(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
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`1111111111111111 IIIIII IIIII 111111111111111 II Ill 111111111111111 lllll lllll 11111111111111111111111
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`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`12 May 2011 (12.05.2011)
`
`PCT
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`(51) International Patent Classification:
`C12Q 1/68 (2006.01)
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`(21) International Application Number:
`PCT/US20l0/055604
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`(22) International Filing Date:
`5 November 2010 (05.l l.2010)
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`(25) Filing Language:
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`(26) Publication Language:
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`English
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`English
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`(30) Priority Data:
`61/280,674
`6 November 2009 (06.11.2009)
`
`us
`Applicant (for all designated States except US): THE
`BOARD OF TRUSTEES OF THE LELAND STAN(cid:173)
`FORD JUNIOR UNIVERSITY [US/US]; 1705 El
`Camino Real, Palo Alto, California 94306-1106 (US).
`
`Inventors; and
`Inventors/Applicants (for US only): QUAKE, Stephen
`R. [US/US]; James H. Clark Center, 318 Campus Drive,
`Room E350Q, Stanford, California 94305 (US). SNY(cid:173)
`DER, Thomas M. [US/US]; James H. Clark Center, 318
`Campus Drive, Room E300, Stanford, California 94305
`(US). V ALANTINE, Hannah [US/US]; 300 Pasteur
`Drive, Dean's Office, Room Ml2l, Stanford, California
`94305 (US).
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`(71)
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`(72)
`(75)
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`(10) International Publication Number
`WO 2011/057061 Al
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`(74) Agent: SHERWOOD, Pamela J.; Bozicevic, Field &
`Francis LLP, 1900 University Avenue, Suite 200, East
`Palo Alto, California 94303 (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, 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, 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, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, VA, VG, 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, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, 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).
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`Published:
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`with international search report (Art. 21 (3))
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`(54) Title: NON-INVASIVE DIAGNOSIS OF GRAFT REJECTION IN ORGAN TRANSPLANT PATIENTS
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`(57) Abstract: The disclosure provides methods, devices, compositions and kits for diagnosing or predicting transplant status or
`0
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`outcome in a subject who has received a transplant. The methods comprise determining the presence or absence of one or more
`0
`nucleic acids from a donor transplant, wherein said one or more nucleic acids from said donor are identified based on a predetem -
`ined marker profile, and diagnosing or predicting transplant status or outcome based on the presence or absence of said one or
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`more nucleic acids.
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`NON-INVASIVE DIAGNOSIS OF GRAFT REJECTION IN ORGAN TRANSPLANT
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`PATIENTS
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`BACKGROUND OF THE INVENTION
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`[0001] Organ transplantation is an important medical procedure which saves lives in cases where a
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`patient has organ failure or disablement, and it is now possible to transplant many organs including
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`heart, lungs, kidney, and liver. In some cases, the transplanted organ is rejected by the recipient
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`patient, which creates a life-threatening situation. Monitoring the patient for rejection is difficult and
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`expensive, often requiring invasive procedures. Furthermore, current surveillance methods lack
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`adequate sensitivity.
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`[0002] The present invention resolves these problems by providing non-invasive methods of
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`monitoring organ transplant patients for rejection that are sensitive, rapid and inexpensive.
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`SUMMARY OF THE INVENTION
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`[0003] The invention provides methods, devices, compositions and kits for diagnosing and/or
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`predicting transplant status or outcome in a subject who has received a transplant. In some
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`embodiments, the invention provides methods of diagnosing or predicting transplant status or
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`outcome comprising the steps of: (i) providing a sample from a subject who has received a transplant
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`from a donor; (ii) determining the presence or absence of one or more nucleic acids from the donor
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`transplant, where the one or more nucleic acids from the donor are identified based on a
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`predetermined marker profile; and (iii) diagnosing or predicting transplant status or outcome based
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`on the presence or absence of the one or more nucleic acids.
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`[0004] In some embodiments, the transplant status or outcome comprises rejection, tolerance, non(cid:173)
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`rejection based allograft injury, transplant function, transplant survival, chronic transplants injury, or
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`titer pharmacological immunosuppression.
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`In some embodiments, the non-rejection based allograft
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`injury is selected from the group of ischemic injury, virus infection, peri-operative ischemia,
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`reperfusion injury, hypertension, physiological stress, injuries due to reactive oxygen species and
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`injuries caused by pharmaceutical agents.
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`[0005] In some embodiments, the sample is selected from the group consisting of blood, serum,
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`urine, and stool. In some embodiments, the marker profile is a polymorphic marker profile. In some
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`embodiments, the polymorphic marker profile comprises one or more single nucleotide
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`polymorphisms (SNP's), one or more restriction fragment length polymorphisms (RFLP's), one or
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`more short tandem repeats (STRs), one or more variable number of tandem repeats (VNTR's), one or
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`more hypervariable regions, one or more minisatellites, one or more dinucleotide repeats, one or
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`more trinucleotide repeats, one or more tetranucleotide repeats, one or more simple sequence repeats,
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`or one or more insertion elements. In some embodiments, the polymorphic marker profile comprises
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`one or more SNPs
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`[0006] In some embodiments, the marker profile is determined by genotyping the transplant donor.
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`In some embodiments, the methods further comprise genotyping the subject receiving the transplant.
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`In some embodiments, the methods further comprise establishing a profile of markers, where the
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`markers are distinguishable between the transplant donor and the subject receiving the transplant. In
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`some embodiments, the genotyping is performed by a method selected from the group consisting of
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`sequencing, nucleic acid array and PCR.
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`[0007] In any of the embodiments described herein, the transplant graft maybe any solid organ and
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`skin transplant. In some embodiments, the transplant is selected from the group consisting of kidney
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`transplant, heart transplant, liver transplant, pancreas transplant, lung transplant, intestine transplant
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`and skin transplant.
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`[0008] In some embodiments, the nucleic acid is selected from the group consisting of double(cid:173)
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`stranded DNA, single-stranded DNA, single-stranded DNA hairpins, DNA/RNA hybrids, RNA and
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`RNA hairpins. In some embodiments, the nucleic acid is selected from the group consisting of
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`double-stranded DNA, single-stranded DNA and cDNA. In some embodiments, the nucleic acid is
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`mRNA. In some embodiments, the nucleic acid is obtained from circulating donor cells. In some
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`embodiments, the nucleic acid is circulating cell-free DNA.
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`[0009] In some embodiments, the presence or absence of the one or more nucleic acids is
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`determined by a method selected from the group consisting of sequencing, nucleic acid array and
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`PCR. In some embodiments, the sequencing is shotgun sequencing. In some embodiments, the array
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`is a DNA array. In some embodiments, the DNA array is a polymorphism array. In some
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`embodiments, the polymorphism array is a SNP array.
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`[0010] In some embodiments, the methods further comprise quantitating the one or more nucleic
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`acids. In some embodiments, the amount of the one or more nucleic acids is indicative of transplant
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`status or outcome. In some embodiments, the amount of the one or more nucleic acids above a
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`predetermined threshold value is indicative of a transplant status or outcome. In some embodiments,
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`the threshold is a normative value for clinically stable post-transplantation patients with no evidence
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`of transplant rejection or other pathologies. In some embodiments, there are different predetermined
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`threshold values for different transplant outcomes or status. In some embodiments, temporal
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`differences in the amount of the one or more nucleic acids are indicative of a transplant status or
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`outcome.
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`[0011] In some embodiments, the methods described herein have at least 56 % sensitivity. In some
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`embodiments, the methods described herein have at least 78 % sensitivity. In some embodiments,
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`the methods described herein have a specificity of about 70% to about 100%. In some
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`embodiments, the methods described herein have a specificity of about 80% to about 100%. In some
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`embodiments, the methods described herein a specificity of about 90% to about 100%. In some
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`embodiments, the methods described herein have a specificity of about 100%.
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`[0012] In some embodiments, the invention provides computer readable mediums comprising: a set
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`of instructions recorded thereon to cause a computer to perform the steps of: (i) receiving data from
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`one or more nucleic acids detected in a sample from a subject who has received transplant from a
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`donor, where the one or more nucleic acids are nucleic acids from the donor transplant, and where
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`the one or more nucleic acids from the donor are identified based on a predetermined marker profile;
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`and (ii) diagnosing or predicting transplant status or outcome based on the presence or absence of
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`the one or more nucleic acids.
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`[0013] In some embodiments, the invention provides reagents and kits thereof for practicing one or
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`more of the methods described herein.
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`INCORPORATION BY REFERENCE
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`[0014] All publications and patent applications mentioned in this specification are herein
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`incorporated by reference to the same extent as if each individual publication or patent application
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`was specifically and individually indicated to be incorporated by reference.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0015] The novel features of the invention are set forth with particularity in the appended claims. A
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`better understanding of the features and advantages of the present invention will be obtained by
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`reference to the following detailed description that sets forth illustrative embodiments, in which the
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`principles of the invention are utilized, and the accompanying drawings of which:
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`[0016] Figure 1 shows patient survival after diagnosis of CAV.
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`[0017] Figure 2 shows detection of donor DNA in patients receiving gender mismatched
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`transplants.
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`[0018] Figure 3 shows a time course study for detection of donor DNA in a transplant patient that
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`received a gender mismatched transplant and suffered a 3A rejection episode.
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`[0019] Figure 4 shows a time course study for detection of donor DNA in a transplant patient that
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`received a gender mismatched transplant and suffered a 3A rejection episode.
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`[0020] Figure 5 depicts in one embodiments of the invention a general strategy to monitor all
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`transplant patients
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`[0021] Figure 6 shows sequencing results comparing four levels of substitutions of donor DNA into
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`recipient DNA.
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`DETAILED DESCRIPTION OF THE INVENTION
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`[0022] Reference will now be made in detail to particularly preferred embodiments of the invention.
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`Examples of the preferred embodiments are illustrated in the following Examples section.
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`[0023] Unless defined otherwise, all technical and scientific terms used herein have the same
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`meaning as is commonly understood by one of skill in the art to which this invention belongs. All
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`patents and publications referred to herein are incorporated by reference in their entirety.
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`[0024] Methods, devices, compositions and kits are provided for diagnosing or predicting transplant
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`status or outcome in a subject who has received a transplant. The transplant status or outcome may
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`comprise rejection, tolerance, non-rejection based transplant injury, transplant function, transplant
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`survival, chronic transplant injury, or titer pharmacological immunosuppression.
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`[0025] This invention describes sensitive and non-invasive methods, devices, compositions and kits
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`for monitoring organ transplant patients, and/or for diagnosing or predicting transplant status or
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`outcome (e.g. transplant rejection). In some embodiments, the methods, devices, compositions and
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`kits are used to establish a genotype for both the donor and the recipient before transplantation to
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`enable the detection of donor-specific nucleic acids such as DNA or RNA in bodily fluids such as
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`blood or urine from the organ recipient after transplantation.
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`[0026] In some embodiments, the invention provides methods of determining whether a patient or
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`subject is displaying transplant tolerance. The term "transplant tolerance" includes when the subject
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`does not reject a graft organ, tissue or cell(s) that has been introduced into/onto the subject. In other
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`words, the subject tolerates or maintains the organ, tissue or cell(s) that has been transplanted to it.
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`The term "patient" or "subject" as used herein includes humans as well as other mammals.
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`[0027] In some embodiments the invention provides methods for diagnosis or prediction of
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`transplant rejection. The term "transplant rejection" encompasses both acute and chronic transplant
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`rejection. "Acute rejection or AR" is the rejection by the immune system of a tissue transplant
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`recipient when the transplanted tissue is immunologically foreign. Acute rejection is characterized by
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`infiltration of the transplanted tissue by immune cells of the recipient, which carry out their effector
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`function and destroy the transplanted tissue. The onset of acute rejection is rapid and generally occurs
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`in humans within a few weeks after transplant surgery. Generally, acute rejection can be inhibited or
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`suppressed with immunosuppressive drugs such as rapamycin, cyclosporin A, anti-CD40L
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`monoclonal antibody and the like.
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`[0028] "Chronic transplant rejection or CR" generally occurs in humans within several months to
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`years after engraftment, even in the presence of successful immunosuppression of acute rejection.
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`Fibrosis is a common factor in chronic rejection of all types of organ transplants. Chronic rejection
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`can typically be described by a range of specific disorders that are characteristic of the particular
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`organ. For example, in lung transplants, such disorders include fibroproliferative destruction of the
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`airway (bronchiolitis obliterans); in heart transplants or transplants of cardiac tissue, such as valve
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`replacements, such disorders include fibrotic atherosclerosis; in kidney transplants, such disorders
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`include, obstructive nephropathy, nephrosclerorsis, tubulointerstitial nephropathy; and in liver
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`transplants, such disorders include disappearing bile duct syndrome. Chronic rejection can also be
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`characterized by ischemic insult, denervation of the transplanted tissue, hyperlipidemia and
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`hypertension associated with immunosuppressive drugs.
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`[0029] In some embodiments, the invention further includes methods for determining an
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`immunosuppressive regimen for a subject who has received a transplant, e.g., an allograft.
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`[0030] Certain embodiments of the invention provide methods of predicting transplant survival in a
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`subject that has received a transplant. The invention provides methods of diagnosing or predicting
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`whether a transplant in a transplant patient or subject will survive or be lost. In certain embodiments,
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`the invention provides methods of diagnosing or predicting the presence of long-term graft survival.
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`By "long-term" graft survival is meant graft survival for at least about 5 years beyond current
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`sampling, despite the occurrence of one or more prior episodes of acute rejection. In certain
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`embodiments, transplant survival is determined for patients in which at least one episode of acute
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`rejection has occurred. As such, these embodiments provide methods of determining or predicting
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`transplant survival following acute rejection. Transplant survival is determined or predicted in
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`certain embodiments in the context of transplant therapy, e.g., immunosuppressive therapy, where
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`immunosuppressive therapies are known in the art. In yet other embodiments, methods of
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`determining the class and/or severity of acute rejection (and not just the presence thereof) are
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`provided.
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`[0031] In some embodiments, the invention provides methods for diagnosis or prediction of non(cid:173)
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`rejection based transplant injury. Examples of non-rejection based graft injury include, but are not
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`limited to, ischemic injury, virus infection, peri-operative ischemia, reperfusion injury, hypertension,
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`physiological stress, injuries due to reactive oxygen species and injuries caused by pharmaceutical
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`agents.
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`[0032] As in known in the transplantation field, the transplant organ, tissue or cell(s) may be
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`allogeneic or xenogeneic, such that the grafts may be allografts or xenografts. A feature of the graft
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`tolerant phenotype detected or identified by the subject methods is that it is a phenotype which occurs
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`without immunosuppressive therapy, i.e., it is present in a host that is not undergoing
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`immunosuppressive therapy such that immunosuppressive agents are not being administered to the
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`host. The transplant graft maybe any solid organ and skin transplant. Examples of organ transplants
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`that can be analyzed by the methods described herein include but are not limited to kidney transplant,
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`pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, pancreas
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`after kidney transplant, and simultaneous pancreas-kidney transplant.
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`[0033] Where a range of values is provided, it is understood that each intervening value, to the tenth
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`of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and
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`lower limit of that range and any other stated or intervening value in that stated range, is
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`encompassed within the invention. The upper and lower limits of these smaller ranges may
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`independently be included in the smaller ranges and are also encompassed within the invention,
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`subject to any specifically excluded limit in the stated range. Where the stated range includes one or
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`both of the limits, ranges excluding either or both of those included limits are also included in the
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`invention.
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`[0034] Certain ranges are presented herein with numerical values being preceded by the term
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`"about." The term "about" is used herein to provide literal support for the exact number that it
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`precedes, as well as a number that is near to or approximately the number that the term precedes. In
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`determining whether a number is near to or approximately a specifically recited number, the near or
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`approximating unrecited number may be a number which, in the context in which it is presented,
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`provides the substantial equivalent of the specifically recited number.
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`[0035] The practice of the present invention employs, unless otherwise indicated, conventional
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`techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology,
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`genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and
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`Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989);
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`CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the
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`series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL
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`APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds.
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`(1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI.
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`Freshney, ed. (1987)).
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`Introduction
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`[0036] Methods, devices, compositions and kits are provided for diagnosing or predicting transplant
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`status or outcome in a subject who has received a transplant.
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`[0037] As mention above, monitoring transplant patients for transplant status or outcome is difficult
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`and expensive, often requiring non-sensitive and invasive procedures. For instance, in heart
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`transplant patients acute rejection surveillance requires serial endomyocardial biopsies that are
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`routinely performed at weekly and monthly intervals during the initial year after transplant, with a
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`total of 6-8 biopsies in most patients. Advances in immunosuppression, rejection surveillance, and
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`early recognition and treatment of life-threatening infections have led to continuous improvements in
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`early outcomes after cardiac transplantation. (Taylor, D.O., et al., J Heart Lung Transplant, 27, 943-
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`956 (2008)) However, there has not been a similar improvement in late mortality, which is largely
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`attributable to cardiac allograft vasculopathy (CAV). (Figure 1) Today, CAV remains the major
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`cause of late graft failure and death amongst the nearly 22,000 living heart transplant recipients in the
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`United States. Early detection of CAV, prior to the development of angiographically apparent
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`disease, graft dysfunction, or symptom onset is important because patient mortality after detection by
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`coronary angiography (the standard of care) is unacceptably high, with 2-year mortality rates of 50%
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`having been reported. Current surveillance methods for CAV lack adequate sensitivity or require
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`invasive procedures and the most commonly applied method, coronary angiography, lacks sensitivity
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`(Kobashigawa, J.A., et al., J Am Coll Cardiol, 45, 1532-1537 (2005)). Delayed diagnosis due to
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`underestimation of disease severity is a feature of coronary angiography that is largely overcome by
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`intravascular ultrasound (IVUS). (Fitzgerald, P.J., et al., Circulation, 86, 154-158 (1992)) However,
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`both of these invasive left-heart, arterial catheter methods are costly, resource intensive, and
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`associated with significant risk of morbidity and patient discomfort. Early detection of CA V, prior to
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`the development of angiographically apparent disease, graft dysfunction, or symptom onset is crucial
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`to guide the appropriate use of emerging therapies that retard and occasionally reverse progression of
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`CAV. The development of markers for early, non-invasive, safe, and cost-effective detection of acute
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`rejection and CA V, and their rapid translation to a practical and reliable test that can be used in the
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`clinic represents a major unmet medical need for the nearly 22,000 living heart transplant recipients
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`in the United States, and a similar number worldwide.
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`[0038] The pressing need for early diagnosis and risk stratification is further underscored by recent
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`studies demonstrating delayed progression and/or reversal of CA V following intervention with newer
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`immunosuppressive regimens. Since the use of these newer therapies are encumbered by adverse
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`effects, drug interactions, and cost, it is important to identity the patients in whom the benefits
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`outweigh the risks. Aside from its impact on mortality and morbidity, CAV surveillance is costly in
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`terms of resource utilization and potential for patient complications. Given the current standard of
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`care to perform annual coronary angiography for the initial five years after heart transplantation, each
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`patient surviving to year 5 will have received 4 angiograms for an average fully loaded cost of
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`$25,000 per angiogram. Since the 5-year survival rate after heart transplantation is 72%,
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`approximately 1,440 patients out of the 2,000 patients receiving heart transplants each year will
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`undergo 4 procedures for a total of at least 5,760 procedures. At an average cost of $25,000 per
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`coronary angiogram, this will amount to $144,000,000 per year in healthcare dollars for monitoring
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`patients after heart transplantation. A non-invasive test that identifies the patients at low risk of CA V
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`would mean that coronary angiography could be safely avoided in this group, thereby considerably
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`reducing the cost of their long-term management.
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`[0039] The same difficulties and expenses are experienced by patients receiving other type of
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`transplants.
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`a. Circulating Nucleic Acids
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`[0040] Circulating, or cell-free, DNA was first detected in human blood plasma in 1948. (Mandel, P.
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`Metais, P., CR Acad. Sci. Paris, 142, 241-243 (1948)) Since then, its connection to disease has been
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`established in several areas. (Tong, Y.K. Lo, Y.M., Clin Chim Acta, 363, 187-196 (2006)) Studies
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`reveal that much of the circulating nucleic acids in blood arise from necrotic or apoptotic cells
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`(Giacona, M.B., et al., Pancreas, 17, 89-97 (1998)) and greatly elevated levels of nucleic acids from
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`apoptosis is observed in diseases such as cancer. (Giacona, M.B., et al., Pancreas, 17, 89-97 (1998);
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`Fournie, G.J., et al., Cancer Lett, 91, 221-227 (1995)) Particularly for cancer, where the circulating
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`DNA bears hallmark signs of the disease including mutations in oncogenes, microsatellite alterations,
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`and, for certain cancers, viral genomic sequences, DNA or RNA in plasma has become increasingly
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`studied as a potential biomarker for disease. For example, Diehl et al recently demonstrated that a
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`quantitative assay for low levels of circulating tumor DNA in total circulating DNA could serve as a
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`better marker for detecting the relapse of colorectal cancer compared with carcinoembryonic antigen,
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`the standard biomarker used clinically. (Diehl, F., et al., Proc Natl Acad Sci, 102, 16368-16373
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`(2005); Diehl, F., et al., Nat Med, 14, 985-990 (2008)) Maheswaran et al reported the use of
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`genotyping of circulating cells in plasma to detect activating mutations in epidermal growth factor
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`receptors in lung cancer patients that would affect drug treatment. (Maheswaran, S., et al., N Engl J
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`Med, 359, 366-377 (2008)) These results collectively establish both circulating DNA, either free in
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`plasma or from circulating cells, as a useful species in cancer detection and treatment. Circulating
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`DNA has also been useful in healthy patients for fetal diagnostics, with fetal DNA circulating in
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`maternal blood serving as a marker for gender, rhesus D status, fetal aneuploidy, and sex-linked
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`disorders. Fan et al recently demonstrated a strategy for detecting fetal aneuploidy by shotgun
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`sequencing of cell-free DNA taken from a maternal blood sample, a methodology that can replace
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`more invasive and risky techniques such as amniocentesis or chorionic villus sampling. (Fan, H.C.,
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`Blumenfeld, Y.J., Chitkara, U., Hudgins, L., Quake, S.R., Proc Natl Acad Sci, 105, 16266-16271
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`(2008))
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`[0041] In all these applications of circulating nucleic acids, the presence of sequences differing from
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`a patient's normal genotype has been used to detect disease. In cancer, mutations of genes are a tell(cid:173)
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`tale sign of the advance of the disease; in fetal diagnostics, the detection of sequences specific to the
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`fetus compared to maternal DNA allows for analysis of the health of the fetus.
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`[0042] In some embodiments, the invention provides non-invasive diagnostics exists for organ
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`transplant patients where sequences from the organ donor, otherwise "foreign" to the patient, can be
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`quantitated specifically. Without intending to be limited to any theory, as cell-free DNA or RNA
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`often arises from apoptotic cells, the relative amount of donor-specific sequences in circulating
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`nucleic acids should provide a predictive measure of on-coming organ failure in transplant patients
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`for many types of solid organ transplantation including, but not limited to, heart, lung, liver, and
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`kidney.
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`b. Circulating Nucleic Acids and Transplant Rejection
`[0043] In some embodiments, the invention provides methods, devices, compositions and kits for
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`detection and/or quantitating circulating nucleic acids, either free in plasma or from circulating cells,
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`for the diagnosis, prognosis, detection and/or treatment of a transplant status or outcome. There have
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`been claims of detection of donor-DNA in sex-mismatched liver and kidney transplant patients;
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`conventional PCR was used to search for Y chromosome sequences from male donors in the blood of
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`female patients. (Lo, Y.M., et al., Lancet, 351, 1329-1330 (1998) However, in a follow-on study Y(cid:173)
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`chromosome specific sequences were not detected above background in 16 out of 18 patients using a
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`more accurate quantitative polymerase chain reaction (qPCR) assay. (Lui, Y.Y., et al., Clin Chem,
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`49, 495-496 (2003)) In renal transplantation, urine samples of similarly sex-mismatched transplant
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`patients were analyzed and Y chromosomal DNA was detected in patients immediately after
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`-8-
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`9
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`Foresight EX1003-p. 9
`Foresight v Personalis
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`WO 2011/057061
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`PCT /0S2010/055604
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`transplantation as well as during graft rejection episodes. (Zhang, J., et al., Clin Chem, 45, 1741-1746
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`(1999) ; Zhong, X.Y., et al., Ann NY Acad Sci, 945, 250-257 (2001))
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`[0044] Example 1 examined gender-mismatched heart transplant recipients and applied digital PCR
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`(Warren, L., Bryder, D., Weissman, I.L., Quake, S.R., Proc Natl Acad Sci, 103, 17807-17812 (2006);
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`Fan, H.C. Quake, S.R., Anal Chem, 79, 7576-7579 (2007)) to detect the level of donor-derived
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`chromosome Y signal in plasma samples taken at the same time that an endomyocardial biopsy
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`determined a grade 3A or 3B rejection episode. While there was not any significant chromosome Y
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`signal detected from four control female-to-female transplant patients, 1.5-8% total genomic fraction
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`for chromosome Y signals at the rejection time points was observed for three male-to-female
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`transplant patients across four rejection episodes (Figure 2). A time-course study for one of these
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`patients revealed that the level of chromosome Y detected in plasma was neglible in plasma at three
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`months prior to rejection, but increased> IO-fold to 2% of total genomic fraction at the time a biopsy
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`determined rejection (See Figures 3 and 4). Collectively, these results establish that for heart
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`transplant patients, donor-derived DNA present in plasma can serve as a potential marker for the
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`onset of organ failure.
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`[0045] While each of these studies demonstrates donor-DNA in bodily fluids for different solid
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`organ transplants, they are all limited to the special case of females receiving organs from males and
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`will not work for females receiving from females, males receiving from males, or males receiving
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`from females. Further problems with this strategy arise from the prevalence of microchimerism in
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`female patients where past male pregnancies or blood transfusions may lead to Y-chromosome
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`specific signals from sources other than the transplanted organ. (Hubacek, J.A., Vymetalova, Y.,
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`Bohuslavova, R., Kocik, M., Malek, I., Transplant Proc, 39, 1593-1595 (2007); Vymetalova, Y., et
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`al., Transplant Proc, 40, 3685-3687 (2008)) The detection of donor-specific human leukocyte
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`antigen (HLA) alleles in circulating DNA has been considered as a signal for organ rejection,
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`specifically for kidney and pancreas transplant patients. (Gadi, V.K., Nelson, J.L., Boespflug, N.D.,
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`Guthrie, K.A., Kuhr, C.S., Clin Chem, 52, 379-382 (2006)) However, this strategy will also be
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`limited by the inability to distinguish HLA alleles between all donors and recip