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Marty Chilberg is a seasoned financial professional with over 30 years of executive leadership, board, consulting and advisory experience.  He began his career as a certified public accountant (CPA). He moved to Silicon Valley in 1981 to begin his career in the software industry, working for... More
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  • Patents By Yuk-Ming Dennis Lo
    Diagnosing fetal chromosomal aneuploidy using massively parallel genomic sequencing

    Patent number: 9051616

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

    Type: Grant

    Filed: July 18, 2014

    Issued: June 9, 2015

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    Methods for assessing liver pathologies

    Patent number: 9051614

    Abstract: The present invention provides a new method for detecting or monitoring a liver disease in a subject that has no indication of any liver pathologies, by measuring the amount of concentration of albumin mRNA in an acellular blood sample from the subject, and then comparing the amount or concentration of albumin mRNA with a standard control.

    Type: Grant

    Filed: September 10, 2010

    Issued: June 9, 2015

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Rebecca Wing Yan Chan

     

    SEQUENCING ANALYSIS OF CIRCULATING DNA TO DETECT AND MONITOR AUTOIMMUNE DISEASES

    Application number: 20150087529

    Abstract: Systems, methods, and apparatuses are provided for diagnosing auto-immune diseases such as systemic lupus erythematosus (SLE) based on the sizes, methylation levels, and/or genomic characteristics of circulating DNA molecules. Patients provide blood or other tissue samples containing cell-free nucleic molecules for analysis. Massively parallel and/or methylation-aware sequencing can be used to determine the sizes and methylation levels of individual DNA molecules and identify the number of molecules originating from different genomic regions.

    Type: Application

    Filed: September 19, 2014

    Issued: March 26, 2015

    Inventors: Yuk-Ming Dennis LO, Rossa Wai Kwun Chiu, Rebecca Wing Yan Chan, Lai Shan Tam

     

    Diagnosing fetal chromosomal aneuploidy using massively parallel genomic sequencing

    Patent number: 8972202

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

    Type: Grant

    Filed: July 18, 2014

    Issued: March 3, 2015

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    Methods for detecting DNA orginating from different individuals

    Patent number: 8962280

    Abstract: In a first aspect, the present invention features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample or to differentiate DNA of an organ donor from DNA of an organ recipient. In preferred embodiments, the DNA species are differentiated by observing epigenetic differences in the DNA species such as differences in DNA methylation. In a second aspect, the present invention features methods of detecting genetic abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. In a third aspect, the present invention features methods for differentiating DNA species originating from an organ donor from those of an organ recipient. In a fourth aspect, the present invention features kits for differentiating DNA species originating from different individuals in a biological sample.

    Type: Grant

    Filed: April 3, 2013

    Issued: February 24, 2015

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Lit Man Poon

     

    NON-INVASIVE DETERMINATION OF METHYLOME OF TUMOR FROM PLASMA

    Application number: 20150011403

    Abstract: Systems, methods, and apparatuses can determine and use methylation profiles of various tissues and samples. Examples are provided. A methylation profile can be deduced for fetal/tumor tissue based on a comparison of plasma methylation (or other sample with cell-free DNA) to a methylation profile of the mother/patient. A methylation profile can be determined for fetal/tumor tissue using tissue-specific alleles to identify DNA from the fetus/tumor when the sample has a mixture of DNA. A methylation profile can be used to determine copy number variations in genome of a fetus/tumor. Methylation markers for a fetus have been identified via various techniques. The methylation profile can be determined by determining a size parameter of a size distribution of DNA fragments, where reference values for the size parameter can be used to determine methylation levels. Additionally, a methylation level can be used to determine a level of cancer.

    Type: Application

    Filed: September 24, 2014

    Issued: January 8, 2015

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan, Miu Fan Lun, Wai Man Chan, Peiyong Jiang

     

    Fetal methylation markers

    Patent number: 8927216

    Abstract: This application describes the discovery that, in a pregnant woman, certain genes (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD) originated from a fetus are highly methylated, whereas the same genes of maternal origin are unmethylated. This discovery allows the easy detection of one or more of these methylated fetal genes in a biological sample from a pregnant woman, serving as a universal indicator of the presence of fetal DNA in the sample. These fetal methylation markers are particularly useful as positive controls for a non-invasive analytical process during which the quality and quantity of fetal DNA are monitored. These newly identified fetal markers can also be measured directly for diagnosis of certain pregnancy-related conditions.

    Type: Grant

    Filed: September 14, 2012

    Issued: January 6, 2015

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Chunming Ding, Kwan Chee Chan, Hing Nam Ivy Wong, Ka Chun Ryan Yuen

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20140329695

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

    Type: Application

    Filed: July 18, 2014

    Issued: November 6, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20140329696

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

    Type: Application

    Filed: July 18, 2014

    Issued: November 6, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    DETERMINING FETAL GENOMES FOR MULTIPLE FETUS PREGNANCIES

    Application number: 20140315200

    Abstract: Techniques are provided for determining inheritance of maternal and paternal haplotypes in preganncies with multiple fetuses. Maternal inheritance can be determined at loci where the mother is heterozygous and the paternally inherited alleles are known (e.g., the father is homozygous). Two types of loci may be used, where one type has the paternal allele appear on a first maternal haplotype, and another type has the paternal allele appear on a second maternal haplotype. Paternal inheritance can be determined from loci where the father is heterozygous and the maother is homozygous. Amounts of different alleles at each locus can be measured. A comparison of the amounts (e.g., using a fractional concentration of each allele and cutoffs) can be used to determine the haplotype inheritance. A haplotype can be linked to a condition of interest.

    Type: Application

    Filed: March 17, 2014

    Issued: October 23, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Wai Kwun Rossa Chiu, Kwan Chee Chan

     

    METHODS AND KITS FOR SELECTIVELY AMPLIFYING, DETECTING OR QUANTIFYING TARGET DNA WITH SPECIFIC END SEQUENCES

    Application number: 20140302506

    Abstract: Disclosed herein are methods and kits for selectively amplifying, detecting or quantifying a DNA fragment with a specific end sequence, especially generated following restriction enzyme digestion. This method can be used, for example, to detect a hypomethylated DNA fragment. This methods and kits are especially useful in detecting or quantifying a hypomethylated fetal DNA fragment in a maternal plasma sample containing a corresponding hypermethylated maternal DNA fragment.

    Type: Application

    Filed: April 29, 2014

    Issued: October 9, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Yu Kwan Tong, Wai Kwun Rossa Chiu, Chunming Ding

     

    DIAGNOSTIC METHOD

    Application number: 20140272975

    Abstract: The present invention concerns a method for the detection or monitoring of cancer using a biological sample selected from blood, plasma, serum, saliva, urine from an individual, said method comprising: (NYSE:A) obtaining DNA from the said biological sample; (NYSE:B) digesting the DNA sample with one or more methylation-sensitive restriction enzymes; (NYSE:C) quantifying or detecting a DNA sequence of interest after step , wherein the target sequence of interest contains at least two methylation-sensitive restriction enzyme recognition sites; and (NYSE:D) comparing the level of the DNA sequence from the individual to a normal standard, to detect, prognosticate or monitor cancer.

    Type: Application

    Filed: May 22, 2014

    Issued: September 18, 2014

    Assignee: THE CHINESE UNIVERSITY OF HONG KONG

    Inventors: Yuk Ming Dennis LO, Kwan Chee Allen CHAN, Chunming DING

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20140256559

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

    Type: Application

    Filed: October 18, 2013

    Issued: September 11, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20140256560

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

    Type: Application

    Filed: November 22, 2013

    Issued: September 11, 2014

    Assignee: THE CHINESE UNIVERSITY OF HONG KONG

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    MATERNAL PLASMA TRANSCRIPTOME ANALYSIS BY MASSIVELY PARALLEL RNA SEQUENCING

    Application number: 20140243212

    Abstract: Methods are provided for diagnosing pregnancy-associated disorders, determining allelic ratios, determining maternal or fetal contributions to circulating transcripts, and/or identifying maternal or fetal markers using a sample from a pregnant female subject. Also provided is use of a gene for diagnosing a pregnancy-associated disorder in a pregnant female subject.

    Type: Application

    Filed: February 28, 2014

    Issued: August 28, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan, Peiyong Jiang, Bo Yin Tsui

     

    DETECTION OF GENETIC OR MOLECULAR ABERRATIONS ASSOCIATED WITH CANCER

    Application number: 20140227699

    Abstract: Systems, apparatus, and methods are provided for determining genetic or molecular aberrations in a biological sample from an organism. Biological samples including cell-free DNA fragments are analyzed to identify imbalances in chromosomal regions, e.g., due to deletions and/or amplifications in a tumor. Multiple loci are used for each chromosomal region. Such imbalances can then be used to diagnose (screen) a patient for cancer, as well as prognosticate a patient with cancer, or to detect the presence or to monitor the progress of a premalignant condition in a patient. The severity of an imbalance as well as the number of regions exhibiting an imbalance can be used. A systematic analysis of non-overlapping segments of a genome can provide a general screening tool for a sample. Additionally, a patient can be tested over time to track severity of each of one or more chromosomal regions and a number of chromosomal regions to enable screening and prognosticating, as well as monitoring of progress (e.g.

    Type: Application

    Filed: April 17, 2014

    Issued: August 14, 2014

    Assignee: THE CHINESE UNIVERSITY OF HONG KONG

    Inventors: Yuk-Ming Dennis Lo, Kwan Chee Chan, Rossa Wai Kwun Chiu, Peiyong Jiang

     

    Methods and kits for selectively amplifying, detecting or quantifying target DNA with specific end sequences

    Patent number: 8748100

    Abstract: Disclosed herein are methods and kits for selectively amplifying, detecting or quantifying a DNA fragment with a specific end sequence, especially generated following restriction enzyme digestion. This method can be used, for example, to detect a hypomethylated DNA fragment. This methods and kits are especially useful in detecting or quantifying a hypomethylated fetal DNA fragment in a maternal plasma sample containing a corresponding hypermethylated maternal DNA fragment.

    Type: Grant

    Filed: August 30, 2007

    Issued: June 10, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Yu Kwan Tong, Wai Kwun Rossa Chiu, Chunming Ding

     

    Detection of genetic or molecular aberrations associated with cancer

    Patent number: 8741811

    Abstract: Biological samples including cell-free DNA fragments are analyzed to identify imbalances in chromosomal regions, e.g., due to deletions and/or amplifications in a tumor. Multiple loci are used for each chromosomal region. Such imbalances can then be used to diagnose (screen) a patient for cancer, as well as prognosticate a patient with cancer, or to detect the presence or to monitor the progress of a premalignant condition in a patient. The severity of an imbalance as well as the number of regions exhibiting an imbalance can be used. A systematic analysis of non-overlapping segments of a genome can provide a general screening tool for a sample. Additionally, a patient can be tested over time to track severity of each of one or more chromosomal regions and a number of chromosomal regions to enable screening and prognosticating, as well as monitoring of progress (e.g. after treatment).

    Type: Grant

    Filed: November 30, 2011

    Issued: June 3, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Peiyong Jiang

     

    Analysis for nucleic acids by digital PCR

    Patent number: 8722334

    Abstract: The present invention provides a method for analyzing nucleic acids for their lengths and relative abundance in a sample, based on digital amplification of individual template molecules. This invention has many applications, including those in noninvasive prenatal diagnosis, transplantation monitoring, and the detection and monitoring of cancers and virus-associated diseases.

    Type: Grant

    Filed: October 28, 2010

    Issued: May 13, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu

     

    Determining a nucleic acid sequence imbalance

    Patent number: 8706422

    Abstract: Methods, systems, and apparatus are provided for determining whether a nucleic acid sequence imbalance exists within a biological sample. One or more cutoff values for determining an imbalance of, for example, the ratio of the two sequences (or sets of sequences) are chosen. The cutoff value may be determined based at least in part on the percentage of fetal DNA in a sample, such as maternal plasma, containing a background of maternal nucleic acid sequences. The cutoff value may also be determined based on an average concentration of a sequence per reaction. In one aspect, the cutoff value is determined from a proportion of informative wells that are estimated to contain a particular nucleic acid sequence, where the proportion is determined based on the above-mentioned percentage and/or average concentration. The cutoff value may be determined using many different types of methods, such as sequential probability ratio testing (NASDAQ:SPRT).

    Type: Grant

    Filed: July 23, 2008

    Issued: April 22, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan, Benny Chung Ying Zee, Ka Chun Chong

     

    MUTATIONAL ANALYSIS OF PLASMA DNA FOR CANCER DETECTION

    Application number: 20140100121

    Abstract: A frequency of somatic mutations in a biological sample (e.g., plasma or serum) of a subject undergoing screening or monitoring for cancer, can be compared with that in the constitutional DNA of the same subject. A parameter can derived from these frequencies and used to determine a classification of a level of cancer. False positives can be filtered out by requiring any variant locus to have at least a specified number of variant sequence reads (NYSEARCA:TAGS), thereby providing a more accurate parameter. The relative frequencies for different variant loci can be analyzed to determine a level of heterogeneity of tumors in a patient.

    Type: Application

    Filed: March 13, 2013

    Issued: April 10, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan, Peiyong Jiang

     

    FETAL GENOMIC ANALYSIS THAT ACCOUNTS FOR GC BIAS

    Application number: 20140080720

    Abstract: Systems, methods, and apparatuses for performing a prenatal diagnosis of a sequence imbalance are provided. A shift (e.g. to a smaller size distribution) can signify an imbalance in certain circumstances. For example, a size distribution of fragments of nucleic acids from an at-risk chromosome can be used to determine a fetal chromosomal aneuploidy. A size ranking of different chromosomes can be used to determine changes of a rank of an at-risk chromosome from an expected ranking. Also, a difference between a statistical size value for one chromosome can be compared to a statistical size value of another chromosome to identify a significant shift in size. A genotype and haplotype of the fetus may also be determined using a size distribution to determine whether a sequence imbalance occurs in a maternal sample relative to a genotypes or haplotype of the mother, thereby providing a genotype or haplotype of the fetus.

    Type: Application

    Filed: November 25, 2013

    Issued: March 20, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Wenli Zheng

     

    DETERMINING PERCENTAGE OF FETAL DNA IN MATERNAL SAMPLE

    Application number: 20140045181

    Abstract: Methods, systems, and apparatus are provided for determining whether a nucleic acid sequence imbalance exists within a biological sample. One or more cutoff values for determining an imbalance of, for example, the ratio of the two sequences (or sets of sequences) are chosen. The cutoff value may be determined based at least in part on the percentage of fetal DNA in a sample, such as maternal plasma, containing a background of maternal nucleic acid sequences. The percentage of fetal DNA can be calculated from the same or different data used to determine the cutoff value, and can use a locus where the mother is homozygous and the fetus is heterozygous. The cutoff value may be determined using many different types of methods, such as sequential probability ratio testing .

    Type: Application

    Filed: September 18, 2013

    Issued: February 13, 2014

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan, Benny Chung-Ying Zee, Ka Chun Chong

     

    NONINVASIVE PRENATAL GENOTYPING OF FETAL SEX CHROMOSOMES

    Application number: 20140019064

    Abstract: Methods, apparatuses, and system are provided for analyzing a maternal sample to determine whether a male fetus of a pregnant female has inherited an X-linked mutation from the mother. A percentage of fetal DNA in the sample is obtained, and cutoff values for the two possibilities (fetus inherits mutant or normal allele) are determined. A proportion of mutant alleles relative to a normal allele on the X-chromosome can then be compared to the cutoff values to make a classification of which allele is inherited. Alternatively, a number of alleles from a target region on the X-chromosome can be compared to a number of alleles from a reference region on the X-chromosome to identify a deletion or amplification. The fetal DNA percentage can be computed by counting reactions with a fetal-specific allele, and correcting the number to account for a statistical distribution among the reactions.

    Type: Application

    Filed: January 5, 2012

    Issued: January 16, 2014

    Assignee: THE CHINESE UNIVERSITY OF HONG KONG

    Inventors: Yuk Ming Dennis Lo, Wai kwun Rossa Chiu, Kwan Chee Chan, Bo Yin Tsui

     

    Size-based genomic analysis

    Patent number: 8620593

    Abstract: Systems, methods, and apparatuses for performing a prenatal diagnosis of a sequence imbalance are provided. A shift (e.g. to a smaller size distribution) can signify an imbalance in certain circumstances. For example, a size distribution of fragments of nucleic acids from an at-risk chromosome can be used to determine a fetal chromosomal aneuploidy. A size ranking of different chromosomes can be used to determine changes of a rank of an at-risk chromosome from an expected ranking. Also, a difference between a statistical size value for one chromosome can be compared to a statistical size value of another chromosome to identify a significant shift in size. A genotype and haplotype of the fetus may also be determined using a size distribution to determine whether a sequence imbalance occurs in a maternal sample relative to a genotypes or haplotype of the mother, thereby providing a genotype or haplotype of the fetus.

    Type: Grant

    Filed: November 5, 2010

    Issued: December 31, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Wenli Zheng

     

    METHODS FOR DETECTING DNA PRIOGINATING FROM DIFFERENT INDIVIDUALS

    Application number: 20130337443

    Abstract: In a first aspect, the present invention features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample or to differentiate DNA of an organ donor from DNA of an organ recipient. In preferred embodiments, the DNA species are differentiated by observing epigenetic differences in the DNA species such as differences in DNA methylation. In a second aspect, the present invention features methods of detecting genetic abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. In a third aspect, the present invention features methods for differentiating DNA species originating from an organ donor from those of an organ recipient. In a fourth aspect, the present invention features kits for differentiating DNA species originating from different individuals in a biological sample.

    Type: Application

    Filed: April 3, 2013

    Issued: December 19, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis LO, Lit Man Poon

     

    DETERMINATION OF THE DEPTH COVERAGE OF THE FETAL GENOME

    Application number: 20130323731

    Abstract: Systems, methods, and apparatus for determining at least a portion of fetal genome are provided. DNA fragments from a maternal sample (maternal and fetal DNA) can be analyzed to identify alleles at certain loci. The amounts of DNA fragments of the respective alleles at these loci can be analyzed together to determine relative amounts of the haplotypes for these loci and determine which haplotypes have been inherited from the parental genomes. Loci where the parents are a specific combination of homozygous and heterozygous can be analyzed to determine regions of the fetal genome. Reference haplotypes common in the population can be used along with the analysis of the DNA fragments of the maternal sample to determine the maternal and paternal genomes. Determination of mutations, a fractional fetal DNA concentration in a maternal sample, and a proportion of coverage of a sequencing of the maternal sample can also be provided.

    Type: Application

    Filed: May 15, 2013

    Issued: December 5, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Charles Cantor

     

    DIAGNOSING CANCER USING GENOMIC SEQUENCING

    Application number: 20130310263

    Abstract: Methods, systems, and apparatus determine whether a first chromosomal region exhibits a deletion or an amplification associated with cancer in a sample from a subject (e.g., where the sample includes a mixture of cell-free DNA from tumor cells and non-malignant cells. Nucleic acid molecules of the biological sample are sequenced. Respective amounts of a clinically-relevant chromosomal region and of background chromosomal region(s) are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether first chromosomal region exhibits a deletion or an amplification associated with cancer.

    Type: Application

    Filed: July 8, 2013

    Issued: November 21, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    Method for detecting chromosomal aneuploidy

    Patent number: 8563242

    Abstract: The present invention relates to a new, non-invasive method for detecting chromosomal aneuploidy by analyzing a sample from a pregnant woman. The detection is based on the ratio between the amount of a fetal methylation marker located on a chromosome relevant to the aneuploidy and the amount of a fetal genetic marker located on a reference chromosome, offering improved accuracy.

    Type: Grant

    Filed: July 9, 2010

    Issued: October 22, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Yu Kwan Tong, Shengnan Jin, Siu Chung Stephen Chim, Wai Yi Tsui

     

    NONINVASIVE PRENATAL DIAGNOSIS OF FETAL TRISOMY BY ALLELIC RATIO ANALYSIS USING TARGETED MASSIVELY PARALLEL SEQUENCING

    Application number: 20130267425

    Abstract: Whether a fetus has an aneuploidy associated with a first chromosome is detected using ratios of alleles detected in a maternal sample having a mixture of maternal and fetal DNA. DNA from the sample is enriched for target regions associated with polymorphic loci and then sequenced. Polymorphic loci (e.g., single nucleotide polymorphisms) in the target regions with fetal-specific alleles are identified on a first chromosome and on one or more reference chromosomes. A first ratio of the fetal-specific alleles and shared alleles is determined for the loci on the first chromosome. A second ratio of the fetal-specific alleles and shared alleles is determined for the loci on the reference chromosome(s). A third ratio of the first and second ratio can be compared to a cutoff to determine whether an aneuploidy is present, and whether the aneuploidy is maternally-derived or paternally-derived.

    Type: Application

    Filed: April 8, 2013

    Issued: October 10, 2013

    Assignee: THE CHINESE UNIVERSITY OF HONG KONG

    Inventors: Jiawei Liao, Kwan Chee Chan, Wai Kwun Rossa Chiu, Yuk Ming Dennis Lo

     

    IDENTIFYING A DE NOVO FETAL MUTATION FROM A MATERNAL BIOLOGICAL SAMPLE

    Application number: 20130253844

    Abstract: Systems, methods, and apparatus for determining at least a portion of fetal genome are provided. DNA fragments from a maternal sample (maternal and fetal DNA) can be analyzed to identify alleles at certain loci. The amounts of DNA fragments of the respective alleles at these loci can be analyzed together to determine relative amounts of the haplotypes for these loci and determine which haplotypes have been inherited from the parental genomes. Loci where the parents are a specific combination of homozygous and heterozygous can be analyzed to determine regions of the fetal genome. Reference haplotypes common in the population can be used along with the analysis of the DNA fragments of the maternal sample to determine the maternal and paternal genomes. Determination of mutations, a fractional fetal DNA concentration in a maternal sample, and a proportion of coverage of a sequencing of the maternal sample can also be provided.

    Type: Application

    Filed: May 15, 2013

    Issued: September 26, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Charles Cantor

     

    METHODS FOR ANALYZING MASSIVELY PARALLEL SEQUENCING DATA FOR NONINVASIVE PRENATAL DIAGNOSIS

    Application number: 20130245961

    Abstract: This invention provides several ways of managing GC bias that occurs during seequencing and analysis of genomic DNA. Maternal plasma can be used as a source of fetal DNA for analysis. DNA segments or tags obtained from the plasma can be aligned with a chromosomal region of interest and with an artificial reference chromosome assembled from regions of the genome having matching GC content.

    Type: Application

    Filed: March 13, 2013

    Issued: September 19, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Wai Kwun Rossa Chiu, Kwan Chee Chan, Wenli Zheng, Hao Sun, Zhang Chen

     

    SIZE-BASED ANALYSIS OF FETAL DNA FRACTION IN MATERNAL PLASMA

    Application number: 20130237431

    Abstract: A fractional concentration of clinically-relevant DNA in a mixture of DNA from a biological sample is determined based on amounts of DNA fragments at multiple sizes. For example, the fractional concentration of fetal DNA in maternal plasma or tumor DNA in a patient's plasma can be determined. The size of DNA fragments in a sample is shown to be correlated with a proportion of fetal DNA and a proportion of tumor DNA, respectively. Calibration data points (e.g., as a calibration function) indicate a correspondence between values of a size parameter and the fractional concentration of the clinically-relevant DNA. For a given sample, a first value of a size parameter can be determined from the sizes of DNA fragments in a sample. A comparison of the first value to the calibration data points can provide the estimate of the fractional concentration of the clinically-relevant DNA.

    Type: Application

    Filed: March 7, 2013

    Issued: September 12, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Wai Kwun Rossa Chiu, Kwan Chee Chan, Wenli Zheng, Peiyong Jiang, Jiawei Liao

     

    Fetal genomic analysis from a maternal biological sample

    Patent number: 8467976

    Abstract: Systems, methods, and apparatus for determining at least a portion of fetal genome are provided. DNA fragments from a maternal sample (maternal and fetal DNA) can be analyzed to identify alleles at certain loci. The amounts of DNA fragments of the respective alleles at these loci can be analyzed together to determine relative amounts of the haplotypes for these loci and determine which haplotypes have been inherited from the parental genomes. Loci where the parents are a specific combination of homozygous and heterozygous can be analyzed to determine regions of the fetal genome. Reference haplotypes common in the population can be used along with the analysis of the DNA fragments of the maternal sample to determine the maternal and paternal genomes. Determination of mutations, a fractional fetal DNA concentration in a maternal sample, and a proportion of coverage of a sequencing of the maternal sample can also be provided.

    Type: Grant

    Filed: November 5, 2010

    Issued: June 18, 2013

    Assignees: The Chinese University Of Hong Kong, Sequenom Inc.

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Charles Cantor

     

    Diagnosing fetal chromosomal aneuploidy using paired end

    Patent number: 8442774

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Grant

    Filed: March 28, 2012

    Issued: May 14, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    Methods for detecting DNA originating from different individuals

    Patent number: 8431343

    Abstract: In a first aspect, the present invention features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample or to differentiate DNA of an organ donor from DNA of an organ recipient. In preferred embodiments, the DNA species are differentiated by observing epigenetic differences in the DNA species such as differences in DNA methylation. In a second aspect, the present invention features methods of detecting genetic abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. In a third aspect, the present invention features methods for differentiating DNA species originating from an organ donor from those of an organ recipient. In a fourth aspect, the present invention features kits for differentiating DNA species originating from different individuals in a biological sample.

    Type: Grant

    Filed: April 2, 2012

    Issued: April 30, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Lit Man Poon

     

    FETAL METHYLATION MARKERS

    Application number: 20130084566

    Abstract: This application describes the discovery that, in a pregnant woman, certain genes (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD) originated from a fetus are highly methylated, whereas the same genes of maternal origin are unmethylated. This discovery allows the easy detection of one or more of these methylated fetal genes in a biological sample from a pregnant woman, serving as a universal indicator of the presence of fetal DNA in the sample. These fetal methylation markers are particularly useful as positive controls for a non-invasive analytical process during which the quality and quantity of fetal DNA are monitored. These newly identified fetal markers can also be measured directly for diagnosis of certain pregnancy-related conditions.

    Type: Application

    Filed: September 14, 2012

    Issued: April 4, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: YUK MING DENNIS LO, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Chunming Ding, Kwan Chee Chan, Hing Nam Ivy Wong, Ka Chun Ryan Yuen

     

    MOLECULAR TESTING OF MULTIPLE PREGNANCIES

    Application number: 20130059733

    Abstract: Methods, systems, and apparatus are provided for determining zygosity of a multiple-fetus pregnancy using a biological sample taken from the mother. The fetal and maternal DNA in the sample (e.g. plasma) can be analyzed for a particular chromosomal region to identify genetic differences in the fetuses. For example, a normalized parameter for the measure of a primary or secondary allele can show variances for different chromosomal regions when fetuses are dizygotic. Such a variance can be determined relative to an expected value if the fetuses were genetically identical. Statistical methods are provided for analyzing the variation of the normalized parameters to determine fetal DNA concentration and the maternal-fetal mixed genotype at various loci. Parental genotype and haplotype information can also be used to identify inheritance of different parental haplotypes to indicate genetic differences among the fetuses.

    Type: Application

    Filed: February 24, 2012

    Issued: March 7, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Wai Kwun Rossa Chu, Kwan Chee Chan, Tak Yeung Leung, Peiyong Jiang

     

    DETECTION OF GENETIC OR MOLECULAR ABERRATIONS ASSOCIATED WITH CANCER

    Application number: 20130040824

    Abstract: Biological samples including cell-free DNA fragments are analyzed to identify imbalances in chromosomal regions, e.g., due to deletions and/or amplifications in a tumor. Multiple loci are used for each chromosomal region. Such imbalances can then be used to diagnose (screen) a patient for cancer, as well as prognosticate a patient with cancer, or to detect the presence or to monitor the progress of a premalignant condition in a patient. The severity of an imbalance as well as the number of regions exhibiting an imbalance can be used. A systematic analysis of non-overlapping segments of a genome can provide a general screening tool for a sample. Additionally, a patient can be tested over time to track severity of each of one or more chromosomal regions and a number of chromosomal regions to enable screening and prognosticating, as well as monitoring of progress (e.g. after treatment).

    Type: Application

    Filed: November 30, 2011

    Issued: February 14, 2013

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Peiyong Jiang

     

    Methods for detecting fetal DNA in a plasma or serum sample from a pregnant woman

    Patent number: 8288100

    Abstract: This application describes the discovery that, in a pregnant woman, certain genes (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD) originated from a fetus are highly methylated, whereas the same genes of maternal origin are unmethylated. This discovery allows the easy detection of one or more of these methylated fetal genes in a biological sample from a pregnant woman, serving as a universal indicator of the presence of fetal DNA in the sample. These fetal methylation markers are particularly useful as positive controls for a non-invasive analytical process during which the quality and quantity of fetal DNA are monitored. These newly identified fetal markers can also be measured directly for diagnosis of certain pregnancy-related conditions.

    Type: Grant

    Filed: June 1, 2010

    Issued: October 16, 2012

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Chunming Ding, Kwan Chee Chan, Hing Nam Ivy Wong, Ka Chun Ryan Yuen

     

    METHOD FOR NON-INVASIVE PRENATAL DIAGNOSIS

    Application number: 20120225798

    Abstract: The present invention is directed to methods of detecting nucleic acids in a biological sample. The method is based on a novel combination of a base extension reaction, which provides excellent analytical specificity, and a mass spectrometric analysis, which provides excellent specificity. The method can be used, for example, for diagnostic, prognostic and treatment purposes. The method allows accurate detection of nucleic acids that are present in very small amounts in a biological sample. For example, the method of the present invention is preferably used to detect fetal nucleic acid in a maternal blood sample; circulating tumor-specific nucleic acids in a blood, urine or stool sample; and donor-specific nucleic acids in transplant recipients. In another embodiment, one can detect viral, bacterial, fungal, or other foreign nucleic acids in a biological sample.

    Type: Application

    Filed: February 8, 2012

    Issued: September 6, 2012

    Assignee: THE TRUSTEES OF BOSTON UNIVERSITY

    Inventors: Charles R. Cantor, Chunming Ding, Yuk Ming Dennis Lo, Rossa Wai Kwum Chiu

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20120208708

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Application

    Filed: March 9, 2012

    Issued: August 16, 2012

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING PAIRED END

    Application number: 20120190559

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Application

    Filed: March 28, 2012

    Issued: July 26, 2012

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    Methods for detecting DNA originating from different individuals

    Patent number: 8168382

    Abstract: In a first aspect, the present invention features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample or to differentiate DNA of an organ donor from DNA of an organ recipient. In preferred embodiments, the DNA species are differentiated by observing epigenetic differences in the DNA species such as differences in DNA methylation. In a second aspect, the present invention features methods of detecting genetic abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. In a third aspect, the present invention features methods for differentiating DNA species originating from an organ donor from those of an organ recipient. In a fourth aspect, the present invention features kits for differentiating DNA species originating from different individuals in a biological sample.

    Type: Grant

    Filed: October 13, 2008

    Issued: May 1, 2012

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Lit Man Poon

     

    Methods and kits for diagnosis, prognosis or monitoring of epstein-barr virus (EBV)-associated cancer

    Patent number: 8124383

    Abstract: Disclosed is a non-invasive method for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer by detecting and/or quantifying EBV associated nucleic acid fragments in a urine sample from an individual. Kits for diagnosis, prognosis or monitoring of cancer are also disclosed.

    Type: Grant

    Filed: October 21, 2010

    Issued: February 28, 2012

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Allen Chan

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20120003635

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Application

    Filed: March 23, 2011

    Issued: January 5, 2012

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20120003636

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Application

    Filed: March 23, 2011

    Issued: January 5, 2012

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan-Chee Chan

     

    MARKER FOR PRENATAL DIAGNOSIS AND MONITORING

    Application number: 20120003650

    Abstract: The present invention relates to new methods for diagnosing a pregnancy-associated disorder by analyzing fetal DNA present in the mother's blood. More specifically, this invention relies on the discovery that the maspin gene is differentially methylated in fetal DNA and in maternal DNA and provides these new diagnostic methods, which distinguish fetal DNA from maternal DNA and detect prenatal disorders based on abnormalities in fetal DNA level and methylation status.

    Type: Application

    Filed: August 22, 2011

    Issued: January 5, 2012

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis LO, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Yu-Kwan Tong, Chunming Ding

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20120003637

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Application

    Filed: March 23, 2011

    Issued: January 5, 2012

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20110318734

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Application

    Filed: March 23, 2011

    Issued: December 29, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    SIZE-BASED GENOMIC ANALYSIS

    Application number: 20110276277

    Abstract: Systems, methods, and apparatuses for performing a prenatal diagnosis of a sequence imbalance are provided. A shift (e.g. to a smaller size distribution) can signify an imbalance in certain circumstances. For example, a size distribution of fragments of nucleic acids from an at-risk chromosome can be used to determine a fetal chromosomal aneuploidy. A size ranking of different chromosomes can be used to determine changes of a rank of an at-risk chromosome from an expected ranking. Also, a difference between a statistical size value for one chromosome can be compared to a statistical size value of another chromosome to identify a significant shift in size. A genotype and haplotype of the fetus may also be determined using a size distribution to determine whether a sequence imbalance occurs in a maternal sample relative to a genotypes or haplotype of the mother, thereby providing a genotype or haplotype of the fetus.

    Type: Application

    Filed: November 5, 2010

    Issued: November 10, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Wenli Zheng

     

    Marker for prenatal diagnosis and monitoring

    Patent number: 8026067

    Abstract: The present invention relates to new methods for diagnosing a pregnancy-associated disorder by analyzing fetal DNA present in the mother's blood. More specifically, this invention relies on the discovery that the maspin gene is differentially methylated in fetal DNA and in maternal DNA and provides these new diagnostic methods, which distinguish fetal DNA from maternal DNA and detect prenatal disorders based on abnormalities in fetal DNA level and methylation status.

    Type: Grant

    Filed: March 15, 2010

    Issued: September 27, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Yu-kwan Tong, Chunming Ding

     

    Analysis for Nucleic Acids by Digital PCR

    Application number: 20110183330

    Abstract: The present invention provides a method for analyzing nucleic acids for their lengths and relative abundance in a sample, based on digital amplification of individual template molecules. This invention has many applications, including those in noninvasive prenatal diagnosis, transplantation monitoring, and the detection and monitoring of cancers and virus-associated diseases.

    Type: Application

    Filed: October 28, 2010

    Issued: July 28, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis LO, Rossa Wai Kwun Chiu

     

    NEW FETAL METHYLATION MARKERS

    Application number: 20110143342

    Abstract: This application describes the discovery that, in a pregnant woman, certain genes (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD) originated from a fetus are highly methylated, whereas the same genes of maternal origin are unmethylated. This discovery allows the easy detection of one or more of these methylated fetal genes in a biological sample from a pregnant woman, serving as a universal indicator of the presence of fetal DNA in the sample. These fetal methylation markers are particularly useful as positive controls for a non-invasive analytical process during which the quality and quantity of fetal DNA are monitored. These newly identified fetal markers can also be measured directly for diagnosis of certain pregnancy-related conditions.

    Type: Application

    Filed: June 1, 2010

    Issued: June 16, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Chunming Ding, Kwan Chee Chan, Hing Nam Ivy Wong, Ka Chun Ryan Yuen

     

    Fetal Genomic Analysis From A Maternal Biological Sample

    Application number: 20110105353

    Abstract: Systems, methods, and apparatus for determining at least a portion of fetal genome are provided. DNA fragments from a maternal sample (maternal and fetal DNA) can be analyzed to identify alleles at certain loci. The amounts of DNA fragments of the respective alleles at these loci can be analyzed together to determine relative amounts of the haplotypes for these loci and determine which haplotypes have been inherited from the parental genomes. Loci where the parents are a specific combination of homozygous and heterozygous can be analyzed to determine regions of the fetal genome. Reference haplotypes common in the population can be used along with the analysis of the DNA fragments of the maternal sample to determine the maternal and paternal genomes. Determination of mutations, a fractional fetal DNA concentration in a maternal sample, and a proportion of coverage of a sequencing of the maternal sample can also be provided.

    Type: Application

    Filed: November 5, 2010

    Issued: May 5, 2011

    Assignee: The Chinese University of Hong Kong c/o Technology Licensing Office

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Chan, Wai Kwun Rossa Chiu, Charles Cantor

     

    METHODS FOR ASSESSING LIVER PATHOLOGIES

    Application number: 20110098192

    Abstract: The present invention provides a new method for detecting or monitoring a liver disease in a subject that has no indication of any liver pathologies, by measuring the amount of concentration of albumin mRNA in an acellular blood sample from the subject, and then comparing the amount or concentration of albumin mRNA with a standard control.

    Type: Application

    Filed: September 10, 2010

    Issued: April 28, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Rebecca Wing Yan Chan

     

    Methods for Evaluating a Disease Condition by Nucleic Acid Detection and Fractionation

    Application number: 20110086357

    Abstract: This invention relates to the discovery that both non-particle and particle associated nucleic acids are present in blood plasma and serum and can be used to evaluate disease conditions.

    Type: Application

    Filed: November 8, 2010

    Issued: April 14, 2011

    Inventors: Yuk Ming Dennis Lo, Kai On Ng, Bo Yin Tsui, Wai Kwun Rossa Chiu, Yuen Shan Lisa Chan, Timothy Hudson Rainer, Yuk Lan Lam

     

    Markers for prenatal diagnosis and monitoring

    Patent number: 7901884

    Abstract: This application provides the use of novel fetal markers for prenatal diagnosis and monitoring of certain pregnancy-related conditions. More specifically, the invention resides in the discovery that certain CpG islands located on fetal chromosome 21 demonstrate a methylation profile that is distinct from that of the corresponding CpG islands located on maternal chromosome 21. This application also provides kits for diagnosing or monitoring of the relevant conditions.

    Type: Grant

    Filed: April 6, 2007

    Issued: March 8, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Chunming Ding, Shengnan Jin, Tracy Yuen Han Lee, Fiona Miu Fun Lun

     

    METHOD FOR DETECTING CHROMOSOMAL ANEUPLOIDY

    Application number: 20110039724

    Abstract: The present invention relates to a new, non-invasive method for detecting chromosomal aneuploidy by analyzing a sample from a pregnant woman. The detection is based on the ratio between the amount of a fetal methylation marker located on a chromosome relevant to the aneuploidy and the amount of a fetal genetic marker located on a reference chromosome, offering improved accuracy.

    Type: Application

    Filed: July 9, 2010

    Issued: February 17, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Yu Kwan Tong, Shengnan Jin, Siu Chung Stephen Chim, Wai Yi Tsui

     

    METHODS AND KITS FOR DIAGNOSIS, PROGNOSIS OR MONITORING OF EPSTEIN-BARR VIRUS (EBV)-ASSOCIATED CANCER

    Application number: 20110033841

    Abstract: Disclosed is a non-invasive method for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer by detecting and/or quantifying EBV associated nucleic acid fragments in a urine sample from an individual. Kits for diagnosis, prognosis or monitoring of cancer are also disclosed.

    Type: Application

    Filed: October 21, 2010

    Issued: February 10, 2011

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Allen Chan

     

    Marker for Prenatal Diagnosis and Monitoring

    Application number: 20100323352

    Abstract: The present invention relates to new methods for diagnosing a pregnancy-associated disorder by analyzing fetal DNA present in the mother's blood. More specifically, this invention relies on the discovery that the maspin gene is differentially methylated in fetal DNA and in maternal DNA and provides these new diagnostic methods, which distinguish fetal DNA from maternal DNA and detect prenatal disorders based on abnormalities in fetal DNA level and methylation status.

    Type: Application

    Filed: March 15, 2010

    Issued: December 23, 2010

    Assignee: THE CHINESE UNIVERSITY OF HONG KONG

    Inventors: YUK MING DENNIS LO, ROSSA WAI KWUN CHIU, STEPHEN SIU CHUNG CHIM, YU-KWAN TONG, CHUNMING DING

     

    METHOD FOR THE DETECTION OF CHROMOSOMAL ANEUPLOIDIES

    Application number: 20100311046

    Abstract: The non-invasive detection of fetal chromosomal aneuploidies is demonstrated. Alleles of fetal RNA-SNPs present in a biological sample (e.g. maternal blood) containing fetal RNA are detected and quantified in order to determine the ratio of the alleles. This ratio is compared to a standard control consisting of euploid fetuses. Deviation of allele ratio indicates the presence of chromosomal aneuploidy.

    Type: Application

    Filed: November 16, 2009

    Issued: December 9, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Bo Yin Tsui, Chunming Ding, Charles Cantor

     

    Methods and kits for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer

    Patent number: 7842482

    Abstract: Disclosed is a non-invasive method for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer by detecting and/or quantifying EBV associated nucleic acid fragments in a urine sample from an individual. Kits for diagnosis, prognosis or monitoring of cancer are also disclosed.

    Type: Grant

    Filed: February 26, 2007

    Issued: November 30, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Allen Chan

     

    Circulating mRNA as diagnostic markers

    Patent number: 7829285

    Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting the conditions of pre-eclampsia, fetal chromosomal aneuploidy, and pre-term labor in a pregnant woman, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more mRNA species encoding human chorionic gonadotropin ? subunit (hCG-?), human placental lactogen (hPL), human corticotropin releasing hormone (hCRH), KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2 (TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and comparing the amount of the mRNA species with a standard control.

    Type: Grant

    Filed: May 24, 2007

    Issued: November 9, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Kai On Ng, Bo Yin Tsui, Wai Kwun Rossa Chiu

     

    MARKERS FOR PRENATAL DIAGNOSIS AND MONITORING

    Application number: 20100267034

    Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting preeclaimpsia in a pregnant woman, trisomy 18 and trisomy 21 in a fetus, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more RNA species derived from a set of genetic loci and comparing the amount of the RNA species with a standard control.

    Type: Application

    Filed: April 5, 2010

    Issued: October 21, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis LO, Rossa Wai Kwun CHIU, Stephen Siu Chung CHIM, Nancy Bo Yin TSUI

     

    Fetal methylation markers

    Patent number: 7754428

    Abstract: This application describes the discovery that, in a pregnant woman, certain genes (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD) originated from a fetus are highly methylated, whereas the same genes of maternal origin are unmethylated. This discovery allows the easy detection of one or more of these methylated fetal genes in a biological sample from a pregnant woman, serving as a universal indicator of the presence of fetal DNA in the sample. These fetal methylation markers are particularly useful as positive controls for a non-invasive analytical process during which the quality and quantity of fetal DNA are monitored. These newly identified fetal markers can also be measured directly for diagnosis of certain pregnancy-related conditions.

    Type: Grant

    Filed: April 6, 2007

    Issued: July 13, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Chunming Ding, Kwan Chee Chan, Hing Nam Ivy Wong, Ka Chun Ryan Yuen

     

    Markers for prenatal diagnosis and monitoring

    Patent number: 7718367

    Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting preeclaimpsia in a pregnant woman, trisomy 18 and trisomy 21 in a fetus, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more RNA species derived from a set of genetic loci and comparing the amount of the RNA species with a standard control.

    Type: Grant

    Filed: March 17, 2006

    Issued: May 18, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Nancy Bo Yin Tsui

     

    Diagnosing Fetal Chromosomal Aneuploidy Using Genomic Sequencing With Enrichment

    Application number: 20100112590

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

    Type: Application

    Filed: November 6, 2009

    Issued: May 6, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    Marker for prenatal diagnosis and monitoring

    Patent number: 7709194

    Abstract: The present invention relates to new methods for diagnosing a pregnancy-associated disorder by analyzing fetal DNA present in the mother's blood. More specifically, this invention relies on the discovery that the maspin gene is differentially methylated in fetal DNA and in maternal DNA and provides these new diagnostic methods, which distinguish fetal DNA from maternal DNA and detect prenatal disorders based on abnormalities in fetal DNA level and methylation status.

    Type: Grant

    Filed: June 3, 2005

    Issued: May 4, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Yu-kwan Tong, Chunming Ding

     

    Method for the detection of chromosomal aneuploidies

    Patent number: 7645576

    Abstract: The non-invasive detection of fetal chromosomal aneuploidies is demonstrated. Alleles of fetal RNA-SNPs present in a biological sample (e.g. maternal blood) containing fetal RNA are detected and quantified in order to determine the ratio of the alleles. This ratio is compared to a standard control consisting of euploid fetuses. Deviation of allele ratio indicates the presence of chromosomal aneuploidy.

    Type: Grant

    Filed: March 17, 2006

    Issued: January 12, 2010

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Bo Yin Tsui, Chunming Ding, Charles Cantor

     

    METHODS FOR DETECTING DNA ORIGINATING FROM DIFFERENT INDIVIDUALS

    Application number: 20090170102

    Abstract: In a first aspect, the present invention features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample or to differentiate DNA of an organ donor from DNA of an organ recipient. In preferred embodiments, the DNA species are differentiated by observing epigenetic differences in the DNA species such as differences in DNA methylation. In a second aspect, the present invention features methods of detecting genetic abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. In a third aspect, the present invention features methods for differentiating DNA species originating from an organ donor from those of an organ recipient. In a fourth aspect, the present invention features kits for differentiating DNA species originating from different individuals in a biological sample.

    Type: Application

    Filed: October 13, 2008

    Issued: July 2, 2009

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis LO, Lit Man Poon

     

    Circulating mRNA as diagnostic markers

    Application number: 20090162842

    Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting the conditions of pre-eclampsia, fetal chromosomal aneuploidy, and pre-term labor in a pregnant woman, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more mRNA species encoding human chorionic gonadotropin ? subunit (hCG-?), human placental lactogen (hPL), human corticotropin releasing hormone (hCRH), KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2 (TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and comparing the amount of the mRNA species with a standard control.

    Type: Application

    Filed: May 24, 2007

    Issued: June 25, 2009

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Kai On Ng, Bo Yin Tsui, Wai Kwun Chiu

     

    Fetal methylation markers

    Application number: 20090155776

    Abstract: This application describes the discovery that, in a pregnant woman, certain genes (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD) originated from a fetus are highly methylated, whereas the same genes of maternal origin are unmethylated. This discovery allows the easy detection of one or more of these methylated fetal genes in a biological sample from a pregnant woman, serving as a universal indicator of the presence of fetal DNA in the sample. These fetal methylation markers are particularly useful as positive controls for a non-invasive analytical process during which the quality and quantity of fetal DNA are monitored. These newly identified fetal markers can also be measured directly for diagnosis of certain pregnancy-related conditions.

    Type: Application

    Filed: April 6, 2007

    Issued: June 18, 2009

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun Chiu, Stephen Siu Chung Chim, Chunming Ding, Kwan Chee Chan, Hing Nam Ivy Wong, Ka Chun Ryan Yuen

     

    DETERMINING A NUCLEIC ACID SEQUENCE IMBALANCE

    Application number: 20090087847

    Abstract: Methods, systems, and apparatus are provided for determining whether a nucleic acid sequence imbalance exists within a biological sample. One or more cutoff values for determining an imbalance of, for example, the ratio of the two sequences (or sets of sequences) are chosen. The cutoff value may be determined based at least in part on the percentage of fetal DNA in a sample, such as maternal plasma, containing a background of maternal nucleic acid sequences. The cutoff value may also be determined based on an average concentration of a sequence per reaction. In one aspect, the cutoff value is determined from a proportion of informative wells that are estimated to contain a particular nucleic acid sequence, where the proportion is determined based on the above-mentioned percentage and/or average concentration. The cutoff value may be determined using many different types of methods, such as sequential probability ratio testing .

    Type: Application

    Filed: July 23, 2008

    Issued: April 2, 2009

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis LO, Rossa Wai Kwun Chiu, Kwan Chee Chan, Benny Chung Ying Zee, Ka Chun Chong

     

    Methods and kits for selectively amplifying, detecting or quantifying target DNA with specific end sequences

    Application number: 20090061425

    Abstract: Disclosed herein are methods and kits for selectively amplifying, detecting or quantifying a DNA fragment with a specific end sequence, especially generated following restriction enzyme digestion. This method can be used, for example, to detect a hypomethylated DNA fragment. This methods and kits are especially useful in detecting or quantifying a hypomethylated fetal DNA fragment in a maternal plasma sample containing a corresponding hypermethylated maternal DNA fragment.

    Type: Application

    Filed: August 30, 2007

    Issued: March 5, 2009

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Yu Kwan Tong, Wai Kwun Rossa Chiu, Chunming Ding

     

    ANALYSIS OF NUCLEIC ACIDS BY DIGITAL PCR

    Application number: 20090053719

    Abstract: The present invention provides a method for analyzing nucleic acids for their lengths and relative abundance in a sample, based on digital amplification of individual template molecules. This invention has many applications, including those in noninvasive prenatal diagnosis, transplantation monitoring, and the detection and monitoring of cancers and virus-associated diseases.

    Type: Application

    Filed: July 31, 2008

    Issued: February 26, 2009

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Rossa Wai Kwun CHIU

     

    DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING

    Application number: 20090029377

    Abstract: Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

    Type: Application

    Filed: July 23, 2008

    Issued: January 29, 2009

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Rossa Wai Kwun Chiu, Kwan Chee Chan

     

    COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING SEVERE ACUTE RESPIRATORY SYNDROME (SARS)

    Application number: 20080286756

    Abstract: The present invention relates to the fields of immunochemistry and pharmacology. Methods and compositions are described for the diagnosis and treatment of SARS CoV infection. More specifically, the application discloses nucleic acids and peptides of the spike glycoprotein of SARS CoV that provide prognostic and therapeutic compositions in treatment of individuals contracting, or in danger of contracting SARS CoV. The peptides of the invention are also useful in producing antibodies against the SARS CoV glycoprotein.

    Type: Application

    Filed: April 3, 2008

    Issued: November 20, 2008

    Assignee: THE CHINESE UNIVERSITY OF HONG KONG

    Inventors: Kwok Wing TSUI, Kwok Pui Fung, Mary Miu Yee Waye, Yuk Ming Dennis Lo, Siu Chung Stephen Chim, Wai Kwun Rossa Chiu, Siu Lun John Tam, Kay Sheung Paul Chan

     

    Methods and kits for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer

    Application number: 20080206749

    Abstract: Disclosed is a non-invasive method for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer by detecting and/or quantifying EBV associated nucleic acid fragments in a urine sample from an individual. Kits for diagnosis, prognosis or monitoring of cancer are also disclosed.

    Type: Application

    Filed: February 26, 2007

    Issued: August 28, 2008

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kwan Chee Allen Chan

     

    Circulating mRNA as diagnostic markers

    Application number: 20080153090

    Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting the conditions of pre-eclaimpsia, fetal chromosomal aneuploidy, and pre-term labor in a pregnant woman, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more mRNA species encoding human chorionic gonadotropin ? subunit (hCG-?), human placental lactogen (hPL), human corticotropin releasing hormone (hCRH), KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2 (TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and comparing the amount of the mRNA species with a standard control.

    Type: Application

    Filed: May 24, 2007

    Issued: June 26, 2008

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Kai On Ng, Bo Yin Tsui, Wai Kwun Rossa Chiu

     

    Compositions and methods for diagnosing and treating severe acute respiratory syndrome (SARS)

    Patent number: 7371525

    Abstract: The present invention relates to the fields of immunochemistry and pharmacology. Methods and compositions are described for the diagnosis and treatment of SARS CoV infection. More specifically, the application discloses nucleic acids and peptides of the spike glycoprotein of SARS CoV that provide prognostic and therapeutic compositions in treatment of individuals contracting, or in danger of contracting SARS CoV. The peptides of the invention are also useful in producing antibodies against the SARS CoV glycoprotein.

    Type: Grant

    Filed: July 28, 2004

    Issued: May 13, 2008

    Assignee: The Chinese University of Hong Kong

    Inventors: Kwok Wing Tsui, Siu Chung Stephen Chim, Mary Miu Yee Waye, Kwok Pui Fung, Yuk Ming Dennis Lo, Wai Kwun Rossa Chiu, Siu Lun John Tam, Kay Sheung Paul Chan

     

    Method for diagnosing preeclampsia by detecting hCRH mRNA

    Patent number: 7235359

    Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting the conditions of pre-eclaimpsia, fetal chromosomal aneuploidy, and pre-term labor in a pregnant woman, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more mRNA species encoding human chorionic gonadotropin ? subunit (hCG-?), human placental lactogen (hPL), human corticotropin releasing hormone (hCRH), KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2 (TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and comparing the amount of the mRNA species with a standard control.

    Type: Grant

    Filed: January 16, 2004

    Issued: June 26, 2007

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Kai On Ng, Bo Yin Tsui, Wai Kwun Rossa Chiu

     

    Methods for evaluating stroke or cardiac ischemia by nucleic acid detection

    Patent number: 7022478

    Abstract: This invention relates to the use of a blood sample from a patient for evaluating stroke or cardiac ischemia in the patient.

    Type: Grant

    Filed: July 11, 2002

    Issued: April 4, 2006

    Assignee: The Chinese University of Hong Kong

    Inventors: Timothy Hudson Rainer, Yuk Ming Dennis Lo, Yuk Lan Lam, Lawrence Ka Sing Wong

     

    Circulating mRNA as diagnostic markers

    Application number: 20040203037

    Abstract: Methods and kits are provided for diagnosing, monitoring, or predicting the conditions of pre-eclaimpsia, fetal chromosomal aneuploidy, and pre-term labor in a pregnant woman, as well as for detecting pregnancy in a woman, by quantitatively measuring in the maternal blood the amount of one or more mRNA species encoding human chorionic gonadotropin &bgr; subunit (hCG-&bgr;), human placental lactogen (hPL), human corticotropin releasing hormone (hCRH), KiSS-1 metastasis-suppressor (KISS1), tissue factor pathway inhibitor 2 (TPFI2), placenta-specific 1 (PLAC1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and comparing the amount of the mRNA species with a standard control.

    Type: Application

    Filed: January 16, 2004

    Issued: October 14, 2004

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk-Ming Dennis Lo, Kai On Ng, Bo Yin Tsui, Wai Kwun Rossa Chiu

     

    Circulating epstein-barr virus DNA in the serum of patients with gastric carcinoma

    Patent number: 6753137

    Abstract: The present invention features methods for diagnosing, detecting, monitoring and determining the prognosis of gastric cancer, non-head and neck and lymphoid malignancies, and gastritis in a patient by detecting or measuring EBV DNA present in the serum or plasma of the patient. The present invention also features diagnostic kits comprising suitable reagents for detecting EBV DNA in the serum or plasma of a patient.

    Type: Grant

    Filed: January 25, 2002

    Issued: June 22, 2004

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Wing Yee Chan, Kwok Wai Ng

     

    Novel classification methods for pleural effusions

    Application number: 20040086864

    Abstract: This invention relates to the detection of nucleic acids in the pleural fluids of a patient suffering from a pleural effusion for the classification of the pleural effusion.

    Type: Application

    Filed: October 22, 2002

    Issued: May 6, 2004

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Michael Ho-Ming Chan

     

    Methods for evaluating a disease condition by nucleic acid detection and fractionation

    Application number: 20040009518

    Abstract: This invention relates to the discovery that both non-particle and particle associated nucleic acids are present in blood plasma and serum and can be used to evaluate disease conditions.

    Type: Application

    Filed: May 13, 2003

    Issued: January 15, 2004

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Kai On Ng, Bo Yin Tsui, Wai Kwun Rossa Chiu, Yuen Shan Lisa Chan, Timothy Hudson Rainer, Yuk Lan Lam

     

    Circulating epstein-barr virus DNA in the serum or plasma of patients for the prediction and detection of epstein-barr virus associated cancers apart from head, neck and lymphoid malignancies

    Application number: 20040005551

    Abstract: The present invention features methods for diagnosing, detecting, monitoring and determining the prognosis of Epstein Barr virus associated cancers apart from head, neck and lymphoid malignancies in a patient by detecting or measuring EBV DNA present in the serum or plasma of the patient. The present invention also features diagnostic kits comprising suitable reagents for detecting EBV DNA in the serum or plasma of a patient.

    Type: Application

    Filed: June 3, 2003

    Issued: January 8, 2004

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Wing Yee Chan, Kwok Wai Ng

     

    Non-invasive prenatal monitoring

    Patent number: 6664056

    Abstract: Embodiments of the present invention are directed to the detection of fetal or maternal RNA in a blood sample from a pregnant subject, and may involve subjecting the sample to a test for fetal or maternal analysis indicative of a fetal or maternal condition or characteristics. For instance, the RNA analysis may involve the assessment of the gene expression pattern of an unborn fetus by analyzing a blood sample from the mother. The prenatal monitoring technology allows, for the first time, the detection of genes which are expressed by the fetus, just by analysis of a sample of maternal blood. In specific embodiments, the prenatal monitoring technology is based on the discovery of circulating RNA of fetal origin in the plasma of pregnant women. In general, the detection method performed on a maternal serum or plasma sample from a pregnant female comprises detecting the presence of RNA of fetal or maternal original in the sample.

    Type: Grant

    Filed: June 6, 2001

    Issued: December 16, 2003

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Lit Man Poon

     

    Combination of circulating epstein-barr virus (EBV) DNA in the serum or plasma of patients and a method to assess EBV subtypes for the prediction and detection of epstein-barr virus associated cancers

    Application number: 20030228575

    Abstract: The present invention features methods for diagnosing, detecting, monitoring and determining the prognosis of Epstein Barr virus associated cancers in a patient by detecting or measuring EBV DNA present in the serum or plasma of the patient followed by EBV subtyping of polymorphisms. The sensitivity of the circulating EBV DNA serves as a good screening tool while the EBV subtyping of polymorphism confirms the prognosis or diagnosis of EBV associated malignancies. The present invention also features diagnostic kits comprising suitable reagents for the above tests.

    Type: Application

    Filed: August 8, 2003

    Issued: December 11, 2003

    Assignees: Yuk Ming Dennis Lo, The Chinese University of Hong Kong

    Inventors: Wah Hin Alex Yeung, Yuk Ming Dennis Lo

     

    Methods for evaluating stroke or cardiac ischemia by nucleic acid detection

    Application number: 20030219759

    Abstract: This invention relates to the use of a blood sample from a patient for evaluating stroke or cardiac ischemia in the patient.

    Type: Application

    Filed: July 11, 2002

    Issued: November 27, 2003

    Assignee: The Chinese University of Hong Kong

    Inventors: Timothy Hudson Rainer, Yuk Ming Dennis Lo, Yuk Lan Lam, Lawrence Ka Sing Wong

     

    Circulating epstein-barr virus DNA in the serum of patients with gastric carcinoma

    Application number: 20020192642

    Abstract: The present invention features methods for diagnosing, detecting, monitoring and determining the prognosis of gastric cancer, non-head and neck and lymphoid malignancies, and gastritis in a patient by detecting or measuring EBV DNA present in the serum or plasma of the patient. The present invention also features diagnostic kits comprising suitable reagents for detecting EBV DNA in the serum or plasma of a patient.

    Type: Application

    Filed: January 25, 2002

    Issued: December 19, 2002

    Assignee: The Chinese University of Hong Kong

    Inventors: Yuk Ming Dennis Lo, Wing Yee Chan, Kwok Wai Ng

     

    Non-invasive prenatal monitoring

    Application number: 20020045176

    Abstract: Embodiments of the present invention are directed to the detection of fetal or maternal RNA in a blood sample from a pregnant subject, and may involve subjecting the sample to a test for fetal or maternal analysis indicative of a fetal or maternal condition or characteristics. For instance, the RNA analysis may involve the assessment of the gene expression pattern of an unborn fetus by analyzing a blood sample from the mother. The prenatal monitoring technology allows, for the first time, the detection of genes which are expressed by the fetus, just by analysis of a sample of maternal blood. In specific embodiments, the prenatal monitoring technology is based on the discovery of circulating RNA of fetal origin in the plasma of pregnant women. In general, the detection method performed on a maternal serum or plasma sample from a pregnant female comprises detecting the presence of RNA of fetal or maternal original in the sample.

    Type: Application

    Filed: June 6, 2001

    Issued: April 18, 2002

    Inventors: Yuk Ming Dennis Lo, Lit Man Poon

     

    Non-invasive prenatal diagnosis

    Application number: 20010051341

    Abstract: The invention relates to a detection method performed on a maternal serum or plasma from a pregnant female, which method comprises the presence of a nucleic acid of fetal origin in the sample. The invention enables non-invasive prenatal diagnosis including, for example, sex determination, blood typing and other genotyping, and detection of pre-eclampsia in the mother.

    Type: Application

    Filed: June 1, 2001

    Issued: December 13, 2001

    Assignee: ISIS Innovation Limited

    Inventors: Yuk-Ming Dennis Lo, James Stephen Wainscoat

     

    Non-invasive prenatal diagnosis

    Patent number: 6258540

    Abstract: The invention relates to a detection method performed on a maternal serum or plasma sample from a pregnant female, which method comprises detecting the presence of a nucleic acid of foetal origin in the sample. The invention enables non-invasive prenatal diagnosis including for example sex determination, blood typing and other genotyping, and detection of pre-eclampsia in the mother.

    Type: Grant

    Filed: November 29, 1999

    Issued: July 10, 2001

    Assignee: Isis Innovation Limited

    Inventors: Yuk-Ming Dennis Lo, James Stephen Wainscoat

    Jul 30 1:38 PM | Link | Comment!
  • Natera: Morgan Stanley Initiates With Equal Weight Rating

    Morgan Stanley came out with their initial coverage report on Natera (NASDAQ:NTRA) today as the quiet period from their IPO ended. As lead investment bank on the offering, Morgan Stanley's insights are particularly insightful. Their report: Natera Inc Born Ready states: NTRA should grow at 25% CAGR over the next 3 years into a $15bn TAM despite price pressure on the core business. We believe the technology and commercial organization are top-tier but valuation suggests expectations are high. Equal-weight, PT $20.

    Morgan provides three valuation assessments as follows:

    • Bull Case $40 6.4x Bullish '17E Revenues Panorama differentiates capturing ~30% of average and low risk pregnancy market by '20E.
    • Base Case $20 3.6x Base '17E Revenues The transition to average and low risk pregnancies reimbursement begins in '16 reaching penetration of ~20% for NIPS in average & low risk pregnancies and ~7-8% in high risk by '20E in the US.
    • Bear Case $8 1.8x Bear '17E Revenues Payers hesitant to reimburse NIPS in average & low risk groups for NIPS with sub-10% penetration by '20E in the US.

    Exhibit 38 provides the trailing quarterly income statement and their projections looking forward

     Mar-2014Jun-2014Sep-2014Dec-2014Mar-2015Jun-2015Sep-2015Dec-2015
    Panorama Revenue19.527.833.835.034.8$32.330.425.9
    Total Revenue27.335.846.349.947.444.543.440.7
    Gross Profit %41.7%46.9%55.0%54.6%47.6%41.1%34.6%24.6%
    R&D %15.7%11.5%9.4%9.0%11.9%15.0%16.0%18.0%
    SG&A %52.6%38.8%35.3%36.7%49.0%62.0%63.0%67.0%
    Net Income$(9.6)$(0.5)$3.7$1.3$(10.0)$(16.9)$(20.2)$(25.2)
    Dil EPS$(0.31)$(0.02)$0.12$0.04$(0.27)$(0.44)$(0.42)$(0.44)

    Exhibit 18 was interesting as it provides a Sum-of-the-parts revenue analysis

     

    C14 $M

    C15 $M

    C16 $M

    C17 $M

    US NIPT High Risk39241615
    US NIPT Microdeletions28304256
    US NIPT Low Risk284582115
    Intl NIPT21253362
    All other43535765
    Total Revenue159176230313

    These tables do well to frame their investment thesis in Natera:

    1. The combined US high risk market (including Microdeletions) for Panorama is viewed to be a no growth business through C17.
    2. International NIPT is showing minimal growth until C17. This is a curious projection given how under-penetrated the international markets are and also that this line includes low risk outside the US.
    3. US low risk appears to be the only growth driver for Natera in C15 and C16 based upon average and low risk reimbursement in C16.
    4. Liquid Biopsy opportunity is large but not anticipated to generate any material revenues in the forecast period. They consider this to be worth approximately $100m in their valuation approach as a "call option" balancing execution risk and the size of the TAM.
    Jul 27 11:55 AM | Link | Comment!
  • Liquid Biopsy For Cancer Screening, Patient Stratification And Monitoring

     

    Graham Brock, Elena Castellanos-Rizaldos, Lan Hu, Christine Coticchia, Johan Skog

    Exosome Diagnostics, Cambridge, MA 02139, USA

    www.thetcr.org/article/view/4546/html


    Abstract: Molecular characterization of a patient's tumor to guide treatment decisions is increasingly being applied in clinical care and can have a significant impact on disease outcome. These molecular analyses, including mutation characterization, are typically performed on tissue acquired through a biopsy at diagnosis. However, tumors are highly heterogeneous and sampling in its entirety is challenging. Furthermore, tumors evolve over time and can alter their molecular genotype, making clinical decisions based on historical biopsy data suboptimal. Personalized medicine for cancer patients aims to tailor the best treatment options for the individual at diagnosis and during treatment. To fully enable personalized medicine it is desirable to have an easily accessible, minimally invasive way to determine and follow the molecular makeup of a patient's tumor longitudinally. One such approach is through a liquid biopsy, where the genetic makeup of the tumor can be assessed through a biofluid sample. Liquid biopsies have the potential to help clinicians screen for disease, stratify patients to the best treatment and monitor treatment response and resistance mechanisms in the tumor. A liquid biopsy can be used for molecular characterization of the tumor and its non-invasive nature allows repeat sampling to monitor genetic changes over time without the need for a tissue biopsy. This review will summarize three approaches in the liquid biopsy field: circulating tumor cells , cell free DNA (cfDNA) and exosomes. We also outline some of the analytical challenges encountered using liquid biopsy techniques to detect rare mutations in a background of wild-type sequences.

    Keywords: Liquid biopsy; exosome; circulating tumor cell (NYSE:CTC); cell free DNA (cfDNA); nucleic acids


    IntroductionOther Section

    The science of noninvasive disease monitoring has advanced greatly since circulating cell free DNA (cfDNA) was first reported in body fluids by Mandel and Metais (1). Since then, the evolution of sensitive cfDNA detection technologies has enabled the development of liquid biopsies with many clinical applications. For example, in oncology, the use of liquid biopsy allows for patient stratification (companion diagnostics), screening, monitoring treatment response and detection of minimal residual disease after surgery/recurrence.

    Liquid biopsies have grown in importance because, the genetic profile of tumors can affect how well they respond to a certain treatment. However, this characterization is currently achieved through a biopsy despite the inherent problems in procurement of tissue samples and the limitations of tumor analyses. For example, the invasive nature of a biopsy poses a risk to patients and can have a significant cost. Tumor sampling from some cancer types also remains difficult resulting in inadequate amount of tissue for genetic testing. In the case of advanced or metastatic non-small cell lung cancers (NSCLC) as many as 31% of cases do not have accessible tissue (2). Even when tissue can be collected, preservation methods such as formalin fixation can cause C > T transitions through deamination of cytosine, potentially leading to false positive results for genetic tests (3). Finally, due to tumor heterogeneity, biopsies often suffer from sample bias (4).

    More concerning with respect to guiding treatment decisions; biopsies will only inform of the genotype at that time-point. However, it is known that tumors are very dynamic and can change their dominant mutation pattern or acquire new mutations, especially after the selective pressure of drug treatment. This could be particularly unfavorable when stratifying patients to a specific targeted therapy based on historical mutation profiles of past tumor biopsies. In another example, approximately 50% of NSCLC patients become resistant to tyrosine kinase inhibitor therapy through an epidermal growth factor receptor (EGFR) T790M mutation (5,6), significantly only <5% of NSCLC patient have this mutation detectable in the primary biopsy (7). Another study showed that 38% of colorectal cancers with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS) developed mutations in this gene after anti-EGFR therapy as rapidly as 6 months after treatment (8).

    Several reports have indicated there are difficulties in detecting tumor derived mutations in plasma, while others have been able to efficiently isolate circulating tumor derived nucleic acid in both metastatic and non-metastatic disease (9-11). This discrepancy is likely due to the methodologies used for detection of the mutation, as the allelic fraction of tumor derived circulating DNA varies from less than 0.01% (or undetectable) to over 90% (12,13). In addition, the amount of recoverable DNA varies significantly (over 3 logs) between patients with an average of about 17 ng of DNA per mL of plasma from advanced-stage cancers (14), corresponding to roughly 5,000 haploid genome equivalents.

    Recent technological developments and the downstream analytics being applied to liquid biopsies are now capable of reproducibly detecting mutations at very low allelic frequencies. Advances have also been made in droplet digital PCR (ddPCR) (15), next-generation sequencing (NYSE:NGS) (16), beads, emulsion, amplification and magnetics (BEAMing) (13), amplification of refractory mutation system (ARMS) (17), co-amplification at lower denaturation temperature-PCR (COLD-PCR) and its derivatives (18,19) and PointMan™ DNA enrichment technology (20), to name but a few.

    Ultimately the choice of platforms and required detection limit will depend on the clinical sample being analyzed, as the most sensitive methods are reported to detect allelic frequencies of as little as 0.01%, providing a theoretical lower limit to detect one mutated copy in a background of 10,000 wild-type alleles (13). Thus, this level of sensitivity requires samples/patients where at least 10,000 target alleles enter the downstream analytical assay.

    Although technically challenging, an inherent advantage of liquid biopsies over other traditional tissue-based methodologies is the enablement of longitudinal monitoring which could help clinical oncologists gain a broader molecular understanding of the disease. This review will focus on the application of genetic profiling of tumor associated RNA and DNA derived from biofluids.


    Approaches to liquid biopsy analysisOther SectionCirculating tumor cells

    CTCs are cells shed into the vasculature from a primary tumor and may constitute seeds for subsequent growth of additional tumors (metastasis) in distant organs. They have been detected in various metastatic carcinomas for example breast, prostate, lung, and colorectal cancer (21,22) but are extremely rare in healthy subjects and patients with nonmalignant diseases (23). Clinical evidence indicates that patients with metastatic lesions are more likely to have CTCs amenable to isolation but their frequency is low, often ~1-10 CTCs per mL of whole blood (24). As 1 mL of blood contains ~7×10e6 white blood cells and ~5×10e9 red blood cells (25), technologies capable of reproducibly isolating a single CTC from the background of all other blood components are fundamental. While such levels of sensitivity are challenging, there are several novel developments in this area. These include positive selection, negative selection, physical properties or even enrichment-free assays to efficiently isolate these rare CTCs (26,27).

    Typically, CTCs are defined as cells with an intact viable nucleus, cytokeratin positive, epithelial cell adhesion molecule (EpCAM) positive and with the absence of CD45. Unfortunately EpCAM and other markers are not always expressed on CTCs and are down-regulated by processes such as epithelial to mesenchymal transition (28). In addition, non-tumor epithelial cells are known to circulate in the blood of patients with prostatitis (29) or patients undergoing surgery (30). From a technical standpoint, the heterogeneity of CTCs is a major challenge and this has led to alternative strategies of CTC enrichment, such as the CTC-iChip (31), which do not rely on tumor antigen expression.

    Sequencing the genetic material from CTCs has demonstrated that, even when the isolated cell(s) fit the phenotypic criteria of being a CTC, the majority are not cancer cells. One study developed a protocol to recover the CTC enriched samples from the cartridge of the Veridex platform and found that from 37 NSCLC patients, the mutation allele abundance ranged between 0.02% and 24.79% with a mean of 6.34% (32). The number of CTCs found in the blood is therefore highly dependent on how the platform defines a cell as a CTC.

    Currently, most CTC isolation platforms require that the whole blood is processed soon after collection, negating the option of long-term bio-banking. In addition, CTCs are fragile and tend to degrade when collected in standard evacuated blood collection tubes. The CellSearch CTC test, a Food and Drug Administration (FDA) approved actionable CTC test, requires that samples are processed within 96 hours of collection after being drawn into the Cellsave preservative tube. This test does not analyze the molecular genetics of the tumor; ratherCellsave is a platform for CTC numeration. A positive test (more than five detected CTCs for metastatic breast and prostate cancer and more than three CTCs for metastatic colorectal cancer per 7.5 mL of blood) is associated with decreased progression-free survival and decreased overall survival in these patients (33-37).

    Cell free DNA (cfDNA)

    There is currently an intensive research effort to understand the utility of cfDNA in various clinical fields such as cancer research (38,39), non-invasive prenatal testing (40) and transplant rejection diagnostics (41). Initial studies in cancer patients reported that cfDNA concentration in serum was significantly increased in comparison to healthy individuals (42), and it was suggested that this correlated with malignancy (43).

    Most cfDNA in plasma is reportedly fragmented, around 150-180 bp in length (44) with a higher prevalence of tumor associated mutations in the shorter fragments (9). In fact, when analyzing the mutation abundance with massively parallel sequencing a significant correlation was found between mutations and fragments less than 150 bp (44). Notably, the size of the majority of cfDNA fragments overlaps well with the size of histone DNA (45).

    The entry of cfDNA into the bloodstream is thought to originate from a cell following apoptosis or necrosis. Late stage cancer patients also have an increased level of cfDNA in plasma, however, most of this DNA is wild-type and believed to be from non-malignant cells and tumor stroma (9). It has also been suggested that the mutant fraction of cfDNA is derived from necrotic neoplastic cells phagocytized by macrophages, which then release digested DNA, a phenomena not seen in macrophages that engulf apoptotic cells (14). The extensive background of wild-type DNA limits the ability of downstream analytical platforms to detect tumor-derived mutation, presenting technical challenges for the use of cfDNA in liquid biopsies. While cell-free tumor DNA analyses are capable of examining the genetic or epigenetic changes that originate in tumor DNA (such as mutations, translocations, amplifications, indels and methylation abnormalities), they cannot analyze the tumor RNA transcriptome or proteome.

    However, an advantage of cfDNA is that it can be analyzed from bio-banked biofluids, such as frozen plasma. In addition, a direct comparison of mutation detection on cfDNA vs. CTCs showed a higher abundance of the mutation on the cfDNA from the same patient (39). Finally, recent large studies comparing the effectiveness of cfDNA analysis to tissue biopsy in NSCLC showed the clinical value of the liquid biopsy approach (46). This positive result led to an approval to use cfDNA analysis for EGFR mutation analysis for IRESSA® in Europe (in patients where a tumor sample was not evaluable), making it the first EGFR tyrosine kinase inhibitor for which cfDNA testing is included in the label.

    Although promising, challenges remain when using cfDNA to characterize the mutation status of a tumor. In addition to the low copy number of mutant alleles, the median half-life of cfDNA in circulation ranges from 15 minutes to a few hours (47). Also, the total concentration of cfDNA in the blood of cancer patients varies considerably (48) with tumor specific mutations ranging from undetectable (less than 1 copy per 5 mL of plasma) to patients with over hundred thousand copies of the mutation per ml of plasma (39). Thus, the challenge of how to maximize the yield of the cfDNA and pair this with a platform sensitive enough to detect rare variants in the background of wild-type DNA remains. Optimally, the ability to detect mutations in plasma should not be limited to a subpopulation of patients with very high mutant copy numbers in circulation. While many analytical platforms report the mutation load with an allelic frequency compared to the wild-type DNA, platforms relying solely on the allelic frequency without recording the number of mutations have limitations. The allelic frequency is affected by the amount of wild-type DNA not related to the tumor. Therefore, it is important to consider the processes that affect the amount of wild-type DNA in circulation. For example, exercise increases cfDNA levels 10-fold (49) and other pre-analytical variables such as blood collection and extraction protocols affect the amount and size range of cfDNA fragments in a sample (50). Delays in blood processing, blood storage temperature, agitation of the sample and shipment can all cause wild-type cfDNA release from lysed nucleated blood cells and effect the allelic frequency (51). For the same reason, plasma is often preferred over serum because of the potential for cell lysis during blood coagulation (52).

    Exosomes

    The exosome field has grown exponentially the last few years impacting various areas of research. Studies demonstrating that exosomes are actively released vesicles (carrying RNA, DNA and protein) and can function as inter-cellular messengers, have contributed to their elevated recognition in the scientific community (53-64). A recent review outlining the biological properties of exosomes and other extracellular vesicles (EV's) highlights these developments (65). However, with respect to nomenclature, the exosome field still lags behind as the definition and characterization of EV types are not yet firmly established (66). The majority of exosomes range in size from 30-200 nanometer in diameter and are isolated from all biofluids, including serum (60), plasma, saliva, urine and cerebrospinal fluid (67).

    Exosomes and other EVs are particularly interesting as cancer biomarkers since they are stable carriers of genetic material and proteins from their cell of origin. They are also thought to be part of the disease process, for example, tumor exosomes have been shown to stimulate tumor cells growth, suppress the immune response and induce angiogenesis (60,68) and even be part of the metastatic process (63,69). Exosome release is also an active process and tumor cells can shed tens of thousands of vesicles per day resulting in hundreds of billions of vesicles per mL of plasma (55). The two mechanisms by which exosomes are released, either involve the formation of multivesicular bodies (MVB) and direct budding at the plasma membrane, or a process more akin to a retrovirus particle leaving the cell (Figure 1) (70).

    (click to enlarge)
    Figure 1 Exosome/microvesicle biogenesis. The classical exosome biogenesis pathway begins with the formation of an endosome, followed by inward budding of the endosome resulting in MVB with ILV. These ILV contain a sample of the cell's cytoplasm, including nucleic acids. (NYSE:A) The ILV are then liberated by fusion of the MVB to the plasma membrane; (NYSE:B) the second way of exosome/microvesicle biogenesis is through direct budding at the plasma membrane. MVB, multivesicular bodies; ILV, intraluminal vesicles.

    In the early decades of exosome research, it was thought that they contained only protein and lipids. However, it has since been shown that exosomes are highly stable packages of RNA from the cell of origin (61). The finding that exosomes contain RNA with tumor specific mutations, can be isolated from biofluid samples and stored for many years in the freezer has opened up new opportunities in the field of diagnostics (60,71). Recent publications have also examined the DNA associated with exosomes and shown its utility for detection of gene amplifications as well as mutations (55,64,72).

    Due to the size of an exosome, on average just over 100 nanometers, the entire transcriptome cannot be packaged inside every vesicle. By way of comparison, retrovirus particles with a similar size can package only around 10 kb (73), so it is likely that a single vesicle of that size carries only a limited number of transcripts. However, exosomes are extremely abundant (10e11 per mL of plasma) and when isolating the vesicle fraction, most of the transcriptome can be detected (74). Exosomal RNA can be used for mutation detection (55,60,71,72) as well as global profiling of most types of RNA (74), and the profile alone (without mutation characterization) can be utilized for diagnostics (58,75,76).

    The precipitous release of exosomes by cancer cells seems to correspond to activation of the mitogen-activated protein kinases (MAPK) pathway frequently upregulated in tumor cells (77). Tumor derived mutations can be detected in exosomes from cerebrospinal fluid (67), serum (60), plasma (64) as well as in urine (71). However, as exosomes are released by all cells, they are particularly useful to profile not only mutations in cancer but also RNA profiles in inflammatory (78), metabolic (79), cardiovascular (80), neurodegenerative (81) and other disease processes.

    Exosomes also carry surface markers from the cell of origin, which can be used for enrichment strategies, similar to CTCs (75). For example, characterization and analysis of exosome surface proteins hold great promise for the ability to identify, separate, sort and enrich exosomes originating from diverse cell sources. While the development of methods that allow for the routine analysis of exosome surface proteins has been a challenge, a number of recent advances have demonstrated potential. Immunoaffinity-bead based capture methods, microfluidic chip methods and antibody-based exosome arrays using both label and label-free detection platforms have all successfully exploited specific exosome surface proteins. This has enabled the capture, enrichment and characterization of unique populations of exosomes in the blood of healthy donors and of patients with pancreatic cancer (82), ovarian cancer (83), lung cancer (84,85). Surface protein-based exosome isolation methods combined with exosomal RNA extraction and qPCR detection assays have proven to be rapid and sensitive enough to monitor therapeutic response and resistance using exosomes from the blood of patients with glioblastoma (86,87).

    In addition, the rapid advancement of a novel method of nanoscale fluorescence activated cell sorting call nanoFACS has further advanced methods of exosome isolation and sorting and allowed for the study of discrete, free, individual exosomes from body fluids (88). This technique and variants thereof hold great promise for future diagnostic applications where isolation and examination of individual exosomes is paramount. Finally, in addition to proteins, analysis of exosome protein-to-lipid ratios can be used to further isolate and characterize subpopulations of exosomes in body fluids (89).

    Exosome investigations have focused on the important physiologic and pathophysiologic functions of these vesicles in micro-metastasis, angiogenesis and immune modulation (63,90) and as a means for detection of tumor specific mutations in biofluids. Consequently, in 2012, interest in this new field increased when the National Institute of Health (NIH) dedicated the large strategic Common Fund to study these new entities of extracellular RNA. The goal of this effort is to better understand how exosomes can be utilized for biomarkers and therapeutics as well as understanding this new mechanism of intercellular communication (http://commonfund.nih.gov/Exrna/index).


    Mutation detection and RNA profilingOther Section

    Analysis of nucleic acids present in bodily fluids can provide a better understanding of the disease, as summarized in Table 1.

    (click to enlarge)
    Table 1 Comparison of the analysis capability of CTC's, cfDNA and exosomes
    Full table

    Mutation detection in biofluids is a challenging task and requires highly sensitive analytical platforms. As this field has evolved, the clinical applications of liquid biopsies have improved significantly. Examples of these analytical platforms include BEAMing (13), ARMS (17), and ddPCR (15). These platforms were developed specifically for the detection of extremely rare alleles and are used when the mutation type and position is known. Other platforms such as ice-COLD PCR and targeted resequencing using NGS platforms can detect rare allelic frequencies even when the type and location of the mutation in the gene is undefined. Targeted resequencing is becoming increasingly popular since it can easily accommodate larger panels of genes to cover the actionable mutations in cancer that have significant diagnostic, prognostic or therapeutic implications for a specific therapy. Initially, the inherent error rate of NGS platforms made it difficult to identify very rare alleles (<1%), but strategies using paired-end sequencing and background correction have enabled detection of allelic frequencies at or below 0.1% (91). Incorporation of unique identifiers to each target enables highly sensitive digital sequencing capable of quantifying the number of mutated reads as well as their allelic frequency (92,93).

    RNA profiling from biofluids also poses numerous challenges. However, the discovery that exosomes contained RNA made it possible to separate the fragile RNA from the large amounts of RNases and PCR inhibitors that are present in most biofluids. As cell-free RNA in blood is immediately degraded, RNAs in serum and plasma are either protected inside vesicles like an exosome, in protein complexes with the Ago2 protein (94) or associated with HDL particles (95) as outlined in Figure 2. Most of the early studies were limited to the more abundant short (~22 nt) regulatory microRNAs. The levels of these microRNAs are tightly regulated in normal cells and dysregulation has been implicated in a number of human diseases e.g., cardiovascular (96) neurological and is strongly linked to cancer development and progression as reviewed by Croce (97). However, although robust and readily detectable, microRNAs represent only a minor fraction of the transcriptome. By contrast, if the appropriate methods are used, the nucleic acids in exosomes can be isolated and the entire transcriptome interrogated for effective molecular profiling and mutation detection. Successful RNA profiling from biofluids requires that the contaminants, which could inhibit downstream analysis are removed. The effective purification of the exosomes can remove these contaminants making the exosome isolation platform scalable, where the sample volume input is linear to the RNA output and not affected by the increased amount of RNases that can co-purify (98). This feature is important, since scaling the volume appropriately will enable profiling also of low copy number RNAs.

    (click to enlarge)
    Figure 2 Circulating nucleic acids are coming from a wide range of cellular processes. It is important to optimize the sample processing for the particular target and understand where the RNA and DNA are coming from as well as their abundance. Whole blood as well as cell free plasma has multiple sources containing nucleic acids (shown in A and B respectively). Even components that lack a nucleus (like erythrocytes and thrombocytes) have been shown to carry RNA and can have cfDNA co-isolating in the preparations. *, based on a range of 0-50% of exosome RNA containing the tumor specific mutant allele (67) (and Exosome Diagnostics unpublished data).

    Finally, special precautions need to be taken to prevent degradation during the RNA extraction procedure, as the RNA purified from exosomes and the microRNAs from Ago2 complexes will now be exposed to RNases. Measuring integrity using an exogenous spiked-in sequence of similar size and structure as the RNAs in the exosomes is recommended. Ideally, the 'spike' should itself be protected from RNases, for example using a synthetic vesicle added directly into the biofluid as opposed to the lysed sample.


    DiscussionOther Section

    The most obvious hurdle for all forms of liquid biopsy remains the relative rarity of nucleic acid derived from a tumor against the background of normal material found in most patient samples. In fact, the majority of cell, cell free nucleic acids, microRNAs and exosomes in a liquid biopsy will have originated from normal cells with numbers fluctuating as a consequence of biological variations. Such challenges are addressed using the strategies highlighted in the methods described above. These methods are currently sensitive enough to detect very rare mutation events. However, it is critical that laboratories undertaking such methods must be scrupulous in their methodologies to avoid erroneous results. Although clichéd, the analogy of a needle in a haystack applies and is appropriate for each of these approaches.

    The analysis of CTCs and exosome has benefited from developments in the field of enrichment prior to the analytical readout. While still at an early stage, a number of studies have demonstrated that protein-based isolation and enrichment methods will be an important tool both in enhancing nucleic acid based assays and as stand-alone diagnostics in the future.

    Clearly, exosomes have a number of advantages for diagnostics. They enable high quality RNA to be extracted from fresh or frozen biofluids, thus increasing the scope of detectable mutations to include mutations, splice variants, fusions as well as expression based assays for mRNA, microRNA, lncRNA and other non-coding RNAs. They are also released from living cells as an active process, whereas cfDNA is released through the process of apoptosis and necrosis. On cfDNA, all genes are present at an equal level, whereas RNA originating from a highly expressed gene could occur in thousands of copies/cell. However, as mutations exist on both exosome RNA (living process) and cfDNA (dying process), utilizing a platform that can use both will have obvious advantages for detecting rare mutations. This is especially true in the case of patients who do not have an abundant amount of mutated nucleic acid in circulation.

    Improvements to analytical sensitivity and specificity will address some of the current hurdles, for example, cancer patients who have very few mutations in their biofluids, likely due to biology rather than analytical sensitivity. In many cases, the mutated alleles can occur at less than 1 copy per mL of plasma. So, combining exosome RNA and cfDNA has the advantage of increasing the detection sensitivity for low frequency mutations.

    For the patient there is an obvious and clear advantage to a liquid biopsy in comparison to conventional surgical methods. However, most of the studies to date have focused on detection of actionable mutations in biofluids, and this is arguably only a fraction of the capability of liquid biopsies in enabling personalized medicine. As DNA mutations will only inform of some aspects of the disease, looking at RNA expression in biofluids can help further understand processes within the cancer patient.

    Cancer is a complex and dynamic disease that can change quickly. To fully deliver on the promise of personalized medicine, development of reliable and robust non-invasive platforms for the diagnosis, patient stratification and to monitor treatment response are paramount. The various liquid biopsy platforms described in this review have the potential to add tremendous value to the care of cancer patients.


    AcknowledgementsOther Section

    Disclosure: The authors are employees of Exosome Diagnostics.


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    Cite this article as: Brock G, Castellanos-Rizaldos E, Hu L, Coticchia C, Skog J. Liquid biopsy for cancer screening, patient stratification and monitoring. Transl Cancer Res 2015;4(3):280-290. doi: 10.3978/j.issn.2218-676X.2015.06.05

     

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