(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`
`Organization
`International Bureau
`(43) International Publication Date
`24 October 2013 (24.10.2013)
`
`“
`
`é
`a_
`9’
`WIPOI PCT
`
`(10) International Publication Number
`
`WO 2013/159035 A2
`
`(51)
`
`International Patent Classification:
`CJZQ 1/68 (2006.01)
`
`(2 1)
`
`International Appliea tion Number:
`
`PCT/US2013/03743 9
`
`(22)
`
`International Filing Date:
`
`19 April 2013 (19.04.2013)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`Priority Data:
`61/635,723
`61/700,873
`61/798,421
`
`19 April 2012 (19.04.2012)
`13 September 2012 (13.09.2012)
`15 March 2013 (15.03.2013)
`
`English
`
`English
`
`US
`US
`US
`
`lVIEDICAL COLLEGE OF WISCONSIN,
`Applicant:
`INC. [US/US]; Office Of Technology Development, 8701
`Watertown Plank Road, lVIilwaukee, WI 53226 (US).
`
`13835 Stonefield
`Inventors: MITCHELL, Michael;
`Court, Elm Grove, MN 53122 (US). MITCHELL, Aoy,
`Tomita; 13855 Stonefield Court, Elm Grove, WI 53122
`(US).
`
`(81)
`
`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, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GlVI, 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,
`TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84)
`
`Designated States (unless otherwise indicated for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, sz, TZ,
`UG, ZM, ZVV), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG),
`Published:
`
`(74)
`
`Agent: VATLAND, Janice, A.; Wolf, Greenfield & Sacks,
`P.C., 600 Atlantic AVenue, Boston, MA 02210-2206 (US).
`
`without international search report and to be republished
`upon receipt oft/mt report (Rule 48.2(g))
`
`(54) Title: HIGHLY SENSITIVE SURVEILLANCE USING DETECTION OF CELL FREE DNA
`
`SE vs E} Day“:
`
`(57) Abstract: Provided herein are
`methods and computer-readable stor-
`age media related to cell-free DNA and
`uses thereof to determine risk of a con-
`dition, such as transplant rejection or
`cancer, in a subject.
`
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`
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`
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`
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`cross-match
`
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`
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`
`Fig, 2
`
`
`
`WO2013/159035A2||||||||||||||||||H|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`

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`WO 2013/159035
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`PCT/US2013/037439
`
`HIGHLY SENSITIVE SURVEILLANCE USING DETECTION OF CELL FREE DNA
`
`RELATED APPLICATIONS
`
`This application claims the benefit under 35 U .S.C. 119(e) of the filing date of U .S.
`
`Provisional Application 61/635,723, filed April 19, 2012; the filing date of U.S. Provisional
`
`Application 61/700,873, filed September 13, 2012; and the filing date of US. Provisional Application
`
`61/798,421, filed March 15, 2013; the contents of each which are incorporated herein by reference in
`
`their entirety.
`
`SUMMARY OF THE INVENTION
`
`In one aspect, a method of assessing a risk in a subject is provided. The method may
`
`comprise any of the steps provided herein. In one embodiment, the method comprises analyzing
`
`nucleic acids from cell—free DNA extracted from a biological sample obtained from the subject to
`
`identify a plurality of loci, the nucleic acids comprising first nucleic acids of the subject and second
`
`nucleic acids not native to the subject; determining an allele of each of the plurality of loci; selecting
`
`at least one informative locus from the plurality of loci based on the determining of the allele;
`
`calculating an estimated allele frequency of a first allele at the at least one informative locus using a
`
`statistical distribution; determining an amount of cell-tree DNA not native to the subject in the cell-
`
`free DNA based on the estimated allele frequency; and determining a risk in the subject based on the
`
`determined amount of the cell—free DNA not native to the subject in the cell—free DNA.
`
`In another aspect, a method of treatment of a subject is provided. In one embodiment, the
`
`method comprises determining an amount of cell-free DNA not native to the subject in cell-free DNA
`
`extracted from a biological sample from the subject; determining a risk in the subject based on the
`
`determined amount of the cell—free DNA not native to the subject; and administering a therapy, or
`
`providing information about a therapy, to the subject based on the determined risk. In one
`
`embodiment, the determining an amount of cell-free DNA not native to the subject comprises
`
`analyzing nucleic acids from the extracted cell—free DNA to identify a plurality of loci, the nucleic
`
`acids comprising first nucleic acids of the subject and second nucleic acids not native to the subject;
`
`determining an allele of each of the plurality of loci; selecting at least one informative locus from the
`
`plurality of loci based on the determining of the allele; and calculating an estimated allele frequency
`
`of a first allele at the at least one informative locus using a statistical distribution, wherein the amount
`
`of cell—free DNA not native to the subject is based on the estimated allele frequency.
`
`In another aspect, a method of assessing a risk of a systemic disease in a recipient of a
`
`transplant is provided.
`
`In one embodiment, the method comprises quantifying the amount of cell—free
`
`DNA extracted from a biological sample obtained from the recipient of a transplant; and
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`determining a risk of a systemic disease in the recipient of a transplant based on the determined
`
`amount of the cell—free DNA.
`
`In one embodiment, the risk is indicative of the presence or absence of
`
`a systemic disease. In one embodiment, the method further comprises in a case Where the amount of
`
`the cell-free DNA is greater than a threshold value, determining that the risk is increased. In another
`
`embodiment, the method further comprises based on the determined amount of the cell-free DNA,
`
`administering a therapy or providing information about a therapy to the recipient of a transplant.
`
`In
`
`another embodiment, the method further comprises based on the determined amount of the cell-free
`
`DNA, evaluating an effect of a therapy administered to the recipient of a transplant.
`
`In one
`
`embodiment, a decreased amount of the determined amount of the cell-free DNA is indicative of a
`
`positive effect of the therapy.
`
`In another embodiment, the method further comprises based on the
`
`determined amount of the cell—free DNA, predicting the likely clinical course.
`
`In another aspect, a method of treatment of a recipient of a transplant is provided. In one
`
`embodiment, the method comprises quantifying the amount of cell-free DNA extracted from a
`
`biological sample obtained from the recipient of a transplant; determining a risk of a systemic disease
`
`in the recipient of a transplant based on the determined amount of the cell-free DNA; and
`
`administering a therapy, or providing information about a therapy, to the recipient of a transplant
`
`based on the determined risk.
`
`In one embodiment, the risk is indicative of the presence or absence of
`
`a systemic disease. In one embodiment, the method further comprises in a case Where the amount of
`
`the cell-free DNA is greater than a threshold value, determining that the risk is increased.
`
`In another aspect, a method of evaluating a subject is provided.
`
`In one embodiment, the
`
`method comprises calculating a value for a Predictive Model, and assessing the condition of the
`
`subject. In one embodiment, the Predictive Model is the Predictive Model of Formula 1 using values
`
`for the time post-initiation of therapy (e.g., surgical or pharmaceutical) x non-native cf—DNA.
`
`Predictive Model (Formula 1) = time post initiation of therapy (e. g., surgical or pharmaceutical) X
`
`non—native cf—DNA.
`
`In another embodiment, the Predictive Model is the Predictive Model of
`
`Formula 2 using values for the time post-clamp removal, recipient weight, donor weight, and donor—
`
`specific cell—free DNA. Predictive Model (Formula 2) = time post-clamp removal x (recipient weight
`
`/ donor weight) X donor—specific cell—free DNA. In another embodiment, the Predictive Model is the
`
`Predictive Model of Formula 3 using values for the time post initiation of a therapy (e. g., anti—
`
`rejection therapy, such as an immunosuppressive therapy, a therapy for treating systemic disease or
`
`anti-cancer therapy), recipient weight, donor weight and non-native cell—free DNA. Predictive Model
`
`(Formula 3) = time post initiation of a therapy X (recipient weight/donor weight) X non—native cell-
`
`free DNA. In one embodiment, the non—native cf—DNA is DS cf—DNA, CS cf—DNA or bacterial,
`
`fungal or viral DNA. In one embodiment, the method further comprises determining an amount of
`
`non-native cell-free DNA in a biological sample from the subject. In one embodiment, the
`
`determining an amount of non—native cell-free DNA comprises any of the steps of the methods for
`
`doing so provided herein, including those in the Examples and Figures. In one embodiment,
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`determining an amount of non—native cell—free DNA comprises analyzing nucleic acids from extracted
`
`cell-free DNA from the biological sample to identify a plurality of loci, the nucleic acids comprising
`
`first nucleic acids of the subject and second nucleic acids of the donor; determining an allele of each
`
`of the plurality of loci; selecting at least one informative locus from the plurality of loci based on the
`
`determining of the allele; and calculating an estimated allele frequency of a first allele at the at least
`
`one informative locus using a statistical distribution, wherein the amount of non—native cell—free DNA
`
`is based on the estimated allele frequency. In another embodiment, the method further comprises
`
`determining or obtaining the time post initiation of therapy (e.g., as in Formula 1, 2 or 3), recipient
`
`weight and/or donor weight. In another embodiment, the method further comprises comparing the
`
`value for the Predictive Model (e. g., as in Formula 1, 2 or 3) with a threshold value to assess the
`
`condition of the subject. In one embodiment, the assessing the condition comprises determining a risk
`
`associated with a transplant or cancer or predicting the likely clinical course. In another embodiment,
`
`the method further comprises, based on the assessing, administering a therapy or providing
`
`information about a therapy to the subject. In another embodiment, the method further comprises,
`
`based on the assessing, evaluating an effect of a therapy administered to subject. In one embodiment,
`
`the amount of the therapy administered to the subject is increased or decreased based on the
`
`evaluation. In another embodiment, a different therapy is administered to the subject based on the
`
`evaluation. In one embodiment, a value of the Predictive Model (e. g., as in Formula 1, 2 or 3) is
`
`determined at one point in time to assess the condition of the subject. In another embodiment, a value
`
`of the Predictive Model (e. g., as in Formula 1, 2 or 3) is determined at at least two points in time to
`
`assess the condition of the subject. Values for the Predictive Model (e.g., as in Formula 1, 2 or 3) can
`
`be determined over a period of time to assess the condition of the subject.
`
`In another aspect, a method of monitoring over a time period a risk in a subject is provided.
`
`In one embodiment, the method comprises determining/assessing/evaluating the risk in the subject at
`
`least twice. The method for determining/assessing/evaluating the risk may comprise any of the
`
`methods provided herein, including those in the Examples and Figures. In one embodiment, the
`
`method comprises performing any of the other methods provided herein at least twice. In another
`
`embodiment, the method comprises evaluating the subject at least twice. The method for evaluating
`
`the subject may comprise the steps of any of the methods provided herein, including those in the
`
`Examples and Figures. In one embodiment, the method of monitoring over a time period can further
`
`comprise performing an additional test on the subject or a biological sample obtained from the
`
`subject. In another embodiment, the method of monitoring over a time period can further comprise
`
`treating the subject with a therapy or providing information about a therapy to the subject.
`
`In another aspect, at least one computer—readable storage medium storing computer—
`
`executable instructions that, when executed by at least one processor, cause a computing device to
`
`perform any of the methods, or one or more of the steps thereof, provided herein, including those in
`
`the Examples and Figures, is provided. In one embodiment, the method comprises determining an
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`allele of each of a plurality of loci; selecting at least one informative locus from the plurality of loci
`
`based on the determining of the allele; calculating an estimated allele frequency of a first allele at the
`
`at least one informative locus using a statistical distribution; and determining an amount of cell—free
`
`DNA not native to a subject in the cell-free DNA based on the estimated allele frequency. In one
`
`embodiment, the method further comprises determining a risk in the subject based on the determined
`
`amount of the cell—free DNA not native to the subject in the cell—free DNA.
`
`In another embodiment, the method comprises determining an amount of cell-free DNA not
`
`native to a subject in cell-free DNA extracted from a biological sample from the subject; and
`
`determining a risk in the subject based on the determined amount of the cell-free DNA not native to
`
`the subject. In one embodiment, determining an allele of each of a plurality of loci; selecting at least
`
`one informative locus from the plurality of loci based on the determining of the allele; and calculating
`
`an estimated allele frequency of a first allele at the at least one informative locus using a statistical
`
`distribution, wherein the amount of cell-free DNA not native to the subject is based on the estimated
`
`allele frequency. In one embodiment, the method further comprises determining an amount of cell—
`
`free DNA not native to the subject comprises analyzing nucleic acids from the extracted cell-free
`
`DNA to identify a plurality of loci, the nucleic acids comprising first nucleic acids of the subject and
`
`second nucleic acids not native to the subject.
`
`In another embodiment, the method comprises quantifying an amount of cell—free DNA
`
`extracted from a biological sample obtained from a recipient of a transplant; and determining a risk of
`
`a systemic disease in the recipient of a transplant based on the determined amount of the cell-free
`DNA.
`
`In another embodiment, the method comprises calculating a value for a Predictive Model
`
`(e. g., as in Formula 1, 2 or 3). In one embodiment, the method further comprises assessing the
`
`condition of the subject. In another embodiment, the method further comprises determining an
`
`amount of non-native cell-free DNA in a biological sample from the subject.
`
`In one embodiment,
`
`determining an amount of non—native cell-free DNA comprises analyzing nucleic acids from extracted
`
`cell-free DNA from the biological sample to identify a plurality of loci, the nucleic acids comprising
`
`first nucleic acids of the subject and second nucleic acids not native to the subject; determining an
`
`allele of each of the plurality of loci; selecting at least one informative locus from the plurality of loci
`
`based on the determining of the allele; and calculating an estimated allele frequency of a first allele at
`
`the at least one informative locus using a statistical distribution, wherein the amount of non—native
`
`cell-free DNA is based on the estimated allele frequency.
`
`In another embodiment, the method further
`
`comprises comparing the value for the Predictive Model (e.g., as in Formula 1, 2 or 3) with a
`
`threshold value to assess the condition of the subject.
`
`In one embodiment of any of the methods provided herein, the subject is a recipient of a
`
`transplant, and the risk is a risk associated With the transplant.
`
`In one embodiment, the risk associated
`
`With the transplant is risk of transplant rejection, an anatomical problem with the transplant or injury
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`to the transplant. In another embodiment, the injury to the transplant is initial or ongoing injury. In
`
`another embodiment, the risk associated With the transplant is indicative of the severity of the injury.
`
`In another embodiment, the risk associated With the transplant is a risk of having or developing a
`
`systemic disease.
`
`In one embodiment, the systemic disease is inflammation, infection or sepsis.
`
`In
`
`another embodiment, the risk associate with the transplant is indicative of the bacterial, fungal and/or
`
`viral load.
`
`In one embodiment of any of the methods provided herein, the cell-free DNA is total cell—free
`
`DNA or cell—free DNA native to the subject.
`
`In one embodiment of any of the methods provided herein, the cell-free DNA not native to the
`
`subject is donor-specific cell-free DNA.
`
`In one embodiment of any of the methods provided herein, the subject has or is at risk of
`
`having a cancer, and the risk is a risk associated with the cancer. In one embodiment, the risk
`
`associated With the cancer is the presence or absence of the cancer, recurrence of the cancer or
`
`metastasis of the cancer.
`
`In another embodiment, the risk associated with the cancer is indicative of
`
`the cancer load in the subject.
`
`In one embodiment of any of the methods provided herein, the cell-free DNA not native to the
`
`subject is cancer-specific cell-free DNA.
`
`In one embodiment of any of the methods provided herein, the method further comprises
`
`extracting the cell—free DNA from the biological sample.
`
`In one embodiment of any of the methods provided herein, the first allele comprises a minor
`
`allele.
`
`In one embodiment of any of the methods provided herein, the at least one informative locus
`
`is selected by detecting the first allele and a second allele at a locus; and determining that the first
`
`nucleic acids are homozygous for the second allele at the at least one informative locus and the second
`
`nucleic acids are heterozygous or homozygous for the first allele at the at least one informative locus.
`
`In one embodiment of any of the methods provided herein, the first allele comprises a minor
`
`allele and the second allele comprises a major allele.
`
`In one embodiment of any of the methods provided herein, the first allele comprises a minor
`
`allele; and the estimated allele frequency of the minor allele is calculated using a statistical
`
`distribution.
`
`In one embodiment, the statistical distribution is a binomial distribution.
`
`In one embodiment of any of the methods provided herein, the first allele comprises a minor
`
`allele; and the estimated allele frequency of the minor allele is calculated using an expectation-
`
`maximization algorithm. In one embodiment, the expectation—maximization algorithm is a maximum
`
`likelihood method.
`
`In one embodiment, of any of the methods provided herein, the estimated allele frequency is
`
`calculated using a combination of a statistical distribution, such as a binomial distribution, and an
`
`expectation-maximixation algorithm, such a maximum likelihood method.
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`Preferably, in some embodiments, the number of informative reads is at least 100, 200, 300,
`
`400, 500, 600, 700, 800, 900, 1000, 1100 or 1200. In one embodiment, the method also comprises
`
`correcting the count or number of reads of the major and minor alleles of the at least one informative
`locus.
`
`In one embodiment of any of the methods provided herein, the nucleic acids are analyzed
`
`using high—throughput DNA sequencing. In one embodiment of any of the methods provided herein,
`
`the nucleic acids are analyzed using quantitative genotyping.
`
`In one embodiment of any of the
`
`methods provided herein, the nucleic acids are analyzed using next generation sequencing.
`
`In one embodiment of any of the methods provided herein, the method further comprises in a
`
`case where the amount of the cell-free DNA not native to the subject in the cell-free DNA is greater
`
`than a threshold value, determining that the risk is increased. In one embodiment, when the subject is
`
`a recipient of a transplant, the threshold value comprises 1%.
`
`In one embodiment of any of the methods provided herein, the method further comprises in a
`
`case where the amount of the cell—free DNA not native to the subject in the cell—free DNA is equal to
`
`or less than a threshold value, determining that the risk is decreased. In one embodiment, when the
`
`subject is a recipient of a transplant, the threshold value comprises 1%.
`
`In one embodiment of any of the methods provided herein, when the subject is a recipient of a
`
`transplant, the method is performed within 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day of receiving the transplant.
`
`ln one embodiment, when the subject is a recipient of a transplant, the method is perfbrmed within 10
`
`days of receiving the transplant.
`
`In another embodiment, when the subject is a recipient of a
`
`transplant, the method is performed within 5 days of receiving the transplant. In another embodiment,
`
`when the subject is a recipient of a transplant, the method is performed within 3 days of receiving the
`
`transplant.
`
`In one embodiment of any of the methods provided herein, when the subject is a recipient of a
`
`transplant the method is performed at a time of a scheduled endomyocardial biopsy (EMB).
`
`In one embodiment of any of the methods provided herein, the method further comprises,
`
`based on the determined amount of the cell-free DNA not native to the subject, administering a
`
`therapy or providing information about a therapy to the subject.
`
`In one embodiment of any of the methods provided herein, the method further comprises,
`
`based on the determined amount of the cell—free DNA, such as cell—free DNA not native to the subject,
`
`evaluating an effect of a therapy administered to the subject.
`
`In one embodiment, a decreased amount
`
`of the determined amount of the cell-free DNA, such as the cell—free DNA not native to the subject, is
`
`indicative of a positive effect of the therapy. In another embodiment, the amount of the therapy
`
`administered to the subject is increased or decreased based on the evaluation. In another embodiment,
`
`a different therapy is administered to the subject based on the evaluation.
`
`In one embodiment of any of the methods provided herein, the therapy is anti-rej ection
`
`therapy.
`
`In one embodiment of any of the methods provided herein, the therapy comprises a
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`therapeutic agent that treats a systemic disease. In one embodiment of any of the methods provided
`
`herein, the therapy comprises an anti-cancer therapy.
`
`In one embodiment of any of the methods provided herein, the method further comprises
`
`performing an additional test on the subject or biological sample.
`
`In one embodiment, the additional
`
`test is a test for assessing a risk associated with a transplant. In another embodiment, the additional
`
`test is a test for assessing the presence or absence of a cancer, or a recurrence or metastasis thereof.
`
`In one embodiment of any of the methods provided herein, the method further comprises,
`
`based on the determined amount of the cell-free DNA not native to the subject, predicting the likely
`
`clinical course.
`
`In one embodiment, when the subject is a recipient of a transplant predicting the
`
`likely clinical course comprises predicting a length of hospital stay after the subject received the
`
`transplant, the likelihood of mortality, likelihood of a risk or the likelihood of a problem with the
`
`transplant. In one embodiment, predicting the likely clinical course comprises calculating a value for
`
`a Predictive Model (e. g., as in Formula 1, 2 or 3).
`
`In one embodiment of any of the methods provided herein, wherein based on the predicted
`
`likely clinical course, a course of action is selected for the subject or information about a course of
`
`action is provided to the subject.
`
`In one embodiment of any of the methods provided herein, when the subject is a recipient of a
`
`transplant, the transplant comprises a heart transplant.
`
`In one embodiment of any of the methods provided herein, the subject is a pediatric patient.
`
`In one embodiment of any of the methods provided herein, the method further comprises
`
`obtaining a biological sample from the subject.
`
`In one embodiment of any of the methods provided herein, the biological sample comprises,
`
`blood, plasma, serum or urine.
`
`In one embodiment of any of the methods provided herein, the method further comprises
`
`determining a value of a Predictive Model (e.g., as in Formula 1, 2 or 3).
`
`In one embodiment, the
`
`method further comprises assessing the condition of the subject.
`
`In one embodiment of any of the methods provided herein, the method comprises a step of
`
`spiking in an internal standard at known quantities to aid in the quantification of the cell-free DNA,
`
`such as cell-free DNA not native to the subject.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Fig. 1 is a diagram showing the data analysis method performed in Example 1.
`
`Fig. 2 is a graph showing log percent donor specific (DS) cell-free (cf) DNA versus percent
`
`donor cf—DNA in patient samples taken during surveillance biopsy (SB) and rejection (Rj).
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`Fig. 3 is a bar graph showing percent donor cf—DNA in surveillance biopsy (SB) patient
`
`samples and rejection (Rj) patient samples taken the day of clinical diagnosis of rejection (Rj d1) and
`
`at day 4 (Rj d4) and day 8 (Rj d8) after clinical diagnosis of rejection.
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`Fig. 4 is a graph showing percent donor cf—DNA in surveillance biopsy (SB) samples and
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`rejection (Rj) samples taken the day of clinical diagnosis of rejection (Rj d1) and at day 2 (Rj d2) and
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`day 3 (Rj d3) after clinical diagnosis of rejection.
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`Fig. 5 shows two graphs showing DS cf—DNA post-surgery (panel A) and pre- and post—
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`biopsy (panel B). Panel A) Levels of DS cf—DNA in plasma from pediatric heart transplant patients at
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`three post-operative time points between days 1—10 (11 patients, 33 samples). Panel B) Levels of DS
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`cf—DNA pre— and postendomyocardial biopsy (EMB) (6 patients, 12 samples) (post-biopsy, range 8—35
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`minutes). The sample indicated by the arrow had the shortest collection time (8 minutes) after biopsy.
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`For both panels, statistical significance was calculated by the Wilcoxon rank sum test for paired data.
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`Fig. 6 is a graph of the length of stay Predictive Model (Formula 1). A significant correlation
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`between length of hospitalization after transplantation surgery and a formula that includes three
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`parameters was found to exist. These parameters include time since cross clamp removal,
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`donor/recipient weight ratio, and the concentration of DS cf—DNA. The graph is plotted versus the log
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`value of length of stay and each circle in the graph represents one patient. The circle with the square
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`around it indicates the single patient who died prior to discharge.
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`Fig. 7 is a series of graphs showing percent DS cf—DNA. total (T ) cf—DNA, and DS cf—DNA
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`in scheduled surveillance biopsies (panels A-C) and unscheduled diagnostic biopsies (panels D-F).
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`Panels A & D) percent DS cf—DNA, panels B & E) T cf—DNA, and panels C & F) DS cf—DNA. Each
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`data point represents a sample collected with the clinical data and biopsy findings indicated by the
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`legend in panels A and D. Data in all six panels are sorted on the X-aXis according to increasing
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`percent DS cf—DNA so that T cf—DNA and DS cf—DNA from each sample align vertically. The dashed
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`line in panels A and D highlights the 1 % DS cf—DNA level, and the vertical solid lines in panels A-C
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`orient the picture so all samples containing less than 1 % DS cf—DNA are on the left-hand side and all
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`samples greater than 1% are on the right.
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`Fig. 8 is a series of graphs showing that percent DS cf—DNA level in plasma is an indicator of
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`rejection. In each panel, surveillance biopsy results are compared with samples taken during biopsy
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`proven rejection at three timepoints; before, during and following intravenous (IV)
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`immunosuppressive treatment. Panel A) percentage DS cf—DNA, panel B) T cf—DNA, and panel C)
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`DS cf—DNA. Surveillance biopsy: 25 plasma samples from 25 patients taken at first study—enrolled
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`surveillance EMB. Rejection samples: 12 samples from 4 patients with biopsy proven rejection, (pre
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`IV therapy = 3 to 44 hours prior to IV steroids), (post IV therapy = 43—98 hours after the last IV
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`steroid dose). Patients found to have antibody—mediated rejection (AM R) not only received IV
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`steroids but were also treated with RituXimab (375mg/M2 weekly X 4) and IV immunoglobulin. The
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`single sample collected at the time of surveillance EMB (which had very high DS cf—DNA percent
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`and was associated with a clinically unsuspected positive EMB for rejection) was excluded because it
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`represented successful detection of subclinical rejection. The brackets with gray p—values indicate a
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`significant difference between surveillance biopsy and rejection samples (pre IV therapy samples) as
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`determined by the Mann-Whitney test for unpaired data. Statistical significance between pre, during,
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`and post IV steroid therapy was calculated by Friedman’s two way test of variance of ranks indicating
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`that there is a significant difference between the three sample groups (p-value displayed on upper non-
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`bracketed line). To identify differences between specific groups, the Wilcoxon rank sum test for
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`paired data was used. The Wilcoxon test results are indicated by brackets with the lowest p-values
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`displayed in black.
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`Fig. 9 is a schematic of an illustrative implementation of a computer system that may be used
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`in connection with any of the embodiments of the invention.
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`Fig. 10 is a series of bar graphs showing the percent DS cf—DNA (A-D) and T cf—DNA (E-II)
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`at three time points post—surgery, in a surveillance biopsy, at 3 times points during rejection, and pre—
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`and post—biopsy.
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`Fig. 11 is a series of graphs showing percent DS ef-DNA and T cf—DNA (GE/mL) during
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`rejection episodes with extended sample collection in five patients.
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`Fig. 12 is a series of graphs showing percent DS ef—DNA and T cf—DNA (GE/InL) in samples
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`from surveillance biopsy (SB). on the day of clinical diagnosis of rejection (Rj D1) and post-operation
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`day 1 (PODl) in several patients.
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`Fig. 13 is a series of graphs showing percent DS ef-DNA (A) and T cf-DNA (B) in samples
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`collected on different post—operative days in patients.
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`Fig. 14 is a series of graphs showing percent DS ef-DNA (A) and T ef-DNA (GE/mL) in
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`samples collected on different post-operative days in patients.
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`Fig. 15 is a series of graphs showing percent DS ef-DNA and T ef-DNA pre and post biopsy.
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`Fig. 16 describes an exemplary method for collecting and analyzing cf—DNA.
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`Fig. 17 is a series of graphs of minor allele frequencies post-error correction.
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`Fig. 18 is a series of graphs of minor allele frequencies post-error correction in SB samples.
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`Fig. 19 is a series of graphs of minor allele frequencies post-error correction in post-operative
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`day 1, 4, and 8 samples (PODl, POD4, and PODS).
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`Fig. 20 provides an example of a distribution of minor allele frequencies from informative
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`loci: Each triangle in the plot represents the minor allele frequency (MAF) at one loci and the circles
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`represents the background noise measured in loci were recipient and donor are homozygous for the
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`same allele. The background errors are subtracted from informative loci at the sequencing read level
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`prior to determination of percent. The probability that a loci contains a certain MAF is calculated
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`from the distribution of all informative loci and plotted on the y—axis as the probability density for
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`probes to contain X % of the minor allele. The peak probability indicated by arrow is used as the
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`percent MAF corresponding to the per