containing over <NUM> single nucleotide polymorphisms. <NPL>)
discloses a loss of heterozygosity (LOH) panel comprising <NUM> SNPs which are enriched from a genomic sample via a two-step PCR step and then sequenced.

Because LOH and copy number information can be clinically useful, there is a need for improved methods of identifying loci and regions of LOH in samples.

Copy number (including allelic imbalance and LOH) analysis of tumor tissues has been traditionally performed using single nucleotide polymorphism (SNP) arrays. The data quality is often highly variable and, especially for FFPE samples, tends to be poor. The inventors have developed a method of genome-wide copy number analysis that produces high quality data from all sample types that is based on in-solution capture of DNA fragments spanning target loci (e.g., SNPs), followed by parallel sequencing to identify and quantitate the alleles. The resulting data allows high quality LOH and copy number analysis of the sample.

Accordingly, in one aspect of the present invention, a method of detecting allelic imbalance status in a plurality of genomic loci in a tumor sample from a cancer patient is provided, comprising the steps of enriching a genomic DNA sample for DNA molecules each comprising a locus of interest, wherein enriching is performed by in-solution or solid-support based capture method; sequencing said DNA molecules to determine the genotype at each such locus, wherein sequencing uses high throughput parallel platform; determining for each locus whether there is allelic imbalance.

Also disclosed but not part of the invention is a method of detecting LOH status in a plurality of genomic loci in a tumor sample from a cancer patient is provided, comprising the steps of enriching a genomic DNA sample for DNA molecules each comprising a locus of interest; sequencing said DNA molecules to determine the genotype at each such locus; determining for each homozygous locus whether it is homozygous due to LOH.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

It has been surprisingly discovered that determining allelic imbalance (e.g., abnormal copy number, LOH) in formalin-fixed paraffin-embedded ("FFPE") samples using sequencing of genomic regions comprising loci of interest (e.g., SNPs) yields far superior quality data when compared to copy number and allelic imbalance data generated using microarrays. This invention enables largescale (e.g., whole genome) copy number (e.g., allelic imbalance) analysis of samples of varying quality. In particular, it enables high quality data to be produced from FFPE-derived DNA. Current array-based platforms are unable to produce data of sufficient quality from this sample type.

Accordingly, in one aspect of the present invention, a method of detecting allelic imbalance status in a plurality of genomic loci in a tumor sample from a cancer patient is provided, comprising the steps of enriching a genomic DNA sample for DNA molecules each comprising a locus of interest, wherein enriching is performed by in-solution or solid-support based capture method; sequencing said DNA molecules to determine the genotype at each such locus, wherein sequencing uses high throughput parallel platform; determining for each locus whether there is allelic imbalance. "Locus" as used herein has its usual meaning in the art. As used herein, "region" means a plurality of substantially adjacent loci. Unless stated otherwise or unless the context clearly indicates otherwise, statements made about a locus will generally apply to a region.

As used herein, "allelic imbalance" means any instance where the somatic copy number differs from the germline copy number at a genomic locus or region. In some embodiments allelic imbalance is expressed in terms of major copy proportion ("MCP"). Major copy proportion and MCP, as used herein, mean the ratio of the major allele copy number to the major + minor allele copy number, as follows: <MAT> In some embodiments, a locus or region shows allelic imbalance if the MCP at such locus or region is <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

One example of allelic imbalance is loss of heterozygosity ("LOH"), in which a locus is heterozygous in the germline but homozygous in somatic tissue. In this sense, homozygosity can include homozygous loss (i.e., deletion) of the locus in somatic tissue. The different types of possible LOH and allelic imbalance are discussed in more detail below.

Thus in some embodiments the present invention provides a method of detecting LOH status in a plurality of genomic loci in a tumor sample from a cancer patient, comprising enriching a genomic DNA sample for DNA molecules each comprising a locus of interest; sequencing said DNA molecules to determine the genotype at each such locus; determining for each homozygous locus whether it is homozygous due to LOH.

According to the present invention, nucleic acid sequencing techniques can be used to identify loci and/or regions as having allelic imbalance. For example, genomic DNA from a cell sample (e.g., a cancer cell sample) can be extracted and fragmented. Any appropriate method can be used to extract and fragment genomic nucleic acid including, without limitation, commercial kits such as QIAamp DNA Mini Kit (Qiagen), MagNA Pure DNA Isolation Kit (Roche Applied Science) and GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). Once extracted and fragmented, either targeted or untargeted sequencing can be done to determine the sample's genotypes at loci of interest. For example, whole genome, whole transcriptome, or whole exome sequencing can be done to determine genotypes at millions or even billions of base pairs (i.e., base pairs can be "loci" to be evaluated).

In some cases, targeted sequencing of known polymorphic loci (e.g., SNPs and surrounding sequences) can be done as an alternative to microarray analysis. For example, the genomic DNA can be enriched for those fragments containing a locus (e.g., SNP location) to be analyzed using kits designed for this purpose (e.g., Agilent SureSelect, Illumina TruSeq Capture, Nimblegen SeqCap EZ Choice, Raindance Thunderstorm™). For example, genomic DNA containing the loci to be analyzed can be hybridized to biotinylated capture RNA fragments to form biotinylated RNA/genomic DNA complexes. Alternatively, DNA capture probes may be utilized resulting in the formation of biotinylated DNA/genomic DNA hybrids. Streptavidin coated magnetic beads and a magnetic force can be used to separate the biotinylated RNA/genomic DNA complexes from those genomic DNA fragments not present within a biotinylated RNA/genomic DNA complex. The obtained biotinylated RNA/genomic DNA complexes can be treated to remove the captured RNA from the magnetic beads, thereby leaving intact genomic DNA fragments containing a locus to be analyzed. These intact genomic DNA fragments containing the loci to be analyzed can be amplified using, for example, PCR techniques. Alternatively, a multiplex PCR reaction can be employed to enrich for loci of interest. PCR primers can be designed to flank loci of interest and a PCR reaction can be run to amplify sequences comprising such loci.

The enriched genomic DNA fragments can be sequenced using any sequencing technique. Beyond Sanger sequencing, numerous suitable sequencing machines and strategies are well known in the art, including but not limited to those developed by Illumina (the Genome Analyzer; <NPL>; HiSeq; MiSeq); by Applied Biosystems, Inc. (the SOLiD™ Sequencer; solid. appliedbiosystems. com); by Roche (e.g., the <NUM> GS FLX™ sequencer; <NPL>; <CIT>; <CIT>; <CIT>); by Helicos Biosciences (Heliscope™ system, see, e.g., <CIT>); by Oxford Nanopore (e.g., GridION™ and MinION™, see, e.g., International Application No. <CIT>, pub. <CIT>); and by others.

The sequencing results from the genomic DNA fragments can be used to identify loci as having allelic imbalance. In some cases, an analysis of the allelic imbalance status of loci over a length of a chromosome can be performed to determine the length of regions of allelic imbalance. For example, a stretch of SNP locations that are spaced apart (e.g., spaced about <NUM> kb to about <NUM> kb apart) along a chromosome can be evaluated by sequencing, and the sequencing results used to determine not only the presence of a region of allelic imbalance ()e.g., somatic homozygosity) along a chromosome but also the length of that region of imbalance. Obtained sequencing results can be used to generate a graph that plots allele dosages along a chromosome. Allele dosage di for SNP i can be calculated from the adjusted number of captured probes for two alleles (Ai and Bi): di =. Ai/(Ai + Bi). An example of such a graph is presented in <FIG>.

Once a sample's genotype (e.g., homozygosity) has been determined for a plurality of loci (e.g., SNPs), common techniques can be used to identify loci and regions of allelic imbalance due to somatic change (e.g., LOH). One way to determine whether imbalance is due to somatic change is to compare the somatic genotype to the germline. For example, the genotype for a plurality of loci (e.g., SNPs) can be determined in both a germline (e.g., blood) sample and a somatic (e.g., tumor) sample. The genotypes for each sample can be compared (typically computationally) to determine where the genome of the germline cell was, e.g., heterozygous and the genome of the somatic cell is, e.g., homozygous. Such loci are LOH loci and regions of such loci are LOH regions.

Computational techniques can also be used to determine whether allelic imbalance is somatic (e.g., due to LOH). Such techniques are particularly useful when a germline sample is not available for analysis and comparison. For example, algorithms such as those described elsewhere can be used to detect allelic imbalance regions using information from SNP arrays (<NPL>)). Typically these algorithms do not explicitly take into account contamination of tumor samples with benign tissue. International Application No. <CIT>; <NPL>. This contamination is often high enough to make the detection of allelic imbalance regions challenging. Improved analytical methods according to the present invention for identifying allelic imbalance, even in spite of contamination, include those embodied in computer software products as described below.

The following is one example. If the observed ratio (e.g., MCP) of the signals of two alleles, A and B, is two to one, there are two possibilities. The first possibility is that cancer cells have LOH with deletion of allele B in a sample with <NUM>% contamination with normal cells. The second possibility is that there is no LOH but allele A is duplicated in a sample with no contamination with normal cells. An algorithm can be implemented as a computer program as described herein to reconstruct LOH regions based on genotype (e.g., SNP genotype) data. One point of the algorithm is to first reconstruct allele specific copy numbers (ASCN) at each locus (e.g., SNP). ASCNs are the numbers of copies of both paternal and maternal alleles. An LOH region is then determined as a stretch of SNPs with one of the ASCNs (paternal or maternal) being zero. The algorithm can be based on maximizing a likelihood function and can be conceptually akin to a previously described algorithm designed to reconstruct total copy number (rather than ASCN) at each locus (e.g., SNP). See International Application No. <CIT> (pub. The likelihood function can be maximized over ASCN of all loci, level of contamination with benign tissue, total copy number averaged over the whole genome, and sample specific noise level. The input data for the algorithm can include or consist of (<NUM>) sample-specific normalized signal intensities for both allele of each locus and (<NUM>) assay-specific (specific for different SNP arrays and for sequence based approach) set of parameters defined based on analysis of large number of samples with known ASCN profiles.

In some cases, a selection process can be used to select loci (e.g., SNP loci) to be evaluated using an assay configured to identify loci as having allelic imbalance (e.g., SNP array-based assays and sequencing-based assays). For example, any human SNP location can be selected for inclusion in a SNP array-based assay or a sequencing-based assay configured to identify loci as having allelic imbalance within the genome of cells. In some cases, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> million or more loci (e.g., SNP locations) present within the human genome can be evaluated to identify those loci that (a) are not present on the Y chromosome, (b) are not mitochondrial loci, (c) have a minor allele frequency of at least about <NUM>% in the population of interest (e.g., Caucasians), (d) have a minor allele frequency of at least about <NUM>% in three populations other than the population of interest (e.g., Chinese, Japanese, and Yoruba), and/or (e) do not have a significant deviation from Hardy-Weinberg equilibrium in any of these populations. In some cases, more than <NUM>,<NUM>, <NUM>,<NUM>, or <NUM>,<NUM> human loci can be selected that meet criteria (a) through (e). Of the human loci meeting criteria (a) through (e), a group of loci (e.g., top <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, or <NUM>,<NUM> loci) can be selected such that the loci have a high degree of allele frequency in the population of interest, cover the human genome in a somewhat evenly spaced manner (e.g., at least one locus of interest every about 5kb, 10kb, 25kb, 50kb, 75kb, 100kb, 150kb, 200kb, 300kb, 400kb, 500kb or more), and are not in linkage disequilibrium with another selected locus in any of the populations used for analysis. In some cases, about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> thousand or more loci can be selected as meeting each of these criteria and included in an assay configured to identify allelic imbalance regions across a human genome. For example, between about <NUM>,<NUM> and about <NUM>,<NUM> (e.g., about <NUM>,<NUM>) SNPs can be selected for analysis with a SNP array-based assay, and between about <NUM>,<NUM> and about <NUM>,<NUM> (e.g., about <NUM>,<NUM>) SNPs can be selected for analysis with a sequencing-based assay.

Accordingly, in one aspect of the present invention, a method of detecting allelic imbalance status in a plurality of genomic loci in a sample from a patient is provided, comprising the steps of enriching a genomic DNA sample for DNA molecules each comprising a locus of interest; sequencing said DNA molecules to determine the genotype at each such locus; determining for each locus whether it has allelic imbalance.

Also disclosed but not part of the invention is a method of detecting LOH status in a plurality of genomic loci in a sample from a patient is provided, comprising the steps of enriching a genomic DNA sample for DNA molecules each comprising a locus of interest; sequencing said DNA molecules to determine the genotype at each such locus; determining for each homozygous locus whether it is homozygous due to LOH.

Also disclosed but not part of the invention is a method of detecting copy number status in a plurality of genomic loci in a sample from a patient is provided, comprising the steps of enriching a genomic DNA sample for DNA molecules each comprising a locus of interest; sequencing said DNA molecules; and quantitating each allele at each such locus to determine its copy number.

In some embodiments at least <NUM>, <NUM>, <NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>,<NUM>, <NUM>,<NUM>,<NUM> or more loci are evaluated. In some embodiments these loci are spaced evenly along the genome. As used herein, loci are "evenly spaced along the genome" when the percentage difference between the distanceAB between any two loci A and B and the distanceCD between any other two loci C and D (i.e., <NUM>*(distanceAB - distanceCD)/distanceAB or <NUM>*(distanceAB - distanceCD)/distanceCD) is less than or equal to <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%. Such percentage difference is referred to herein as the "genomic spacing" of loci. In some embodiments the sample is an FFPE tissue sample. In some embodiments the sample is a tumor sample from the patient.

Another aspect of the invention provides a system for determining allelic imbalance status in a plurality of loci in a sample comprising: a sample analyzer for (<NUM>) enriching a genomic DNA sample for DNA molecules each comprising a locus of interest and (<NUM>) sequencing said DNA molecules to produce a plurality of quantitative signals about each such locus; a computer program for analyzing said plurality of quantitative signals to determine whether each such locus has allelic imbalance.

Also disclosed but not part of the invention is a system for determining LOH status in a plurality of loci in a sample comprising: a sample analyzer for (<NUM>) enriching a genomic DNA sample for DNA molecules each comprising a locus of interest and (<NUM>) sequencing said DNA molecules to produce a plurality of quantitative signals about each such locus; a computer program for analyzing said plurality of quantitative signals to determine whether each such locus is homozygous in the sample; and a computer program for determining for each homozygous locus whether it is homozygous due to LOH.

Also disclosed but not part of the invention is a system for detecting copy number status in a plurality of genomic loci in a sample from a patient comprising: a sample analyzer for (<NUM>) enriching a genomic DNA sample for DNA molecules each comprising a locus of interest and (<NUM>) sequencing said DNA molecules to produce a plurality of quantitative signals about each such locus; and a computer program for analyzing said plurality of quantitative signals to quantitate each allele at each such locus to determine its copy number.

In some embodiments of the systems of the invention, one sample analyzer both enriches the sample for DNA of interest and sequences that DNA. In other embodiments two or more sample analyzers perform these functions. In some embodiments, one software program analyzes the plurality of quantitative signals to determine whether each locus is homozygous in the sample and also determines for each homozygous locus whether it is homozygous due to LOH.

<FIG> is a diagram of an example of a computer device <NUM> and a mobile computer device <NUM>, which may be used with the techniques described herein. Computing device <NUM> is intended to represent various forms of digital computers, such as laptops, desktops, workstations, personal digital assistants, servers, blade servers, mainframes, and other appropriate computers. Computing device <NUM> is intended to represent various forms of mobile devices, such as personal digital assistants, cellular telephones, smart phones, and other similar computing devices.

Also, multiple computing devices <NUM> may be connected, with each device providing portions of the necessary operations (e.g., as a server bank, a group of blade servers, or a multiprocessor system).

The computer program product may also contain instructions that, when executed, perform one or more methods, such as those described herein. The information carrier is a computer- or machine-readable medium, such as the memory <NUM>, the storage device <NUM>, memory on processor <NUM>, or a propagated signal.

The low-speed expansion port, which may include various communication ports (e.g., USB, Bluetooth, Ethernet, or wireless Ethernet) may be coupled to one or more input/output devices, such as a keyboard, a pointing device, a scanner, an optical reader, a fluorescent signal detector, or a networking device such as a switch or router, e.g., through a network adapter.

Computing device <NUM> includes a processor <NUM>, memory <NUM>, an input/output device such as a display <NUM>, a communication interface <NUM>, and a transceiver <NUM>, among other components (e.g., a scanner, an optical reader, a fluorescent signal detector).

For example, expansion memory <NUM> may include instructions to carry out or supplement the processes described herein, and may include secure information also.

The computer program product contains instructions that, when executed, perform one or more methods, such as those described herein. The information carrier is a computer- or machine-readable medium, such as the memory <NUM>, expansion memory <NUM>, memory on processor <NUM>, or a propagated signal that may be received, for example, over transceiver <NUM> or external interface <NUM>.

Various implementations of the systems and techniques described herein can be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs (application specific integrated circuits), computer hardware, firmware, software, and/or combinations thereof.

As used herein, the terms "machine-readable medium" and "computer-readable medium" refer to any computer program product, apparatus and/or device (e.g., magnetic discs, optical disks, memory, and Programmable Logic Devices (PLDs)) used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal.

To provide for interaction with a user, the systems and techniques described herein can be implemented on a computer having a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying information to the user and a keyboard and a pointing device (e.g., a mouse or a trackball) by which the user can provide input to the computer.

The systems and techniques described herein can be implemented in a computing system that includes a back end component (e.g., as a data server), or that includes a middleware component (e.g., an application server), or that includes a front end component (e.g., a client computer having a graphical user interface or a Web browser through which a user can interact with an implementation of the systems and techniques described herein), or any combination of such back end, middleware, or front end components.

In some cases, a system provided herein can be configured to include one or more sample analyzers. A sample analyzer can be configured to produce a plurality of signals about genomic DNA of a cancer cell. For example, a sample analyzer can produce signals that are capable of being interpreted in a manner that identifies the allelic imbalance status of loci along a chromosome. In some cases, a sample analyzer can be configured to carry out one or more steps of a sequencing-based assay and can be configured to produce and/or capture signals from such assays. In some cases, a computing system provided herein can be configured to include a computing device. In such cases, the computing device can be configured to receive signals from a sample analyzer.

The computing device can include computer-executable instructions or a computer program (e.g., software) containing computer-executable instructions for carrying out one or more of the methods or steps described herein. In some cases, such computer-executable instructions can instruct a computing device to analyze signals from a sample analyzer, from another computing device, or from a sequencing-based assay. The analysis of such signals can be carried out to determine genotypes, allelic imbalance at certain loci, regions of allelic imbalance, the number of allelic imbalance regions, to determine the size of allelic imbalance regions, to determine the number of allelic imbalance regions having a particular size or range of sizes, or to determine a combination of these items.

In some cases, a system provided herein can include computer-executable instructions or a computer program (e.g., software) containing computer-executable instructions for formatting an output providing an indication about copy number, allelic imbalance, LOH, or a combination of these items.

In some cases, a system provided herein can include a pre-processing device configured to process a sample (e.g., cancer cells) such that a sequencing-based assay can be performed. Examples of pre-processing devices include, without limitation, devices configured to enrich cell populations for cancer cells as opposed to non-cancer cells, devices configured to lyse cells and/or extract genomic nucleic acid, and devices configured to enrich a sample for particular genomic DNA fragments.

The process described here utilized an Agilent SureSelect Capture system followed by Illumina HiSeq sequencing, however any in solution or solid support based capture method and high throughput parallel sequencing platform could be used.

The initial design selection process utilized the ~<NUM> million SNPs on the Illumina Omni2. <NUM> SNP array. This list of SNPs was chosen because it is the currently the largest list of SNPs from which there is genotyping information available for multiple different population groups. All <NUM>,<NUM>,<NUM> SNP locations were input into the Agilent eArray Sure Select Target Enrichment wizard for Single End Long Reads using the default settings. <NUM>,<NUM>,<NUM> passed the selection criteria and had baits designed.

Then, <NUM>,<NUM> SNPs with high minor allele frequences and evenly covering the genome were selected. In the selection, SNPs in strong linkage disequilibriom and SNPs with stong deviation from Hardy-Weinberg equilibrium were discarded.

Two preliminary library designs were constructed comprised of <NUM>,<NUM> probes each targeting <NUM>,<NUM> different SNP locations. Testing was carried out using a high quality normal DNA sample to check for even capture of both alleles of every SNP. In addition, <NUM> FFPE samples were captured and used to select the most optimally performing probes. We looked for probes that showed robust capture and even sequence depth without over or underrepresentation of sequence reads in the final sequencing library.

The final capture probe library design was comprised of the <NUM>,<NUM> optimal probes identified using the preliminary capture designs.

Claim 1:
An in vitro method of detecting genome-wide allelic imbalance status in a plurality of single nucleotide polymorphism (SNP) genomic loci in a formalin-fixed, paraffin-embedded sample, comprising:
enriching, from a patient tumor sample, genomic DNA for DNA molecules, each DNA
molecule comprising at least one locus from the plurality of SNP loci; wherein enriching is performed by in-solution or solid-support based capture method;
sequencing the DNA molecules to determine the genotype at each locus, wherein sequencing uses high throughput parallel platform; and,
determining, for each locus, whether the locus has allelic imbalance.