(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`\2
`(43) International Publication Date
`WO 2013/132305 A1
`WIPOI PCT
`12 September 201 3 (1 2.09.2013)
`
`(10) International Publication Number
`
`(51)
`
`International Patent Classification:
`CIZQ 1/68 (2006.01)
`G06F 19/20 (2011.01)
`G061" 19/18 (2011.01)
`
`(21)
`
`International Application Number:
`
`PCT/IB2013/000312
`
`(22)
`
`International Filing Date:
`
`8 March 2013 (08.03.2013)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`(74)
`
`Priority Data:
`61/608,623
`61/621,451
`
`8 March 2012 (08.03.2012)
`6 April 2012 (06.04.2012)
`
`US
`US
`
`(81)
`
`Applicant for all designated States except US): THE
`CHINESE UNIVERSITY OF HONG KONG [CN/CN];
`Knowledge Transfer Office, Shatin, New Territorries,
`Hong Kong, SAR (CN).
`
`Inventors: LO, Yuk Ming Dennis; 4th floor, 7 King Tak
`Street, Homantin, Kowloon, Hong Kong SAR (CN).
`CHAN, Kwan Chee; Flat A, 13/F, Block 34, Broadway
`Street, Mei Foo Sun Chuen, Kowloon Hong Kong SAR
`
`(CN). Zl-IENG, Wenli; C/o The Chinese University of
`Hong Kong, R2601, PGH2 Slia Tin, New Territories, Hong
`Kong, SAR (CN). JIANG, Peiyong; Flat 7, 1st floor of
`Block B, Kwong Lam Court, Nos 62-66 Siu Lok Yuen
`Road, Shatin, New Territories, Hong Kong SAR (CN).
`LIAO, Jiawey; Flat 16,
`lO/F, Yat Yan House, Yat Nga
`court, Tai Po Market, New Territories, Hong Kong SAR
`(CN). CHIU, Wai Kwun Rossa; House 31, Double
`Haven, 52 Ma Lok Path, Shatin, New Territories, Hong
`Kong (CN).
`
`Agent: GRIFFITH HACK; Level 10, 161 Collins Street,
`Melbourne, VIC 3000 (AU).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, Bw, BY,
`BZ, CA, CII, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, Ls, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU,
`RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ,
`
`[Continued on next page]
`
`(54) Title: SIZE—BASED ANALYSIS OF FETAL DNA FRACTION IN RIATERNAL PLASMA
`300
`
`Measure amounts of DNA fragments
`corresponding to various sizes
`
`i
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`320
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`330
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`350
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`
`
`
`Y
`Obtain one or more first calibration data points that specify
`a fractional concentration that corresponds to a particular
`value ofthe first parameter.
`
`
`
`
`
`Compare the first value to a calibration value ofat least
`one first calibration data point
`
`
`
`V
`Estimate the fractional concentration ofthe
`clinically-relevant DNA in the biological sample
`
`based on the comparison
`
`
`
`FIG. 3
`
`(57) Abstract: A fractional concentration of clinically-relev-
`ant DNA in a mixture of DNA fi‘om a biological sample is
`determined based 011 amounts of DNA fragments at multiple
`sizes. For example, the fractional concentration of fetal DNA
`in matemal 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 propoltion of fetal DNA and a
`proportion of tumor DNA, respectively. Calibration data
`points (e. g, as a calibration function) indicate a correspond-
`ence 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 thc 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 thc clinically-rol-
`evant DNA.
`
`Y.
`Calculate a first value ofa first parameter based on based on
`the amounts of DNA fragments at multiple sizes
`
`
`
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`WO 2013/132305 A1 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`TM, TN, TR, TT, TZ, UA, UG, us, uz, VC, VN, ZA,
`EE, Es, E1, ER, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`ZM, ZW.
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`.
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`.
`,
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`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`(84) Des1gnated States (unless otherwzse ma’zcated, for every
`GW, ML, MR, NE, SN, TD, TG).
`kind afregionalprotectz’on available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Published:
`
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, _
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
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`

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`WO 2013/132305
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`PCT/IB2013/000312
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`SIZE—BASED ANALYSIS OF FETAL DNA FRACTION IN MATERNAL
`
`PLASMA
`
`CROSS-REFERENCES TO RELATED APPLICATION
`
`[0001] This application is a non—provisional of and claims the benefit ofU.S. Provisional
`
`Patent Application No. 61/608,623, entitled “SIZE-BASED ANALYS1S OF FETAL DNA
`
`FRACTION TN MATERNAL PLASMA,” filed on March 8, 2012, and US. Provisional
`
`Patent Application No. 61/621,451, entitled “SIZE-BASED ANALYSIS OF FETAL DNA
`
`FRACTION IN MATERNAL PLASMA,” filed on April 6, 2012, which are herein
`
`incorporated by reference in its entirety for all purposes.
`
`BACKGROUND
`
`[0002] The discovery of cell-free fetal DNA in maternal plasma has opened up new
`possibilities for noninvasive prenatal diagnosis (Lo YMD et al. Lancell9973502485487).
`
`The mean/median fractional fetal DNA concentration has been reported to be approximately
`
`3% to 10% (Lo YMD et al. Am JHum Genet 1998;62:768-775; Lun FMF et al. Clin Chem
`
`2008;54:1664-1672). The fractional fetal DNA concentration, is an important parameter
`
`which affects the performance of noninvasive prenatal diagnostic tests using maternal plasma
`
`DNA. For example, for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies
`
`(e.g. trisomy 21, trisomy 18 or trisomy 13), the higher the fractional fetal DNA concentration
`
`is, the higher will be the overrepresentation ofDNA sequences derived from the aneuploid
`
`chromosome in maternal plasma. Indeed, it has been demonstrated that for every two times
`
`reduction in the fractional fetal DNA concentration in maternal plasma, the number of
`
`molecules that one would need to count to achieve aneuploidy detection would be four times
`
`(L0 YMD et al. Proc Natl Acad Sci USA 2007;104:13116-13121).
`
`[0003]
`
`For the noninvasive prenatal detection of fetal trisomy by random massively parallel
`
`sequencing, the fractional fetal DNA concentration ofa sample would affect the amount of
`
`sequencing that one would need to perform to achieve a robust detection (Fan HC and Quake
`
`SR. PLoS One 2010;52e10439). Indeed, a number of groups have included a quality control
`
`step in which the fractional fetal DNA concentration is first measured and only samples that
`
`contain more than a minimum fractional fetal DNA concentration would be eligible to
`
`generate a diagnostic result (Palomaki GE et al. Genet Med 201 1,131913—920). Other groups
`have included the fractional fetal DNA concentration in their diagnostic algorithm for
`1
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`estimating the risk that a particular maternal plasma sample is obtained from an aneuploid
`
`pregnancy (Sparks AB et al. Am JObstet Gynecol 2012; 206: 319.e1—9).
`
`[0004]
`
`In addition to aneuploidy detection, the fractional fetal DNA concentration also
`
`similarly affects noninvasive prenatal diagnostic tests conducted using maternal plasma DNA
`
`for detecting monogenic diseases, e.g. the hemoglobinopathies (Lun FMF et al. Proc Natl
`
`Acad Sci USA2008; l 05: 19920-19925) and hemophilia (Tsui NBY et al. Blood
`
`201 1;] 17:3684-3691). The fractional fetal DNA concentration also affects the depth of
`
`sequencing that one would need to perform for constructing a fetal genomewide genetic and
`
`mutational map, as well as fetal whole genome sequencing (Lo YMD et al. Sci Trans] Med
`
`2010;2z61ra91 and US. Patent Application 2011/0105353).
`
`[0005] A number of methods have been described for measuring the fractional fetal DNA
`
`concentration. One approach is to measure the concentration of a fetal-specific,
`
`paternally—inherited sequence that is absent from the maternal genome. Examples of such
`
`sequences include the sequences on the Y chromosome that are present in male fetuses and
`
`sequences from the RHD gene in a Rhesus D positive fetus carried by aRhesus D negative
`
`pregnant woman. One could also measure the total maternal plasma DNA using sequences
`
`that are 'present in both the mother and the fetus. To arrive at a fractional fetal DNA
`
`concentration, one could then calculate the ratio of the concentration ofthe fetal-specific,
`
`paternally-inherited sequence over the concentration of the total maternal plasma DNA.
`
`[0006] Another example of sequences that one could use includes the use of single
`
`nucleotide polymorphisms (Lo YMD et al. Sci Trans] Med 2010;226lra91). A disadvantage
`
`of using genetic markers for the measurement of the fractional fetal DNA concentration is
`
`that no single set of genetic markers would be informative for all fetus-mother pair. Yet
`
`another method that one could employ is the use of DNA sequences that exhibit fetal or
`
`placental-specific DNA methylation patterns in maternal plasma (Nygren AO et al. Clin
`
`Chem 2010;56: I 627-1635). The potential disadvantage ofthe use of DNA methylation
`
`markers is that there may be inter—individual variation in the level of DNA methylation.
`
`Furthermore, methods that are used for the detection of DNA methylation markers are
`
`typically complex, including the use of methylation-sensitive restriction enzyme digestion
`
`(Chan KCA et al. Clin Chem 2008;52:22l l—2218) or bisulfite conversion (Chim SSC et al..
`
`Proc Natl Acad Sci USA 2005;102:14753-14758) or methylated DNA immunoprecipitation
`
`(MeDlP) (Papageorgiou EA et al. Nat Med 201 1; 17: 510—513).
`
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`[0007]
`
`Since the fractional fetal DNA concentration is an important value, it is desirable to
`
`have additional methods and systems for determining the value.
`
`BRIEF SUMMARY
`
`[0008]
`
`Embodiments can provide methods and systems for estimating a fractional
`
`concentration of clinical ly-relevant DNA in a mixture of DNA from a biological sample
`
`based amounts of DNA fragments at various 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 is shown to be correlated with a proportion of fetal DNA and a
`
`proportion of tumor DNA. Calibration data points (e.g., as a calibration function) indicate a
`
`correspondence between values ofa size parameter and the fractional concentration of the
`
`clinically-relevant DNA. For a given sample, a first value ofa size parameter can be
`
`determined from the sizes of DNA fragments in a sample. A comparison ofthe first value
`
`to the calibration data points provides the estimate of the fractional concentration of the
`
`clinically-relevant DNA.
`
`[0009] According to one embodiment, a method estimates a fractional concentration of
`
`clinically-relevant DNA in a biological sample, the biological sample including the clinically-
`
`relevant DNA and other DNA. For each size ofa plurality of sizes, an amount ofa plurality
`
`of DNA fragments from the biological sample corresponding to the size is measured. A
`
`computer system calculates a first value ofa first parameter based on the amounts of DNA
`
`fragments at multiple sizes. The first parameter provides a statistical measure ofa size
`
`profile of DNA fragments in the biological sample. One or more first calibration data points
`
`are obtained. Each first calibration data point specifies a fractional concentration of
`
`clinically-relevant DNA corresponding to a calibration value of the first parameter. The one
`
`or more calibration data points are determined from a plurality of calibration samples. The
`
`first value is compared to a calibration value of at least one calibration data point. The
`
`fractional concentration of the clinically-relevant DNA in the biological sample is estimated
`
`based on the comparison.
`
`[0010] According to another embodiment, a method analyzes a biological sample of an
`
`organism. The biological sample includes DNA originating from normal cells and potentially
`
`from cells associated with cancer. At least some of the DNA is cell-free in the biological
`
`sample. For each size ofa plurality of sizes, an amount ofa plurality of DNA fragments from
`
`the biological sample corresponding to the size is measured. A computer system calculates a
`
`first value ofa first parameter based on the amounts of DNA fragments at multiple sizes. The
`
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`first parameter provides a statistical measure of a size profile of DNA fragments in the
`
`biological sample. The first value is compared to a reference value. A classification ofa
`
`level ofcancer in the organism is determined based on the comparison.
`
`[0011] Other embodiments are directed to systems, portable consumer devices, and
`
`computer readable media associated with methods described herein.
`
`[0012] A better understanding of the nature and advantages of the present invention may be
`
`gained with reference to the following detailed description and the accompanying drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0013]
`
`FIG. 1 shows a plot 100 ofa size distribution of circulating cell—free DNA in
`
`maternal plasma according to embodiments ofthe present invention.
`
`[0014]
`
`FIG. 2A shows a plot 200 of size distributions of fetal DNA in two maternal plasma
`
`samples (15' trimester pregnancies) with different fractional fetal DNA concentrations
`
`according to embodiments of the present invention.
`
`[0015]
`
`Figure 2B shows a plot 250 of size distributions of DNA fragments in two maternal
`
`plasma samples (2”dtrimester pregnancies) with different fractional fetal DNA concentrations
`
`according to embodiments of the present invention.
`
`[0016']
`
`FIG. 3 is a flowchart ofa method 300 illustrating a method of estimating a
`
`fractional concentration of clinically~relevant DNA in a biological sample according to
`
`embodiments of the present invention.
`
`[0017]
`
`FIG. 4 is a plot 400 showing a size distribution (electropherogram) ofmaternal
`
`plasma DNA obtained using electrophoresis according to embodiments of the present
`
`invention.
`
`[0018]
`
`FIG. 5A is a plot 500 showing a proportion of DNA fragments that are 150 bp or
`
`below for samples having various fetal DNA percentage in maternal plasma according to
`
`embodiments of the present invention.
`
`[0019]
`
`FIG. 5B is a plot 550 showing a size ratio ofthe amounts of DNA fragments of
`
`5150 bp and DNA from 163 bp to I69 bp, which labeled as (CF(sizeSlSO)/size(l63-169)).
`
`10
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`[0020]
`
`FIG. 6A is a plot 600 showing a size ratio of the amounts of DNA fragments from
`
`140 bp to [46 bp and DNA from 163 bp to 169 bp, which is labeled as (size(l 40-
`
`30
`
`146)/size(l63-169)).
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`[0021]
`
`FIG. 6B is a plot 650 showing a size ratio of the amounts of DNA fragments from
`
`140 bp to 154 bp and DNA from 163 bp to 169 bp, which is labeled as (size(] 40-
`
`154)/size(163-169)).
`
`[0022]
`
`FIG. 7 is a plot 700 showing a size ratio of the amounts of DNA fragments from
`
`5
`
`I00 bp to I50 bp and DNA from 163 bp to 169 bp, which is labeled as (size( I 00-
`
`150)/size(163-169)).
`
`[0023]
`
`FIG. 8 is a plot 800 showing a proportion of DNA fragments of 150 bp or below for
`
`samples having various fetal DNA percentages in maternal plasma according to embodiments
`
`of the present invention.
`
`[0
`
`[0024]
`
`FIG. 9A is a plot 900 showing a size ratio ofthe amounts of DNA fragments of
`
`5150 bp and DNA from 163 bp to 169 bp, which is labeled as (CF(sizeSISO)/size(l63—l69)).
`
`[0025]
`
`FIG. QB is a plot 950 showing a size ratio ofthe amounts of DNA fragments from
`
`I40 bp to 146 bp and DNA from 163 bp to [69 bp, which is labeled as (size(140-
`
`I46)/size(l63-l69)).
`
`15
`
`[0026]
`
`FIG. 10A is a plot 1000 showing a size ratio ofthe amounts of DNA fragments from
`
`140 bp to 154 bp and DNA from 163 bp to 169 bp, which is labeled as (size(l40-
`
`I 54)/size(163- I 69)).
`
`[0027]
`
`FIG. 108 is a plot 1005 showing a size ratio ofthe amounts of DNA fragments from
`
`100 bp to 150 bp and DNA from 163 bp to 169 bp, which is labeled as (size(IOO-
`
`20
`
`150)/size(163-169)).
`
`[0028]
`
`FIG.
`
`I I
`
`is a plot showing a size ratio plotted vs. fetal DNA percentage for the size
`
`of repeat elements according to embodiments of the present invention.
`
`[0029]
`
`FIG. 12A is an electropherogram 1200 that may be used to determine a size ratio
`
`according to embodiments of the present invention.
`
`25
`
`[0030]
`
`FIG. [28 a plot 1250 showing a size ratio ofthe amounts of DNA fragments from
`
`200 bp to 267 bp and DNA from 290 bp to 294 bp for samples having various fetal DNA
`
`percentage in maternal plasma according to embodiments of the present invention.
`
`[0031]
`
`FIG. 13 is a flowchart ofa method 1300 for determining calibration data points
`
`from measurements made from calibration samples according to embodiments of the present
`invention.
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`[0032]
`
`FIG. 14A is a plot 1400 ofa size ratio against the fractional concentration of fetal
`
`DNA for the training set according to embodiments of the present invention.
`
`[0033]
`
`FIG. 14B is a plot 1450 of fractional concentrations deduced (estimated) from linear
`
`function 1410 of FIG. 14A against the fractional concentrations measured using fetal-specific
`
`sequences according to embodiments of the present invention.
`
`[0034]
`
`FIG. 15A is a plot 1500 showing a proportion of DNA fragments of 150 bp or
`
`below for samples having various tumor DNA percentages in plasma of two hepatocellular
`
`carcinoma (HCC) patients before and after tumor resection according to embodiments of the
`
`present invention.
`
`10
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`[0035]
`
`FIG. 15B is a plot 1550 showing a size ratio of the amounts of DNA fragments of
`
`3150 bp and DNA from 163 bp to 169 bp, which is labeled as (CF(size$150)/size(163-169)),
`
`for two HCC patients before and after tumor resection.
`
`[0036]
`
`FIG. 16A is a plot 1600 showing a size ratio of the amounts of DNA fragments from
`
`140 bp to 146 bp and DNA from 163 bp to 169 bp, which is labeled (size(l 40-146)/size(163—
`
`'15
`
`169)), for two HCC patients before and after tumor resection.
`
`[0037]
`
`FIG. 16B is a plot 1650 showing a size ratio of the amounts of DNA fragments from
`
`140 bp to 154 bp and DNA from 163 bp to 169 bp, which is labeled as (size(140-
`
`154)/size(163-169)), for two HCC patients before and after tumor resection.
`
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`
`[0038]
`
`FIG. 17 is a plot 1700 showing a size ratio ofthe amounts of DNA fragments from
`
`100 bp to 150 bp and DNA from 163 bp to 169 bp, which is labeled as (size(100-
`
`150)/size(163-169)), for two HCC patients before and after tumor resection.
`
`[0039]
`
`FIG. 18A is a plot 1800 showing a proportion of DNA fragments of 150 bp or
`
`below for HCC patients before and after tumor resection.
`
`[0040]
`
`FIG. 188 is a plot 1850 showing a size ratio of the amounts of DNA fragments of
`
`5150 bp and DNA from 163 bp to 169 bp, which is labeled as (CF(size5150)/size(163-169)),
`
`for HCC patients before and after tumor resection.
`
`[0041]
`
`FIG. 19A is a plot 1900 showing a size ratio ofthe amounts of DNA fragments from
`
`140 bp to 146 bp and DNA from 163 bp to 169 bp, which is labeled as (size(l40-
`
`146)/size(163-169)), for HCC patients before and after tumor resection.
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`[0042]
`
`FIG. 19B is a plot 1950 showing a size ratio of the amounts of DNA fragments from
`
`140 bp to 154 bp and DNA from 163 bp to 169 bp, which is labeled as (size(140-
`
`154)/size(163- 169)), for HCC patients before and afier tumor resection.
`
`[0043]
`
`FIG. 20 is a plot 2000 showing a size ratio ofthe amounts of DNA fragments from
`
`100 bp to 150 bp and DNA from 163 bp to 169 bp, which is labeled as (size(100-
`
`lSO)/size(l 63~169)), for HCC patients before and after tumor resection.
`
`[0044]
`
`FIG. 2].
`
`is a flowchart illustrating a method 2100 for analyzing a biological sample
`
`of an organism to determine a classification ofa level of cancer according to embodiments of
`
`the present invention.
`
`[0045]
`
`FIG. 22 is a table 2200 showing some common chromosomal aberrations seen in
`
`various types ofcancers.
`
`[0046]
`
`FIG. 23 shows a block diagram ofan example computer system 2300 usable with
`
`system and methods according to embodiments of the present invention.
`
`DEFlNlTlONS,
`
`10
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`15
`
`[0047] The term "biological sample" as used herein refers to any sample that is taken from
`a subject (e.g.,a human, such as a pregnant woman) and contains one or more nucleic acid
`
`molecule(s) of interest. Examples include plasma, saliva, pleural fluid, sweat, ascitic fluid,
`
`20
`
`bile, urine, serum, pancreaticjuice, stool and cervical smear samples. The biological sample
`may be obtained from a human, an animal, or other suitable organism. A “calibration
`
`sample” corresponds to a biological sample whose clinically-relevant \DNA fraction is known
`or determined via a calibration method, e.g., using an allele specific to the clinically relevant
`
`DNA. Examples ofclinically-relevant DNA are fetal DNA in maternal plasma or tumor
`
`DNA in a patient’s plasma.
`
`25
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`30
`
`[0048] As used herein, the term “locus” or its plural form “loci” is a location or address of
`
`any length of nucleotides (or base pairs) which has a variation across genomes. The term
`
`“sequence read” refers to a sequence obtained from all or part ofa nucleic acid molecule,
`
`e.g., a DNA fragment.
`
`In one embodiment,just one end ofthe fragment is sequenced.
`
`'
`
`Alternatively, both ends (e.g., about 30 bp from each end) of the fragment can be sequenced
`
`to generate two sequence reads. The paired sequence reads can then be aligned to a reference
`
`genome, which can provide a length of the fragment. In yet another embodiment, a linear
`
`DNA fragment can be circularized, e.g., by ligation, and the part spanning the ligation site
`
`can be sequenced.
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`[0049]
`
`The term “universal sequencing” refers to sequencing where adapters are added to
`
`the end of a fragment, and the primers for sequencing attached to the adapters. Thus, any
`
`fragment can be sequenced with the same primer, and thus the sequencing can be random.
`
`[0050]
`
`The term fractional fetal DNA concentration is used interchangeably with the terms
`
`fetal DNA proportion and fetal DNA fraction, and refers to the proportion of fetal DNA
`
`molecules that are present in a biological sample (e.g., maternal plasma or scrum sample) that
`
`is derived from the fetus (Lo YMD et al. Am JHum Genet l998;62:768-775; Lun FMF et al.
`
`Clin Chem 2008;54:1664-1672). Similarly, the terms fractional tumor DNA concentration
`
`may be used interchangeably with the terms tumor DNA proportion and tumor DNA fraction,
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`and refers to the proportion of tumor DNA molecules that are present in a biological sample.
`
`[0051]
`
`The term “Size profile” generally relates to the sizes of DNA fragments in a
`
`biological sample. A size profile may be a histogram that provides a distribution of an
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`amount of DNA fragments at a variety of sizes. Various statistical parameters (also referred
`
`to as size parameters orjust parameter) can be used to distinguish one size profile to another.
`
`One parameter is the percentage of DNA fragment ofa particular size or range of sizes
`
`relative to all DNA fragments or relative to DNA fragments of another size or range.
`
`[0052]
`
`Examples of“clinically-relevant” DNA include fetal DNA in maternal plasma and
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`tumor DNA in the patient’s plasma. Another example include the measurement of the
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`amount of graft-associated DNA in the plasma ofa transplant patient. A further example
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`include the measurement of the relative amounts of hematopoietic and nonhematopoietic
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`DNA in the plasma ofa subject. This latter embodiment can be used for detecting or
`
`monitoring or prognosticating pathological processes or injuries involving hematopoietic
`
`and/or nonhematopoietic tissues.
`
`[0053]
`
`A “calibration data point” includes a “calibration value” and a measured or known
`
`fractional concentration of the DNA of interest (i.e., the clinically-relevant DNA). The
`
`calibration value is a value ofa size parameter as determined for a calibration sample, for
`
`which the fractional concentration of the clinically—relevant DNA is known. The calibration
`
`data points may be defined in a variety of ways, e.g., as discrete points or as a calibration
`
`function (also called a calibration curve or calibrations surface).
`
`[0054] The term “level ofcancer” can refer to whether cancer exists, a stage of a cancer, a
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`size of tumor, how many deletions or amplifications ofa chromosomal region are involved
`
`(eg. duplicated or tripled), and/or other measure ofa severity ofa cancer. The level of
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`cancer could be a number or other characters. The level could be zero. The level of cancer
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`also includes premalignant or precancerous conditions associated with deletions or
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`amplifications.
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`DETAILED DESCRIPTION
`
`[0055]
`
`It is known that cell-free fetal DNA molecules in maternal plasma are generally
`
`shorter than the maternally-derived ones (Chan KCA et al. Clin Chem 2004;50:88-92; Lo
`
`YMD et al. Sci Trans] Med 20] 0;2:6lra9] ). The presence of fetal DNA results in a shift in
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`the overall size distribution of maternal plasma DNA and the degree ofshifting is associated
`
`with the fractional concentration of fetal DNA. By measuring particular values ofthe size
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`profile of maternal plasma DNA, embodiments can obtain the fractional fetal DNA
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`lO
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`concentration in maternal plasma.
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`[0056] Apart from applications in noninvasive prenatal diagnosis, embodiments can also be
`
`used for measuring the fractional concentration ofclinically useful nucleic acid species of
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`different sizes in biological fluids, which can be useful for cancer detection, transplantation,
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`and medical monitoring.
`
`It has previously been shewn that tumor—derived DNA is shorter
`
`than the non-cancer—derived DNA in a cancer patient’s plasma (Diehl F et al. Proc NatlAcad
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`Sci USA 2005; 102: 16368—16373). In the transplantation context, it has been shown
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`hematOpoietic—der‘ived DNA is shorter than non-hematopoietic DNA (Zheng YW et al. Clin
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`Chem 2012;58:549-55 8). For example, ifa patient receives a liver from a donor, then the
`
`DNA derived from the liver (a nonhematopoietic organ in the adult) will be shorter than
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`hematopoietic—derived DNA in the plasma (Zheng YW et al. Clin Chem 2012;58:549-558).
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`Similarly, in a patient with myocardial infarction or stroke, the DNA released by the damaged
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`nonhematopoietic organs (ie. the heart and brain, respectively) would be expected to result in
`
`a shift in the size profile of plasma DNA towards the shorter spectrum.
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`I.
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`SIZE DISTRIBUTION
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`[0057]
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`To demonstrate embodiments, we show in thefollowing examples that one can
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`measure the size profile, for example, by paired-end massively parallel sequencing or by
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`electrophoresis (e.g. using a Bioanalyzer). The latter example is particularly useful because
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`electrophoresis using a Bioanalyzer is a quick and relatively cheap procedure. This would
`
`allow one to rapidly perform this analysis as a quality control measure before one would
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`subject a plasma DNA sample to the relatively expensive sequencing process.
`
`[0058]
`
`FIG.
`
`I shows a plot lOO ofa size distribution of circulating cell-free DNA in
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`maternal plasma according to embodiments ofthe present invention. A size distribution can
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`be obtained by measuring a size of DNA fragments and then counting the number of DNA
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`fragments at various sizes, e.g., within the range of 50 bases to about 220 bases. Plot 100
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`shows two distributions. Distribution 1 10 is for all of the DNA fragments in the maternal
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`plasma sample, and distribution 120 is only for DNA that is from the fetus. The horizontal
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`axis is the size in base pairs (bp) ofthe DNA fragments. The vertical axis is the percentage
`
`of measured DNA fragments
`
`[0059]
`
`In FIG. 1, the size distribution offetal-derived DNA in maternal plasma has been
`
`shown to be shorter than that of the maternally derived ones (Chan KC et al. ClinChem 2004;
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`50:88-92.) Recently, we have used paired-end massively parallel sequencing analysis to
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`determine the high-resolution size distribution of the fetal-specific DNA and total DNA
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`(mainly derived from the mother) in a pregnant woman. We showed that a main difference
`
`between the two species of DNA is that there is a reduction in the fraction of l 66 bp DNA
`
`fragments and an increase proportion ofshorter DNA of below 150 bp for the fetal-derived
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`DNA (Lo YM et a1. Sci Trans] Med 2010 2:61ra9 l).
`
`[0060] Herein, we outline how an analysis ofa size distribution of total DNA fragments in
`
`a maternal plasma sample (an example ofa biological sample) would be useful for
`
`determining the fractional concentration of fetal DNA in maternal plasma. The increased
`
`fractional concentration of fetal DNA in maternal plasma would result in the shortening of
`
`the overall size distribution ofthe total DNA.
`
`In one embodiment, the relative abundance (an
`
`example ofa parameter) of DNA fragments ofapproximately 144 bp and DNA fragments of
`
`approximately 166 bp could be used to reflect the fractional concentration of fetal DNA.
`
`In
`
`another embodiment, other parameters or combination of parameters regarding a size profile
`
`can be used to reflect the size distribution of plasma DNA.
`
`[0061]
`
`FIG. 2A shows a plot 200 of size distributions of fetal DNA in two maternal plasma
`
`samples (lSI trimester pregnancies) with different fractional fetal DNA concentrations
`
`according to embodiments of the present invention. Both ofthese two pregnant women were
`
`carrying male fetuses. The fractional fetal DNA concentrations were determined from the
`
`proportion of sequences from the Y chromosome among the total sequenced DNA fragments.
`Both samples were taken from pregnant women during the first trimester oftheir pregnancies.
`
`Case 338 (solid line, fractional fetal DNA concentration 10%) had a lower fractional fetal
`
`DNA concentration than Case 263 (dotted line, fractional fetal DNA concentration 20%).
`
`When compared with Case 263, Case 338 had a higher peak at 166 bp but the peaks for size
`
`below 150 bp were lower. In other words, DNA fragments shorter than 150 bp were more
`
`abundant in Case 263 whereas the fragments of approximately 166 bp were more abundant in
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`Case 338. These observations are consistent with the hypothesis that the relative amounts of
`
`short and long DNA may be correlated to the fractional fetal DNA concentration.
`
`[0062]
`
`Figure ZB shows a plot 250 of size distributions of DNA fragments in two maternal
`
`plasma samples (2nd trimester pregnancies) with different fractional fetal DNA concentrations
`
`according to embodiments of the present invention. Both samples were taken from pregnant
`
`women during the second trimester. Both of these two pregnant women were carrying male
`
`fetuses. The fractional fetal DNA concentrations were determined from the proportion of
`
`sequences from the Y chromosome among the total sequenced DNA fragments. Similar to the
`
`previous example, case 5415 (dotted line, with higher fractional fetal DNA concentration
`
`19%) had higher peaks for sizes below 150 bp whereas case 5166 (solid line, with lower
`
`fractional fetal DNA concentration 12%) had a higher peak at [66 bp.
`
`[0063]
`
`The correlation of different values ofa size parameter to values of fractional fetal
`
`DNA concentration is shown in data plots below. Additionally, the size of fragments of
`
`tumor DNA is correlated to the percentage of tumor DNA fragments in a sample with tumor
`
`DNA fragments and DNA fragments from normal cells. Thus, the size oftumor fragments
`
`can also be used to determine the percentage oftumor fragments in the sample.
`
`II.
`
`METHOD
`
`[0064]
`
`Since the size of DNA fragments is correlated to a fractional concentration (also
`
`referred to as a percentage), embodiments can use this correlation to determine a fractional
`
`concentration ofa particular type of DNA (e.g., fetal DNA or DNA from a tumor) in a
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`sample. The particular type ofDNA is clinically-relevant as that is the fractional
`
`concentration being estimated. Accordingly, a method can estimate a fractional
`
`concentration ofclinically-relevant DNA in a biological sample based on a measured size of
`
`the DNA fragments.
`
`[0065]
`
`FIG. 3 is a flowchart ofa method 300 illustrating a method ofestimating a
`
`fractional concentration of clini