WO2018000031A1

WO2018000031A1 - Use of cell free dna as a measure of organ health following transplantation

Info

Publication number: WO2018000031A1
Application number: PCT/AU2017/050657
Authority: WO
Inventors: Su Kah Goh; Alexander Dobrovic; Hongdo Do
Original assignee: Olivia Newton-John Cancer Research Institute
Priority date: 2016-06-27
Filing date: 2017-06-27
Publication date: 2018-01-04

Abstract

The present disclosure relates to the use of donor-specific cell-free DNAfor detecting organ transplant rejection, organ dysfunction or organ failure as well as organ transplant health. Donor-specific cell-free DNA is quantified using a probe-free quantitative PCR comprising the use of allele specific forward or reverse primers which fully hybridise to at least one donor specific deletion/insertion (DIP) allelic sequence.

Description

USE OF CELL FREE DNA AS A MEASURE OF ORGAN HEALTH FOLLOWING

TRANSPLANTATION

RELATED APPLICATIONS

This application is related to Australian Provisional Application 2016902514, filed on 27 June 2016, which is incorporated herein by reference in its entirety for all purposes.

All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

The entire content of the electronic submission of the sequence listing is incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present disclosure relates to the use of donor-specific cell-free DNA for detecting organ transplant rejection, organ dysfunction or organ failure as well as organ transplant health.

BACKGROUND OF THE INVENTION

Breakthroughs in surgical techniques and perioperative care have significantly improved the outcomes of liver transplantation (LTx) with acceptable mortality and morbidity. LTx is the only effective treatment for many irreversible liver diseases like end-stage liver failure, fulminant liver failure and hepatocellular carcinoma. Many of these conditions have poor prognoses without a LTx. After transplantation, the long- term outcomes are favourable with 10-year overall survival of 73% (Australia and New Zealand Liver Transplant Registry. ANZLT Registry report 2014. Brisbane; 2014).

Recipients may be affected by a variety of complications after transplantation. Despite improvements in immunosuppressive therapy, hepatic allograft rejection remains an important cause of morbidity and late graft loss in patients undergoing liver transplantation. Organ rejection in the form of acute cellular rejection (ACR) is a common complication that occurs after LTx (Shaked A et al. (2009) Am J Transplant. 9(2):301 -8). Approximately 20-50% of the liver transplant recipients will develop an episode of ACR within the first 12 months (Lerut J et al. (2008) Ann Surg. 248(6):956- 67). Recurrent episodes of ACR, moderate to severe ACR and/or late-occurring ACR (>3 months after LTx) are associated with reduced graft and recipient survival (Wiesner RH et al. (1998) Hepatology Sep 28(3):217-24). In some cases, re- transplantation is required for the treatment of graft failure secondary to rejection. However, salvage re-transplantation (approximately 20% are due to organ rejection) is a technically challenging undertaking. Re-transplantation is high-risk and this procedure can further worsen the shortage of donor organs.

Acceptable practice for the surveillance of graft health after LTx involves the careful assessment and estimation of graft function. Current means for the diagnosis of ACR are inaccurate or invasive. The constellation of clinical symptoms and signs are generally non-specific. The main investigation of surveillance for ACR is based on serum liver function tests which is neither specific nor sensitive. Although liver function tests is economical to monitor graft health, abnormalities are not diagnostic of ACR [area under the receiver-operating-characteristic (ROC) curve of 0.5] and the magnitude of change does not always correlate with the severity of ACR (Hickman PE et al. (1997) Clin Chem 43(8 Suppl.):1546-54). Treatment of suspected ACR is almost always preceded by a liver biopsy for histological diagnosis and the assessment of severity (Yippner C et al. (2001 ) Transplantation 27;72(6):1 122-8).

A liver biopsy entails the retrieval of a small sample of liver tissue through the abdominal wall under local anaesthetic for histological assessment by a liver pathologist. The invasive nature of liver biopsies is associated with a significant (-30%) risk of pain and carries a reasonable (-1 %) risk of bleeding and/or sepsis (Grant A et al. (1999) Gut 1 ;459 Supplement 4):iv1 -1 1 ). The accuracy of liver biopsies is often challenged by inter-observer variation between pathologists and sampling errors (where the core of liver tissue examined is not a true reflection and representation of the overall changes in the graft) (Regev A et al. (2002) Gastroenterol 97(10):2614-8).

Many studies have evaluated the role of biomarkers such as interleukins, inter-cellular adhesion molecules, toll-like receptors and hepatocyte-derived microRNA (Verhelst XPD et al. (2013) Hepatol Res. 43(2):165-78). Most of these published markers have conflicting diagnostic performance for organ rejection and require further validation.

Since the discovery of donor-specific cell-free DNA (dscfDNA) in the circulation of transplant recipients, clinical interest in dscfDNA continues to grow (Lo D et al. (1998) Lancet 351 (91 12):1329-30). dscfDNA is increasingly recognised as a non-invasive and clinically important biomarker to monitor allograft integrity after solid organ transplantation. During physiological cellular turnover, cell-free DNA derived from the donor organ is shed into the circulation of the recipient in low-levels as dscfDNA. The release of cell-free DNA into the circulation of the recipient is thought to arise from apoptosis, necrosis and potentially active secretion (Jahr S et al. (2001 ) Cancer Res 61 (4):1659-65). Cellular injury induced by ACR and other processes will lead to an increased level of dscfDNA quantifiable in the circulation of the recipient after solid- organ transplantation.

Existing tests based on detection of dscfDNA rely on genotypic differences between the donor and the recipient.

Various prior art methodologies have been utilised for detecting dscfDNA. The presence of the Y-chromosome in the circulation of the female recipient after transplantation can be used to differentiate donor- and recipient- specific DNA (Macher HC et al. (2014) PLoS One. Public Library of Science Jan 9; 9(12):e1 13987). The limitation with this methodology is that it can only be used in gender-mismatched transplantation and accordingly, is only available for approximately one in every five transplantations.

Genetic polymorphisms like single-nucleotide polymorphisms (single DNA base substitution) or large deletion/insertion polymorphisms (loss or gain of several thousand consecutive DNA bases) have been proposed as effective markers to distinguish donor- and recipient-specific DNA (Beck J et al. (2013) Clin. Chem. 59(12):1732-1741 ; Snyder TM et al. (201 1 ) Proc Natl Acad Sci 108(15):6229-34). This is because each genetic polymorphism locus has allelic sequences that can differ between individuals in a population. Hence, allelic sequences of a polymorphic locus that are only present in the donor and absent in the recipient are considered informative and will differentiate donor-specific DNA from recipient-specific DNA. Unlike the monitoring of the Y-chromosome, genetic polymorphisms can be used to monitor all transplantation cases (gender matched or mismatched).

The measurement of dscfDNA using large deletion/insertion polymorphisms (sometimes called copy number variations (CNV)) to discriminate donor and recipient DNA is useful. However, the initial step where donor-recipient CNV genotypes are established is laborious, expensive and carries an enormous potential for cross- contamination. Furthermore, the quantification by standard-curve based calibration using real-time PCR can result in inaccuracies especially in the setting of low levels of dscfDNA and is often challenged by reproducibility and inter-sample variability. The use of probe-based droplet digital PCR may improve precision and reproducibility of quantification but probe-based assays are considerably more expensive and significantly more complex to optimize. Thus, more cost-effective and straightforward yet accurate methodologies are required to support clinical implementation.

The quantification of dscfDNA by massive parallel sequencing of plasma to identify single nucleotide polymorphisms (SNPs) that are specific to the donor and the recipient has been described (De Vlaminck I (2014) Sci Transl Med 6(241 ):333-6). Although innovative, sequencing-based methods are expensive and require a few days to generate results. These factors are highly unsatisfactory as a routine clinical tool.

The advent of droplet digital PCR (ddPCR) enables the quantification of rare DNA by limiting dilution and is useful for detecting low levels of dscfDNA. ddPCR was first utilised to measure dscfDNA by Beck and colleagues (Beck J et al. (2013) Clin. Chem. 59(12):1732-1741 ). In order to detect single-base differences between the donor- and recipient DNA and quantify dscfDNA levels, fluorescent probes and PCR pre-amplification were required. However, the use of fluorescent probes leads to added costs and assay complexity. More importantly, the use of PCR pre- amplification has critical limitations that can result in biased amplification, erroneous quantification, the loss of precision and unnecessary added sample handling.

Thus, while the potential for dscfDNA to provide a powerful biopsy-free diagnostic method is attractive, there still remains a need for a rapid, cost-effective, accurate and straightforward dscfDNA-based quantitative methodology that can be readily applied to diagnose graft health following organ transplantation, and monitor the occurrence of transplant rejection and organ dysfunction.

SUMMARY OF THE INVENTION

The present inventors have developed a method for the quantification of dscf DNA that is rapid, robust, cost-effective, probe-free and eliminates PCR pre-amplification and which avoids the limitations associated with the prior art approaches. The present disclosure is based on exploiting the presence of deletion/insertion polymorphisms (DIPs) (including small (<50bp) DIPs) that differ between donor and recipient. DIPs with high heterozygosity can be used for the discrimination of an admixture of DNA from two individuals e.g. genomic chimerism, particularly the chimerism that occurs after transplantation. The variable distribution of genotypes in humans over multiple polymorphic DIP loci enables the choice of donor-specific markers for the distinction of donor-specific DNA from recipient-specific DNA. The ability to readily distinguish polymorphic alleles that are donor-specific and distinct between donor and recipient enables the quantification of the proportion of chimerism.

The presence of small DIP alleles of donor-specific sequences that are present in the donor and not in the recipient is utilized in the present disclosure. More particularly, the present disclosure is based on the amplification of donor-specific allelic breakpoints as a surprising methodology to quantify low levels of donor-specific DNA in an abundance of recipient-specific DNA. Amplification of donor-specific breakpoints is highly-specific and eliminates the need for PCR pre-amplification, costly fluorescent probes and sample handling.

Furthermore, the use of small DIPs will simplify the step where donor and recipient genotypes are established. Unlike the genotyping of SNPs or CNVs which requires expensive and/or laborious methodologies, small DIPs can be readily genotyped by simple techniques such as high-resolution melting analyses.

The methods of the present disclosure are non-invasive and provide for monitoring the success of an organ transplant in a recipient with comparable predictive values of biopsies. In one embodiment, the disclosure provides a method for detecting organ transplant rejection, organ dysfunction or organ failure, the method comprising:

quantifying circulating donor-specific cell-free DNA (dscfDNA) in a transplant recipient using probe-free quantitative PCR comprising the use of a forward or reverse primer which fully hybridises to at least one donor-specific deletion/insertion polymorphism (DIP) allelic sequence present in the donor but which is not present in the recipient, said donor-specific DIP allelic sequence comprising a) one or more allelic breakpoints corresponding to either an insertion or deletion junction of the polymorphism in the donor, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to either an insertion or deletion junction for the same polymorphism in the recipient; or b) donor-specific insertion sequence, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to a deletion junction for the same polymorphism in the recipient; and

diagnosing, predicting or monitoring the transplant status or outcome of the recipient based on the quantification of dscfDNA in the recipient at one or more time points post-transplant, wherein an increase in the quantity of the dscfDNA is indicative of transplant rejection, organ dysfunction or organ failure.

The method may further comprise genotyping a donor and recipient pair against a panel of multi-allelic deletion/insertion polymorphisms (DIPs) to identify one or more donor-specific DIP alleles which are present in the donor but absent in the recipient. In one embodiment the multi-allelic DIPs include bi-allelic small deletion/insertion polymorphisms (DIPs).

The method may also further comprise a corresponding primer which is a common primer located either downstream or upstream or the forward or reverse primer respectively wherein the common primer hybridises to allelic sequences common to both the donor and the recipient.

Depending upon whether the donor-specific DIP allele is an insertion or deletion polymorphism, primer sets will be chosen which provide for selective amplification of donor allelic sequence. Accordingly, in certain aspects:

(i) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the insertion junction of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient; (ii) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the insertion of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient;

(iii) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the deletion junction of the DIP in the donor but does not fully hybridise to allelic sequence comprising the insertion junction of the DIP in the recipient;

(iv) the reverse primer is a common primer and the forward primer hybridises to allelic sequence comprising the insertion junction of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient;

(v) the reverse primer is a common primer and the forward primer hybridises to allelic sequence comprising the insertion of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient; or

(vi) the reverse primer is a common primer and the forward primer hybridises to allelic sequence comprising the deletion junction of the DIP in the donor but does not fully hybridise to allelic sequence comprising the insertion junction of the DIP in the recipient.

It will be appreciated that where the polymorphism results in an insertion of nucleic acid sequence into the donor DNA, allelic breakpoints will be present at both the 5' and 3' junctions of the inserted sequence. Depending on the length of the insertion, the forward or reverse primer may span one of the junctions (allelic breakpoint) or both junctions. Accordingly, in some examples, the forward or reverse primer may span two allelic breakpoints. Accordingly, in one aspect, an allelic breakpoint corresponds to either the 5' or 3' insertion junction of DIP nucleic acid in the allelic sequence of the donor DNA. In other aspect, allelic breakpoints correspond to both the 5' and 3' insertion junctions of DIP nucleic acid in the allelic sequence of the donor DNA. Alternatively, the forward or reverse primer may comprise donor-specific insertion sequence only, and not span either the 5' or the 3' junctions.

In one aspect, specific primer sets are used for one or more donor-specific DIP alleles.

In some aspects, the primer sets for quantification are selected from at least one primer set for a given DIP locus set forth in Table 2 or Table 4. The organ transplant can be any solid organ transplant. Examples of organ transplants that can be analyzed by the methods described herein include but are not limited to, kidney transplant, pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, bowel transplant, or a combination of any solid organ transplantation.

In one aspect, the organ transplant is a liver transplant.

In some embodiments, the transplant status or outcome comprises rejection, tolerance, non-rejection based allograft injury, transplant function, transplant survival, or titer pharmaceutical immunosuppression. In some embodiments, non-rejection based allograft injury is selected from the group of ischemic injury, viral infection, perioperative ischemia, reperfusion injury, hypertension, physiological stress, injuries due to reactive oxygen species, and injuries caused by pharmaceutical agents.

In one aspect, the quantification is performed using digital PCR or other type of quantification methodology known in the art (e.g. real-time PCR). In one aspect, the digital PCR is droplet digital™ PCR (ddPCR™) Technology.

In one aspect, the identification of donor-specific DIP alleles comprises performing high resolution melting analysis (HRMA).

Deletion/insertion polymorphisms (DIPs) are known in the art and can be retrieved from various databases as described herein. A deletion/insertion polymorphism suitable in the present methods is preferably characterized by (i) a deletion or insertion length of between 10 and 100bp, (ii) absence of repetitive sequences and (iii) absence of polymorphism >1 % minor allele frequency flanking 100 nucleotide bases of the polymorphism. In one aspect, the DIP is characterized by high heterozygosity. In another aspect, a high heterozygosity is >0.35, meaning that at least 35% of a given population is heterozygous for the polymorphism.

In another embodiment, a deletion/insertion polymorphism suitable in the present methods is preferably characterized by a small deletion or insertion length of between 10 and 50bp. In one aspect, the insertion or deletion sequence of the DIP consists of a length of between 10 and 100 nucleotide bases, between 10 and 50 nucleotide bases between 10 and 45 nucleotide bases, between 10 and 40 nucleotide bases, between 10 and 35 nucleotide bases, between 10 and 30 nucleotide bases, between 10 and 25 nucleotide bases, between 10 and 20 nucleotide bases or between 10 and 15 nucleotide bases.

In another embodiment, the DIP is selected from one or more of the sequences comprising or consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21 , SEQ ID NO:24 or SEQ ID NO:27.

A "panel of DIPs" according to the present methods may comprise any number of deletion/insertion polymorphisms which are sufficient to identify at least one donor- specific DIP allele in a given donor-recipient pair. In one aspect, a panel of DIPs may comprise any number of DIPs which are sufficient to identify at least one donor- specific DIP allele in at least 50%, in at least 60%, in at least 70%, in at least 80%, in at least 85%, in at least 90%, in at least 95%, in at least 98%, in at least 98.5%, in at least 99%, in at least 99.5% or in at least 99.8% of potential donor-recipient pairs. In some aspects, the panel of DIPs may comprise at least one, two, three, four, five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen at least fifteen, at least twenty or more DIPs.

In some aspects, the genotyping may identify more than one donor-specific DIP allele which is present in the donor but absent in the recipient. In some aspects, two, three, four or five donor-specific DIP alleles may be identified. Subsequent quantification may therefore be based on one donor-specific DIP allele, or more than one donor- specific DIP allele, for example, two or three.

The methods of the disclosure are preferably performed on biological samples obtained from the donor and recipient. The biological sample may be selected from the group consisting of smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, plasma, lymph fluid, saliva and urine. In one aspect, the biological sample is plasma. Quantification of dscfDNA according to the present methods will reveal the postoperative transplant status of the recipient. Preferably, following transplant surgery a number of samples will be drawn from the recipient over time to monitor status of the transplant. A temporal increase in the quantity of dscfDNA (following the usual initial post-transplant surge) is indicative of transplant rejection, organ dysfunction or organ failure. Quantification may be performed at multiple time points following transplantation, typically at the discretion of the clinician. In some aspects, quantification is performed in the recipient at pre-transplant, and post-transplant days 1 , 3, 5, 7, 14, 21 , and at about months 1 , 2, 3, 4, 5, 6, 8, 10, 12, 13, 14, 15, 20, and 24 post-transplant.

In some aspects, transplant rejection, organ dysfunction or organ failure is characterised by one or more of the following in the recipient:

(i) any temporal increase in dscfDNA copies/ml in the biological sample following the initial post-transplant surge;

(ii) an increase in dscfDNA copies/ml in the biological sample of at least 50% between two or more time points between days 3 and 28 post-transplant;

(iii) an at least two-fold increase, or at least three-fold increase, or greater than three-fold increase in dscfDNA copies/ml in the biological sample between days 3 and 28 post-transplant compared to the corresponding dscfDNA copies/ml in the biological sample from post-transplant recipients with no evidence of transplant rejection or other pathologies;

(iv) a dscfDNA copies/ml level in the biological sample that is atypically higher post-transplant compared to the corresponding dscfDNA copies/ml in biological samples from post-transplant recipients with no evidence of transplant rejection or other pathologies; or

(vi) an absolute level of > 500 dscfDNA copies/ml in the biological sample from about 4 weeks post-transplant.

In one aspect, the increase is at least 55%, at least 60%, at least 65%, or at least 70% between days 3 and 28 post-transplant.

In one aspect, the temporal increase of at least 50% dscfDNA copies/ml in the biological sample is between days 3 and 15, between days 3 and 10, or between days 3 and 8 post-transplant. In one aspect, the absolute level is > 250 dscfDNA copies/ml, > 280 dscfDNA copies/ml, > 300 dscfDNA copies/ml, > 350 copies/ml dscfDNA, >400 copies/ml dscfDNA, or >500 dscfDNA copies/ml.

In one aspect, the biological samples is plasma or serum.

In other aspects, transplant status is determined by comparing the level of dscfDNA in the recipient with a pre-determined threshold level obtained from clinically stable posttransplantation recipients with no evidence of transplant rejection or other pathologies. In one aspect, the threshold level is a quantity of dscfDNA above 200 copies/ml, above 300 copies/ml, above 400 copies/ml or above 500 copies/ml from at least four weeks or more post-transplant. However, persons skilled in the art, will appreciate that the threshold value can be determined empirically for an individual laboratory.

In another embodiment, quantifying circulating donor-specific cell-free DNA (dscfDNA) in a transplant recipient using probe-free quantitative PCR comprising the use of a forward or reverse primer which fully hybridises to at least one donor-specific deletion/insertion polymorphism (DIP) allelic sequence present in the donor but which is not present in the recipient, said donor-specific DIP allelic sequence comprising a) one or more allelic breakpoints corresponding to either an insertion or deletion junction of the polymorphism in the donor, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to either an insertion or deletion junction for the same polymorphism in the recipient; or b) donor-specific insertion sequence, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to a deletion junction for the same polymorphism in the recipient; and

monitoring or predicting the transplant status or outcome of the recipient at one or more time points post-transplant, based on the quantification of dscfDNA in the recipient wherein non-rejection is characterised by one or more of the following:

(i) a continuous decline in dscfDNA copies/ml in the biological sample over time post-transplant;

(ii) a greater than 50% reduction in dscfDNA copies/ml in the biological sample by two weeks (day 14) post-transplant compared to the initial post-transplant surge;

(iii) less than 500 dscfDNA copies/ml in the biological sample by four weeks post-transplant; and (iv) less than 500 dscfDNA copies/ml in the biological sample at the stable phase.

In one aspect, non-rejection is characterized by a greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80% reduction in dscfDNA copies/ml in the biological sample by two weeks (day 14) post- transplant.

In one aspect, non-rejection is characterized by less than 500 dscfDNA copies/ml, less than 480 dscfDNA copies/ml, less than 450 dscfDNA copies/ml, less than 370 dscfDNA copies/ml, less than 350 dscfDNA copies/ml, less than 300 dscfDNA copies/ml, or less than 250 dscfDNA copies/ml in the biological sample by four weeks post-transplant.

In one aspect, non-rejection is characterized by between 30 and 100 dscfDNA copies/ml in the biological sample by four weeks post-transplant.

In one aspect, the biological sample is plasma or serum.

The present disclosure also provides for treating organ transplant rejection in a recipient. Such treatment may comprise determining, modifying or maintaining an immunosuppressive regimen.

In another embodiment, there is provided a kit comprising one or more of the forward and reverse primers set forth in Table 2 or Table 4 for amplifying one or more of the DIPs set forth in Table 1 or Table 3, together with suitable reagents and instructions for performing the quantification method described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 . Genotype specific melting profiles for each DIP assayed by high- resolution melting analyses (HRMA). Insertion/insertion (LONG), deletion/deletion (SHORT) and insertion/deletion (heterozygote; HET) genotypes are labelled respectively. Figure 2 Schematic of the two step workflow for the quantification of donor- specific cell-free DNA using allele-specific primers.

Figure 3 Representative 1 D plot derived from the probe-free ddPCR methodology evaluating the dscfDNA levels of a recipient who underwent liver transplantation without any complications. H02 is the positive control (genomic DNA from the matched donor). dscfDNA levels were analysed at pre-transplant (A02), post- transplant day 3 (C02), day 7 (D02), day 14 (E02), day 28 (F02) and day 42 (G02). Threshold selected at 12000 fluorescent units to assign positive droplets (above threshold) containing dscfDNA for absolute quantification.

Figure 4 Genotyping information for three donor-recipient pairs using a panel of 9 deletion/insertion polymorphisms. Informative markers are highlighted by grey shading.

Figure 5 Box plot of dscfDNA levels in six recipients who underwent uneventful liver transplantation. Whiskers represent minimum and maximum values.

Figure 6 Serum liver function tests and dscfDNA levels of a recipient who underwent liver transplantation. The recipient developed an episode of biopsy-proven acute cellular rejection (ACR) at day 7 denoted by persistently elevated alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Following the diagnosis, immunosuppressive therapy was increased. The elevation of dscfDNA correlates with the diagnosis of ACR and improved with further adjustment of immunosuppressive therapy. Serum liver function tests are represented on the left Y-axis and dscfDNA levels are represented on the right Y-axis.

Figure 7 Surveillance of dscfDNA levels in the plasma of a recipient with acute cellular rejection (ACR) after liver transplantation compared to the mean of six recipients who underwent liver transplantation without any complications. The elevation in dscfDNA levels at day 7 coincided with the diagnosis of ACR on liver biopsy. BTR16L: donor-specific allele BTR16-LONG. Mean levels shown for each point. Error bars represent the standard deviation of mean of the normal recipients.

Figure 8 Surveillance of dscfDNA levels in the plasma of a recipient with ischemic reperfusion injury following liver transplantation compared to the mean of six recipients who underwent liver transplantation without any complications. The levels of dscfDNA were markedly raised at day 3 and recovered to levels approximating recipients with healthy grafts by day 28. BTR09L: donor-specific allele BTR09-LONG. Mean levels shown for each point. Error bars represent the standard deviation of mean of the normal recipients.

Figure 9 Schematic of amplification of dscfDNA using primers comprising one or more allelic breakpoints and deletion allele-specific primers . In (a), (b), (d) and (e) a forward or reverse primer which fully hybridises to a donor-specific insertion polymorphism allelic sequence present in the donor but which is not present in the recipient is used. The allelic sequence includes one allelic breakpoint corresponding to an insertion junction of the polymorphism in the donor. In (c) and (f) a forward or reverse primer which fully hybridises to a donor-specific insertion polymorphism allelic sequence present in the donor but which is not present in the recipient is used. The allelic sequence includes allelic breakpoints corresponding to two insertion junctions of the polymorphism in the donor. In (g) and (h) a forward or reverse primer which fully hybridises to a donor-specific deletion polymorphism allelic sequence present in the donor but which is not present in the recipient is used. The allelic sequence includes one allelic breakpoint corresponding to a deletion junction of the polymorphism in the donor.

Figure 10 Schematic of amplification of dscfDNA using primers comprising donor- specific insertion sequence. In a) and b), a forward or reverse primer which fully hybridises to donor-specific insertion sequence which is not present in the recipient is used. The forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to a deletion junction for the same polymorphism in the recipient.

Figure 11 Oligonucleotides for the quantification of dscfDNA using allele-specific primers. BTR20 is a representative 40bp deletion/insertion polymorphism (DIP), (a) shows an example of primers flanking the DIP used for determining the genotypes of the polymorphism, (b) shows two alternative examples; a reverse primer which fully hybridises to donor-specific insertion sequence which is not present in the recipient and does not include an allelic breakpoint (Option 1 "), or a reverse primer which fully hybridises to a donor-specific insertion polymorphism allelic sequence present in the donor which is not present in the recipient and includes one allelic breakpoint corresponding to an insertion junction of the polymorphism in the donor (Option 2"). (c) shows an example of the placement of primers to specifically amplify the SHORT allele. BOLD text represents the insertion allele and the downward arrows indicate the insertion junctions in (a) and (b). BOLD text and downward arrow represents the deletion junction in (c).

Figure 12 DscfDNA levels of recipients after liver transplantation, (a) This data shows that in stable recipients, levels of dscfDNA were consistently low 12 months after transplantation, (b) Shows 8 recipients (at least 3 months after transplantation) with abnormal serum liver biochemistry undergoing liver biopsy. DscfDNA levels remain low in recipients with biopsy evidence of hepatitis without organ rejection. In recipients with biopsy evidence of organ rejection dscfDNA levels are demonstrably higher.

Figure 13 Box plot of dscfDNA levels in 13 recipients who underwent uneventful liver transplantation. Whiskers represent minimum and maximum values.

Figure 14 Specificity of ddPCR assays for (a) BTR03-LONG and BTR03-SHORT Specificity of ddPCR assays for light grey droplets (Ch1 Amplitude of about >10000) indicate amplified templates. Dark grey droplets indicate non-amplifiable templates (Ch1 Amplitude of about 4000). Lane 1 : the LONG (insertion allele) primer set amplified the genomic DNA with an insertion/insertion (INS/INS) genotype. Lane 2, 3 and 4: the LONG (insertion allele) primer set did not amplify the genomic DNA with a deletion/deletion (DEL/DEL) genotypes. Lane 5: the SHORT (deletion allele) primer set amplified the genomic DNA with a DEL/DEL genotype. Lane 6, 7 and 8: the SHORT (deletion allele) primer set did not amplify the genomic DNA with INS/INS genotypes.

Figure 15 Specificity of ddPCR assays for (a) BTR08-LONG and BTR08-SHORT. Light grey droplets indicate amplified templates (Ch1 Amplitude about >7500). Dark grey droplets indicate non-amplifiable templates (Ch1 Amplitude about 5000). Lane 1 : the LONG (insertion allele) primer set amplified the genomic DNA with an insertion/insertion (INS/INS) genotype. Lane 2 and 3: the LONG (insertion allele) primer set did not amplify the genomic DNA with a deletion/deletion (DEL/DEL) genotype. Lane 4: the SHORT (deletion allele) primer set amplified the genomic DNA with a DEL/DEL genotype. Lane 5 and 6: the SHORT (deletion allele) primer set did not amplify the genomic DNA with INS/INS genotypes, (b) Specificity of ddPCR assays for BTR09-LONG and BTR09-SHORT. Light grey droplets indicate amplified templates (Ch1 Amplitude about >10000). Dark grey droplets (Ch1 Amplitude <7000) indicate non-amplifiable templates. Lane 1 : the LONG (insertion allele) primer set amplified the genomic DNA with an insertion/insertion (INS/INS) genotype. Lane 2 and 3: the LONG (insertion allele) primer set did not amplify the genomic DNA with a deletion/deletion (DEL/DEL) genotype. Lane 4: the SHORT (deletion allele) primer set amplified the genomic DNA with a DEL/DEL genotype. Lane 5 and 6: the SHORT (deletion allele) primer set did not amplify the genomic DNA with INS/INS genotypes.

Figure 16 Specificity of ddPCR assays for (a) BTR12-LONG and BTR12- SHORT. Light grey droplets indicate amplified templates (Ch1 Amplitude about >10000). Dark grey droplets (Ch1 Amplitude <7000) indicate non-amplifiable templates. Lane 1 : the LONG (insertion allele) primer set amplified the genomic DNA with an insertion/insertion (INS/INS) genotype. Lane 2 and 3: the LONG (insertion allele) primer set did not amplify the genomic DNA with a deletion/deletion (DEL/DEL) genotype. Lane 4: the SHORT (deletion allele) primer set amplified the genomic DNA with a DEL/DEL genotype. Lane 5 and 6: the SHORT (deletion allele) primer set did not amplify the genomic DNA with INS/INS genotypes.

Figure 17 Surveillance of dscfDNA in the plasma of a recipient T015 with acute cellular rejection (ACR) after liver transplantation compared to the mean of thirteen recipients who underwent liver transplantation without any complications. The elevation in dscfDNA levels (determined using primers specific for alleles of BTR08, BTR03 and BTR17) at day 9 coincided with the diagnosis of ACR on liver biopsy. Immunosuppression was adjusted and a subsequent liver biopsy performed on day 14 confirmed mild improvement in organ health. Mean levels shown for each point. Error bars represent the standard deviation of mean of the normal recipients.

Figure 18 Surveillance of dscfDNA in the plasma of a recipient with acute cellular rejection (ACR) after liver transplantation. Shown is the surveillance of dscfDNA in the plasma of recipient T038 with ACR after liver transplantation compared to the mean of thirteen recipients who underwent liver transplantation without any complications. The elevation in dscfDNA levels (determined using primers specific for the BTR12-LONG) at day 14 coincided with the diagnosis of ACR on liver biopsy and improved with further treatment. Mean levels shown for each point. Error bars represent the standard deviation of mean of the normal recipients.

Figure 19 Shown is the surveillance of dscfDNA in the plasma of recipient T022 who developed cholestasis secondary to biliary complications after transplantation compared to the mean of thirteen recipients who underwent liver transplantation without any complications. Continued improvement of dscfDNA levels (mean determined using primers specific for the BTR02-LONG; BTR03-LONG, BTR08- LONG and BT18-SHORT alleles) indicates that dscfDNA is independent of cholestasis. Mean levels shown for each point. Error bars represent the mean of normal recipients (standard deviation).

Figure 20 A representative 1 D plot derived from the probe-free ddPCR methodology evaluating the dscfDNA levels of a recipient who underwent liver transplantation. A03 is the positive control (genomic DNA from the matched donor). dscfDNA levels were analysed at pre-transplant (B03), post-transplant day 3 (D03), day 7 (E03), day 1 without any complications (F03), day 28 (G03) and day 42 (H03). Threshold selected at 6000 fluorescent units to assign positive droplets (above threshold) containing dscfDNA for absolute quantification. BTR03-SHORT Forward and Reverse primers were used.

Key to Sequence Listing

SEQ ID NO:1 : BTR02 Forward Primer

SEQ ID NO:2: BTR02 Reverse Primer

SEQ ID NO:3: BTR02 DIP Sequence

SEQ ID NO:4: BTR03 Forward Primer

SEQ ID NO:5: BTR03 Reverse Primer

SEQ ID NO:6: BTR03 DIP Sequence

SEQ ID NO:7: BTR06 Forward Primer

SEQ ID NO:8: BTR06 Reverse Primer

SEQ ID NO:9: BTR06 DIP Sequence

SEQ ID NO:10: BTR08 Forward Primer

SEQ ID NO:1 1 : BTR08 Reverse Primer

SEQ ID NO:12: BTR08 DIP Sequence

SEQ ID NO:13: BTR09 Forward Primer

SEQ ID NO :50: BTR12-SHORT Forward Primer

SEQ ID NO :51 : BTR12-SHORT Reverse Primer

SEQ ID NO :52: BTR16-LONG Forward Primer

SEQ ID NO :53: BTR16-LONG Reverse Primer

SEQ ID NO :54: BTR16-SHORT Forward Primer

SEQ ID NO :55: BTR16-SHORT Reverse Primer

SEQ ID NO :56: BTR17-LONG Forward Primer

SEQ ID NO :57: BTR17-LONG Reverse Primer

SEQ ID NO :58: BTR17-SHORT Forward Primer

SEQ ID NO :59: BTR17-SHORT Reverse Primer

SEQ ID NO :60: BTR18-LONG Forward Primer

SEQ ID NO :61 : BTR18-LONG Reverse Primer

SEQ ID NO :62: BTR18-SHORT Forward Primer

SEQ ID NO :63: BTR18-SHORT Reverse Primer

SEQ ID NO :64 BTR20 Forward Primer

SEQ ID NO :65 BTR20 Reverse Primer

SEQ ID NO :66: BTR20 DIP Sequence

SEQ ID NO :67: BTR20 LONG Option #1 Forward Primer

SEQ ID NO :68: BTR20 LONG Option #1 Reverse Primer

SEQ ID NO :69: BTR20 LONG Option #2 Forward Primer

SEQ ID NO :70: BTR20 LONG Option #2 Reverse Primer

SEQ ID NO :71 : BTR20 SHORT Forward Primer

SEQ ID NO :72: BTR20 SHORT Reverse Primer

General Techniques and Selected Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art. (e.g., polymerase chain reaction, Droplet Digital™ PCR (ddPCR™) Technology, or HRMA).

All publications and patent applications mentioned in this document are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, all patents and publications referred to herein are incorporated by reference in their entirety. As used in this specification and the appended claims, terms in the singular and the singular forms "a," "an" and "the," for example, optionally include plural referents unless the content clearly dictates otherwise.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention disclosure.

The term "about", is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. When referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified amount.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. Each example described herein is to be applied mutatis mutandis to each and every other example of the disclosure unless specifically stated otherwise.

Those skilled in the art will appreciate that the disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure.

The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA technology, cell biology and immunology. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81 ; Sproat et al, pp 83-1 15; and Wu et al, pp 135-151 ; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series, Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1 -284, Academic Press, New York. 12. Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer- Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I- IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

The term "consists of" or "consisting of" shall be understood to mean that a method, process or composition of matter has the recited steps and/or components and no additional steps or components.

It is envisaged that the methods of the present disclosure can be performed in vitro.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

The terms "nucleotide bases", "base pairs (bp)", "bases" or "DNA" bases referred to herein may be used interchangeably and refer to the DNA nucleotide bases a (adenine), c (cytosine), t (thymine) and g (guanine).

The term "organ rejection" as used herein refers to the rejection by the immune system of an organ transplant recipient when the transplanted organ is immunologically foreign. As used herein, the terms "organ transplant rejection", "transplant rejection", and "organ rejection", are used interchangeably. The term "transplanted organ" can be used interchangeably with the term "graft". In the present context, a graft denotes an organ in situ in the transplanted recipient. The term "acute cellular rejection" (ACR) as used herein refers to infiltration of the transplanted organ by immune cells of the recipient, which carry out their effector function and destroy the transplanted organ/graft. The onset of acute rejection is rapid and generally occurs in humans within a few weeks after transplant surgery. In the context of liver transplantation, it is defined by Snover's triad of portal hepatitis, endothelialitis (or endotheliitis), and lymphocytic cholangitis. Acute rejection is generally reversible, either spontaneously or with additional immunosuppressive therapy, and can be reliably graded using a system with categories of mild, moderate, and severe rejection, associated with 37%, 48%, and 75% unfavourable short term and 1 %, 12%, and 14% unfavourable long-term outcomes, respectively.

A "polymorphism" is a locus that is variable; that is, within a population, the nucleotide sequence at a polymorphism has more than one version or allele. One example of a polymorphism is an "deletion/insertion polymorphism (DIP)", a type of genetic variation in which a specific nucleotide sequence is present (insertion or INS) or absent (deletion or DEL).

The term "deletion/insertion polymorphism (DIP)" also referred to as an INDEL (INsertion/DELetion) as used herein refers to small insertions or deletions of bases or combination of insertions and deletions of bases in the genomic DNA of a subject. Such polymorphisms are distinct from the more common single nucleotide polymorphism (SNP). As used herein, "DIPs" is the plural of DIP.

A multi-allelic site is a specific locus in a genome that contains three or more observed alleles; a reference allele again and two or more variant alleles. The term "multi-allelic DIP" as used herein, refers to a DIP with three or more possible alleles; for example, a deletion allele and two or more insertion alleles. Accordingly, multi- allelic DIPs relate to a deletion/insertion polymorphism that has numerous potential genotypes. Such multi-allelic DIPs include bi-allelic DIPs, tri-allelic-DIPs, tetra-allelic DIPs etc.

A bi-allelic site is a specific locus in the genome that contains two observed alleles; a reference allele and one variant allele. In one embodiment, the variant allele is the insertion allele and the reference allele is the non-insertion allele. In another embodiment, the variant allele is the deletion allele and the reference allele is the non- deletion allele. The term "bi-allelic DIP" as used herein, refers to a DIP with two possible alleles; a deletion allele or the insertion allele. Accordingly, bi-allelic DIPs relate to a deletion/insertion polymorphism that has three potential genotypes, namely insertion/insertion (referred to as LONG herein), deletion/deletion (referred to as SHORT herein), and insertion/deletion (referred to as heterozygous/HET herein).

The methods of the present disclosure preferably detect deletion/insertion polymorphisms (DIPs) of less than 100bp, less than 50bp, or between 10 and 50bp, between 10 and 30 bp, or between 10 and 15bp. The term "small DIP" as used herein, is intended to refer to a deletion/insertion polymorphism which results in the insertion or the deletion of between 10 and 50 DNA bases, more preferably between 10 and 30 DNA bases into or from the genome respectively.

The term "allele" refers to one of two or more different nucleotide sequences that occur or are encoded at a specific locus, or two or more different polypeptide sequences encoded by such a locus. For example, a first allele can occur on one chromosome, while a second allele occurs on the second homologous chromosome, e.g., as occurs for a heterozygous individual, or between different homozygous or heterozygous individuals in a population.

The term "informative donor-specific DIP allele" refers to a deletion/insertion polymorphism allele that is present in the donor but which is absent in the recipient. For example, a recipient can have the genotype DEL/DEL or INS/INS as long as the donor does not have any of the recipient alleles and vice versa.

A "DIP locus" is a chromosomal position or region in which the DIP is located. For example, a DIP locus is a position or region where a DIP is located or genomic sequence deleted.

The term "donor-specific cell free DNA (dscfDNA)" as used herein refers to DNA that is circulating in the plasma, urine and other bodily fluids of humans. Cell-free DNA comprises single or double-stranded DNA fragments that are relatively short typically 130 to 180 base-pairs) and are normally at low concentration (e.g. 1 -100 ng/ml in plasma). In the context of the present disclosure, dscfDNA is used as a biomarker to distinguish donor from recipient. The term "hybridises" as used herein refers to the ability of a given nucleic acid sequence (primer sequence) to form a double stranded nucleic acid sequence with a complementary sequence. Typically the sequence will be fully complementary however, one or two mismatches may be tolerated to the extent that strand extension and amplification is not prevented. The term "does not fully hybridise" as used herein refers to a nucleic acid sequence (primer) containing sufficient mismatches to the complementary sequence so that strand extension and amplification is prevented.

The term "amplifying" or "amplification" in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.

An "amplicon" is an amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).

A "genotype" is the genetic constitution of an individual (donor or recipient as referred to herein) or group of individuals at one or more genetic loci. Genotype is defined by the allele(s) of one or more known loci of the subject, typically, the compilation of alleles inherited from its parents.

A "panel of DIPs" as used herein, refers to a collection or group of deletion/insertion polymorphisms, or the data derived therefrom, used for the purpose of distinguishing donor and recipient. Databases such as the Marshfield Clinic database referred to herein provide details of over 2,000 human DIPs which are stored electronically. The panel of DIPs may include any number of known human DIPs at the discretion of the clinician which number is sufficient to genotype the donor and recipient for at least one, preferably two informative donor DIP alleles.

The term "allelic breakpoint" as used herein refers to either the deletion junction or insertion junction of the polymorphism in the germline DNA. For example, in the case of the insertion allele of a DIP, there will be two allelic breakpoints which correspond to the 5' and 3' junctions of the inserted polymorphic sequence. In the case of the corresponding deletion allele of a DIP, the allelic breakpoint will be a single breakpoint which occurs at the site of the deleted (e.g. non-inserted) allelic sequence.

The term "insertion sequence" as used herein refers to the sequence inserted in the insertion allele of a DIP.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the present disclosure provides a workflow to quantify dscfDNA in a transplant recipient. Briefly, in step 1 , donor and recipient genomic DNA are genotyped against a panel of multi-allelic or bi-allelic small deletion/insertion polymorphisms to identify informative allelic sequences that are present in the donor and absent in the recipient. These informative sequences can be subsequently used as markers to quantify chimerism. In step 2, selective amplification of donor-specific alleles by primers that hybridise to allelic breakpoints or which amplify allelic breakpoints enables the absolute quantification of dscfDNA by a probe-free dPCR methodology developed by the inventors.

Methods, devices, compositions and kits are provided for diagnosing or predicting transplant status or outcome in a subject who has received a transplant. The transplant status or outcome may comprise rejection (such as acute cellular rejection), tolerance, non-rejection based transplant injury, transplant survival, chronic transplant injury, or titre pharmacological immunosuppression.

The disclosure provides sensitive and non-invasive methods and kits for monitoring organ transplant recipients, and/or for diagnosing or predicting transplant status or outcome (e.g. transplant rejection). The disclosure further provides methods and kits to establish a genotype for both the donor and the recipient before transplantation to enable the detection of donor-specific DNA in bodily fluids such as blood or urine from the organ recipient after transplantation.

In some embodiments, the disclosure provides methods of determining whether a recipient is displaying graft tolerance. The term "graft tolerance" includes when the subject does not reject an organ that has been introduced into the subject. In other words, the subject tolerates or maintains the organ, that has been transplanted to it. The term "donor" as used herein refers to human donors as well as non-human donors.

The term "recipient" as used herein refers to a human recipient or non-human recipient capable of receiving an organ transplant.

Deletion/Insertion polymorphisms (DIPs) and DIP classification/databases

Information regarding deletion/insertion polymorphisms (DIPs) in the human genome can be retrieved from databases such as the Marshfield Clinic database (https://www3.marshfiledclinic.org/mgs/) which has characterised over 2,000 human DIPs ((Weber JL et al (2002) Am J Hum Genet 71 :854-62)), or the 1000 Genomes Project (http://www.internationalgenome.org).

DIPs were selected that were not associated with any traits.

Sequence co-ordinates for DIPs can be retrieved for example from the human genome browser assembly hg38 (Kent WJ et al. (2002) Genome Research 12(6):996- 1006).

The methods disclosed herein are based on exploiting the use of small deletion/insertion polymorphisms (DIPs) that can be used to distinguish donor and recipient. Importantly, the use of small DIPs provide ease and speed of genotyping analysis by a range of methodologies described further herein. Additionally, the inventors found that the use of small DIPs facilitated the accurate quantification of donor-specific alleles by the use of allele-specific primers and DNA intercalating dyes on a digital PCR platform (thus eliminating the need of probes). Accordingly, the present invention provides methods that are rapid, accurate (sensitive), simple to implement and/or cost-effective.

Although a focus of the field, single nucleotide polymorphisms (SNPs) were not used since they are more laborious to genotype and require probe-based assays to genotype. Furthermore, avoiding the quantification of a single SNP locus avoids expensive and complex probe-based quantitative PCR. Alternatively, massive parallel sequencing can be used for both genotyping and quantification but this technique is slow, laborious and clearly more expensive. Copy number variations (CNVs) have also been used in the prior art however these are difficult and laborious to genotype. Typically, the genotyping of CNVs is multi-step comprising PCR amplification using internal primers, PCR amplification using external primers and fragment length analysis by agarose gel electrophoresis.

Any small informative bi-allelic or multi-allelic DIP can be used in the methods of the present disclosure. In one embodiment, the DIP is characterised by a deletion and insertion length of between 10 and 100 bp, absence of repetitive sequence (e.g. Alu repeats), and absence of polymorphisms >1 % minor allele frequency flanking 100 bases of the locus. In another embodiment, the DIP is characterised by a small deletion and insertion length of between 10 and 50 bp. In one example, the DIP is further characterised by high heterozygosity. In another example, the DIP is further characterised by heterozygosity of >0.35%, meaning that greater than 35% of the population are heterozygous for the polymorphism.

Genotyping of donor and recipient

In an embodiment, the donor and recipient are both genotyped prior to transplantation to identify informative deletion/insertion polymorphisms (DIPs) which are capable of distinguishing the donor and the recipient. A DIP is considered informative if one or other of the two allelic sequences for the polymorphism are present in the donor and absent in the recipient.

For example, a DIP locus is considered informative if:

(i) the DONOR is INS/INS and the RECIPIENT is DEL/DEL, then the DIP locus is informative as INS is not in the recipient

(ii) the DONOR is DEL/DEL and the RECIPIENT is INS/INS, then the DIP locus is informative as DEL is not in the recipient Furthermore, a DIP locus is considered informative if:

(iii) the DONOR is INS/DEL and the RECIPIENT is DEL/DEL, then the DIP locus is informative as INS is not in the recipient

(iv) the DONOR is INS/DEL and the RECIPIENT is INS/INS, then the DIP locus is informative as DEL is not in the recipient

On the other hand, a DIP locus is not informative if:

(i) the DONOR is DEL/DEL and the RECIPIENT is DEL/DEL

(ii) the DONOR is INS/INS and the RECIPIENT is INS/INS

(iii) the DONOR is DEL/DEL and the RECIPIENT is INS/DEL

(iv) the DONOR is INS/INS and the RECIPIENT is INS/DEL

(v) the DONOR is INS/DEL and the RECIPIENT is INS/DEL

Examples of methods that can be used to genotype the transplant donor and the transplant recipient include, but are not limited to, whole genome sequencing, exome sequencing, polymorphism arrays, mass spectrometry, droplet digital PCR (ddPCR), microfluidic electrophoresis of PCR products or high resolution melting analysis (HRMA).

A set of relevant DIPs which identifies differences between the donor and the recipient is established from a panel of DIPs. DIPs can be obtained from databases referred to above and in the example or from publications, see, for example (Weber JL et al (2002) Am J Hum Genet 71 (4):854-862; Mullaney JM et al 92010) 19(R2):R131 -R136).

Genotyping primer sets, such as those exemplified in Table 1 herein, are designed to flank the multi-allelic or bi-allelic DIP locus (i.e. are positioned upstream and downstream of the DIP locus) to produce three distinguishable genotypes [insertion/insertion (LONG/homozygous)], insertion/deletion (HET/heterozygous) or deletion/deletion (SHORT/homozygous). An appropriately prepared biological sample obtained from each of the donor and the recipient pair is then amplified using polymerase chain reaction (PCR).

In one example, the forward primers are selected from one of more of SEQ ID NO:1 , SEQ ID No:4, SEQ ID No:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25. In another example, the reverse primers are selected from one or more of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:1 1 , SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23 and SEQ ID NO:26.

PCR amplicons are then analysed using an appropriate method. Typically, a number of DIPs will be assessed between the donor and recipient sufficient to distinguish at least 95%, preferably at least 98%, more preferably at least 99%, still more preferably greater than 99% of any donor-recipient pair.

In one aspect, the PCR amplicons are assessed for donor and recipient differences using high resolution melting analysis (HRMA) as described herein. This determines a personalised set of allelic sequences that are present in the donor and absent in the recipient that are subsequently used as a basis for the quantification step. Preferably, this analysis is performed once for each donor-recipient pair.

In another aspect, genomic DNA from normal (non-transplant) individuals is genotyped to establish unique genotype-specific melting profiles for each DIP and to determine the diversity of coverage of a given panel of DIPs. This will assist in confirming the DIPs have three genotypes (long, short and heterozygous), can be genotyped by HRMA and serve as a useful positive control for genotyping clinical samples.

High resolution melting analysis (HRMA) is a closed tube post-PCR method that allows for analysis of variations in PCR amplicons by use of a dye that fluoresces when intercalated into double-stranded DNA. When the sample is heated to high temperatures, the DNA denatures and the dye stops fluorescing as the double stranded DNA separates, generating a melting curve. Because different genetic sequences melt at slightly different rates, they can be viewed, compared, and detected using these curves. Samples can be discriminated according to their sequence, length, GC content or strand complementarity (Montgomery JL et al. (2010) Expert Rev Mol Diagn 10:219-40; Li M et al (2014) Clin Chem 60:864-72; Dobrovic A (2015) Clin Chem 61 :684-5). The fluorescent dyes that are suitable for performing HRMA include, but are not limited to LC Green, SYT09, Eva Green, Chromofy, BEBO, SYBR Green or RAZOR probe.

Various instrumentation for performing HRMA is available as described in the examples. Non-limiting examples of such instruments include HR-1 (Idaho Technology), the Rotor-Gene Q (Qiagen), the LightScanner (Idaho Technology), the LightCycler 480 (Roche Molecular Systems), the CFX Connect thermocycler (BioRad) and the magnetic induction cycler (MIC).

There are two ways HRMA curve plots can distinguish between samples, either by shape (i.e. using detail in the shape of the melt curve itself) or by shift (i.e. the thermal offset of a curve from other curves). Before HRMA curves are plotted, the raw data is first normalised. Melt curves are normally plotted with fluorescence on the Y axis and temperature on the X axis. The fluorescence axis of HRMA plots is normalised onto a 0 to 100% scale. If required, normalisation can also be applied to the temperature axis which is designed to compensate for well to well temperature measurement variations between samples. This is referred to as "temperature shifting".

Following HRMA, normalised melting curves for each genotype are distinct and can be readily distinguished. Heterozygotes show multiphasic melting profiles owing to the denaturation of heteroduplexes. Heteroduplexes (double-stranded DNA comprising sense strand insertion alleles with anti-sense deletion alleles or vice versa) are generally less stable and dissociate before the homoduplexes. Since the two alleles of each bi-allelic DIP have substantial sequence differences, or the alternative alleles of multi-allelic DIPs have substantial sequence differences, these DIPs are attractive genomic loci for genotyping by HRMA (Wittwer CT et al (2009) Hum Mutat 30:857-9). Deletion polymorphisms result in shorter amplicons which melt at lower temperatures compared to insertion alleles generating longer amplicons.

A donor allele that is not present in the recipient is considered informative. By way of example, a donor-recipient pair in which the donor is heterozygous for the deletion/insertion polymorphism and the recipient is homozygous for the deletion polymorphism (SHORT) is considered informative. A donor-recipient pair in which the donor is homozygous for the insertion polymorphism (LONG) and the recipient is homozygous for the deletion polymorphism (SHORT) is considered informative. Alternatively, a donor-recipient pair in which the donor is homozygous for the deletion polymorphism (SHORT) and the recipient is homozygous for the insertion polymorphism is considered informative.

A donor allele of a multi-allelic polymorphism that is not present in the recipient is also considered informative.

A donor may contain only one informative donor allele or more than one informative donor allele, for example, two or three as described in the examples herein.

HRMA provides a number of distinct advantages. It provides an alternative to other methods of genotyping such as dHPLC sequencing screening or digital PCR. It is fast and accurate which is particularly important in the transplant setting where potential recipients are very sick. Furthermore, no or minimal post-PCR sample handling is required.

However, other genotyping methodologies known in the art are suitable for use in the methods of the present disclosure. Such methodologies include PCR (quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), real-time PCR (RT-PCR), hot-start PCR, droplet digital PCR (ddPCR), chips, or high- throughput shotgun sequencing of circulating nucleic acids. Other suitable amplification methods include the ligase chain reaction (LCR), transcription amplification, self-sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), degenerate oligonucleotide-primed PCR (DOP-PCR) and nucleic acid based sequence amplification (NABSA). Other amplification methods that may be used to amplify specific polymorphic loci include those described in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and 6,582,938.

Genotyping donor and recipient nucleic acids, and/or detection, identification and/or quantitation of the dscfDNA after transplantation can be performed by sequencing such as whole genome sequencing or exome sequencing. Sequencing can be accomplished through classic Sanger sequencing methods which are well known in the art. Sequence can also be accomplished using high-throughput systems some of which allow detection of a sequenced nucleotide immediately after or upon its incorporation into a growing strand, i.e., detection of sequence in Real time or substantially real time. In some cases, high throughput sequencing generates at least 1 ,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000 or at least 500,000 sequence reads per hour; with each read being at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120 or at least 150 bases per read. Sequencing can be performed using nucleic acids described herein such as genomic DNA, cDNA derived from RNA transcripts as a template.

The genotyping is preferably performed on a biological sample obtained from the donor and the recipient. Examples of suitable biological samples include, but are not limited to smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, plasma, lymph fluid, saliva, and urine. Preferably, the sample is plasma.

Preferably the donor and recipient are both human. However, in some examples, the donor may be a non-human donor, in the case of xenotransplantation.

It will also be appreciated by person skilled in the art that genotyping of the donor and recipient may be performed in a separate laboratory, clinic or premises from the quantification methodology discussed in detail below.

Quantification of dscfDNA in the recipient

Pre-transplantation and following transplantation, a biological sample such as blood can be drawn from the recipient and the level of dscfDNA present in the recipient determined. Examples of biological samples include, but are not limited to smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, plasma lymph fluid, saliva, and urine.

In one embodiment, the disclosure provides a method for detecting organ transplant rejection, organ dysfunction or organ failure, the method comprising:

quantifying circulating dscfDNA in a transplant recipient using probe-free quantitative PCR comprising the use of a forward or reverse primer which fully hybridises to at least one donor-specific deletion/insertion polymorphism (DIP) allelic sequence present in the donor but which is not present in the recipient, said donor- specific DIP allelic sequence comprising a) one or more allelic breakpoints corresponding to either an insertion or deletion junction of the polymorphism in the donor, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to either an insertion or deletion junction for the same polymorphism in the recipient; or b) donor-specific insertion sequence, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to a deletion junction for the same polymorphism in the recipient; and

The term "recipient" as used herein is preferably a human recipient.

The methods of the present disclosure also provide for quantification of dscfDNA. Methods for quantifying nucleic acids are known in the art and include, but are not limited to, gas chromatography, supercritical fluid chromatography, liquid chromatography (including partition chromatography, adsorption chromatography, ion exchange chromatography, size exclusion chromatography, thin-layer chromatography, and affinity chromatography), electrophoresis (including capillary electrophoresis, capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic capillary chromatography, isotachophoresis, transient isotachophoresis and capillary gel electrophoresis), comparative genomic hybridization (CGH), microarrays, and bead arrays.

The quantification method developed by the inventors and described herein is based on a unique methodology that takes advantage of insertion sequence and/or allelic breakpoints in the germline DNA associated with DIPs. To the inventors' knowledge, this approach has not been utilised in the prior art methods.

The allelic breakpoints correspond to either the deletion or insertion junction of the polymorphism in the germline/allelic DNA as defined herein. By way of example as shown in Table 2 for a number of DIP loci, a given polymorphism may be characterised by insertion of sequences into the germline DNA or by deletion. These insertions or deletions create breakpoints (e.g. two in the case of a single insertion) or one in the case of a deletion. The primers hybridising across these breakpoints are shown in Table 2 where the portion of the sequence in bold and italics corresponds to the complement of the insertion sequence of the polymorphism and the portion of the sequence in bold and underlined corresponds to the complement of bases at the breakpoint (point where the allelic sequence is deleted).

The insertion sequence corresponds to the sequence inserted in the insertion polymorphism allele of the DIP. As discussed above and as shown in Table 2 for a number of DIP loci, a given polymorphism may be characterised by insertion of sequences into the germline DNA or by deletion. These insertions include insertion sequence which is present in only one allele of the polymorphism. In one embodiment, the insertion sequence is present in one allele of the DIP, and therefore is a donor-specific insertion sequence. Primers hybridising to this insertion sequence are shown schematically in Figures 10 and 1 1 . Primers hybridising to the insertion sequence do not include the complement of sequence across the breakpoint or the complement of sequence outside of the insertion (e.g. the insertion sequence common to the insertion and deletion alleles).

When primers hybridising to insertion sequence are used, the allelic breakpoint which is not present in the primer comprising insertion sequence is amplified using that primer and a common primer.

In one embodiment, the methods described herein further comprise a corresponding primer which is a common primer located either downstream or upstream or the forward or reverse primer respectively wherein the common primer hybridises to allelic sequences common to both the donor and the recipient.

In another embodiment, (i) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the insertion junction of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient; (ii) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the insertion of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient; (iii) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the deletion junction of the DIP in the donor but does not fully hybridise to allelic sequence comprising the insertion junction of the DIP in the recipient; (iv) the reverse primer is a common primer and the forward primer hybridises to allelic sequence comprising the insertion junction of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient; (v) the reverse primer is a common primer and the forward primer hybridises to allelic sequence comprising the insertion of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient; or (vi) the reverse primer is a common primer and the forward primer hybridises to allelic sequence comprising the deletion junction of the DIP in the donor but does not fully hybridise to allelic sequence comprising the insertion junction of the DIP in the recipient.

In another embodiment, an allelic breakpoint corresponds to each insertion junction of the insertion sequence of the DIP in the allelic sequence.

When a primer to a donor-specific deletion junction is used, a primer to an insertion sequence in the recipient (a sequence inserted in the insertion polymorphism allele in the recipient) may be used.

With respect to the quantification of dscfDNA in the recipient, this is illustrated for one embodiment shown in Figure 2. The DIP locus BTR08 was determined to be informative since the donor was genotyped as having an INS/INS (LONG) and the recipient was genotyped as a DEL/DEL (SHORT). The informative allele is thus the insertion allele.

In this example, a common forward primer which hybridises to the allelic sequence of both the donor and the recipient is used. A reverse primer spanning the allelic breakpoint with a matched 3' end to the insertion allele (i.e. the 3' end is inside the insertion sequence) is selected, and this will allow the amplification of donor-specific DNA. However, the same reverse primer (spanning the allelic breakpoint) has a mismatched 3' end to the deletion junction, and thus will not amplify the recipient- specific DNA.

Alternatively, a reverse primer comprising only insertion sequence (i.e. the 5' and 3' ends are inside the insertion sequence) is selected, and this will allow the amplification of donor-specific DNA. Accordingly, the methodology is highly specific for amplification of the donor insertion (LONG) allele only (as demonstrated by the non-amplification of circulating free DNA in the recipient prior to transplantation in Figure 3 - lane A02).

In one example, the forward primers are selected from one of more of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60 and SEQ ID NO:62.

In another example, the reverse primers are selected from one or more of SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43 and SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61 , and SEQ ID NO:63.

The advantages of this approach is that, unlike prior art methods which require the use of internal primers in which the recipient must be DEL/DEL and the donor must be INS/DEL or INS/INS for internal primer hybridisation, the present methods allow for the use of either deletion or insertion junctions to be amplified. Accordingly, the present method allows for amplification of donor-specific deletion/insertion polymorphism (DIP) allelic sequence present in the donor which is not present in the recipient. The use of a primer spanning at least one allelic breakpoint or a primer comprising insertion sequence allows for either donor-specific deletion junctions or donor-specific insertion junctions to be amplified.

Typically PCR quantification methodologies of the prior art rely on the use of fluorescent probes. Such dyes are known in the art and may typically be divided into families, such as fluorescein and its derivatives; rhodamine and its derivatives; cyanine and its derivatives; coumarin and its derivatives; Cascade Blue™ and its derivatives; Lucifer Yellow and its derivatives; BODIPY and its derivatives; and the like. Exemplary fluorophores include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor®-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC), carboxy- fluorescein (FAM), phycoerythrin, rhodamine, dichlororhodamine (dRhodamine™), carboxy tetramethylrhodamine (TAMRA™), carboxy- X-rhodamine (ROX.TM.), LIZ™, VIC™., NED™, PET™., SYBR, PicoGreen, RiboGreen, and the like.

While traditional methodologies have employed the use of fluorescent probes, the inventors have developed a digital PCR methodology which does not require use of fluorescent probes for dscfDNA quantification or pre-amplification. Probe-free approaches are more desirable because of the added complexity and costs associated with fluorescent probes. The inventors methodology which is exemplified herein exploits the properties of DNA intercalating dyes such as EvaGreen on a droplet digital PCR platform. EvaGreen fluorescence emission from each droplet directly relates to the amount of DNA encapsulated within and will vary by amplicon length. Examples of intercalating dyes suitable for use according to the methods described herein include SYBR Green, LC Green, EvaGreen, BEBO, BOXTO, and SYT09.

Digital PCR is a technique where a limiting dilution of the sample is made across a large number of separate PCR reactions so that most of the reactions have no template molecules and give a negative amplification result. Those reactions that are positive at the reaction endpoint are counted as individual template molecules present in the original sample in a 1 to 1 relationship. (See, e.g., Kalina et al. NAR 25: 1999- 2004 (1997) and Vogelstein and Kinzler, PNAS 96:9236-9241 (1999); U.S. Pat. Nos. 6,440,706, 6,753, 147, and 7,824,889.) Quantitative partitioning is assumed, and the dynamic range is governed by the number of containers available for stochastic separation. The molecules are then detected by PCR and the number of positive containers is counted. Each successful amplification is counted as one molecule, independent of the actual amount of product, in some embodiments, a digital PCR may be a microfluidics-based digital PCR. In some embodiments, a droplet digital PCR may be employed. One of skill in the art can readily design primers to target a DIP of interest.

In one embodiment, quantification is performed using probe-free digital PCR, more particularly droplet digital PCR. "Droplet digital PCR" (ddPCR) refers to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification (McDermott GP et al (2013) PLoS One 10:e0142572; Hinson et al., 201 1 , Anal. Chem. 83 :8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-101 1 ). A single ddPCR reaction may be comprised of at least 20,000 partitioned droplets per well. In ddPCR, each PCR reaction is compartmentalized to allow the direct measurement of target sequences based on limiting dilution.

A "droplet" or "water-in-oil droplet" refers to an individual partition of the droplet digital PCR assay. A droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for realtime PCR applications (Hinson et al., 201 1 , Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: 1003-101 1 ).

Droplet digital PCR may be performed using any platform that performs a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification. As discussed briefly above, a sample is diluted and partitioned into thousands to millions of separate reaction chambers (water-in-oil droplets) so that each contains one or no copies of the nucleic acid molecule of interest. The number of "positive" droplets detected, which contain the target amplicon (i.e., nucleic acid molecule of interest), versus the number of "negative" droplets, which do not contain the target amplicon (i.e., nucleic acid molecule of interest), may be used to determine the number of copies of the nucleic acid molecule of interest that were in the original sample. Examples of droplet digital PCR systems include the QX100™ Droplet Digital PCR System by Bio-Rad, which partitions samples containing nucleic acid template into 20,000 nanoliter-sized droplets; and the RainDrop™ digital PCR system by RainDance, which partitions samples containing nucleic acid template into 1 ,000,000 to 10,000,000 picoliter-sized droplets.

The use of TaqMan hydrolysis probes in ddPCR assays enables genotyping by allelic discrimination and if necessary, absolute quantification (George D et al. (2013) Chimerism 4:102-8). By "probe-free" it is means without use of a fluorescent probe that hybridises to the target DNA molecule.

In another embodiment, the quantification method of the present disclosure is performed using ddPCR comprising a fluorescent DNA intercalating agent. The inventors use of primers that span allelic breakpoints, or of primers comprising insertion sequence which amplify allelic breakpoints using primers specific to insertion sequence, and which are therefore specific for the donor allele eliminate the need for probes (used in prior art methodologies) to distinguish donor-specific DNA from recipient-specific DNA. Advantageously, the method can be performed using intercalating dyes which avoid the costly use of probes.

The skilled person will appreciate that the use of probes is costly. Considerable time and money is required in the optimisation of probes. For example, analysis of a panel of twenty DIPs could costs thousands of dollars which has flow-on effects in terms of the cost to analyse each donor/recipient pair. Furthermore, the use of probes is often challenged by co-hydrolysis and can be associated with the potential for an increased "rain" effect in scatterplots which makes it difficult to accurately draw the distinction between positive and negative droplets in droplet digital PCR. In contrast, the use of allele-specific primers that hybridise to the allelic breakpoints coupled with the use of intercalating agents in the present methods results in comparatively less rain effect and more accurate analysis by thus minimising false positives.

Organs

The methods of the present invention are preferably performed in the context of solid organ transplants. Examples of organ transplants that can be analysed by the methods described herein include, but are not limited to kidney transplant, pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, pancreas after kidney transplant, or bowel transplant or a combination of any of the foregoing.

The organ may be derived from a human or non-human donor (xenotransplant). Preparation of biological samples

Depending on the biological sample, various methodologies can be employed for extracting and processing a biological sample from a donor and recipient. Such methodologies, include, but are not limited to centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, etc. By using antibodies specific for markers identified with particular cell types, a relatively homogeneous population of cells or cell free material may be obtained. Cells can also be separated by using filters. For example, whole blood can also be applied to filters that are engineered to contain pore sizes that select for the desired cell type or class. Cells can be filtered out of diluted, whole blood following the lysis of red blood cells by using filters with pore sizes between 5 to 10 μηι, as disclosed in U.S. Patent Application No. 09/790,673. Other devices can separate cells from the bloodstream, see Demirci U, Toner M., Direct etch method for microfluidic channel and nanoheight post-fabrication by picoliter droplets, Applied Physics Letters 2006; 88 (5), 0531 17; and Irimia D, Geba D, Toner M., Universal microfluidic gradient generator, Analytical Chemistry 2006; 78: 3472-3477. Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time. Methods to isolate one or more cells or cell free material for use according to the methods of this invention are performed according to standard techniques and protocols well-established in the art.

To obtain a blood sample, any technique known in the art may be used, e.g. a syringe or other vacuum suction device. A blood sample can be optionally pre-treated or processed. Examples of pre-treatment steps include the addition of a reagent such as a stabilizer, a preservative, a fixant, a lysing reagent, a diluent, an anti- apoptotic reagent, an anti-coagulation reagent, an anti-thrombotic reagent, magnetic property regulating reagent, a buffering reagent, an osmolality regulating reagent, a pH regulating reagent, and/or a cross-linking reagent.

When a blood sample is obtained, a preservative such an anti-coagulation agent and/or a stabilizer can be added to the sample. This allows for extended time for analysis/detection. Thus, a sample, such as a blood sample, can be analysed under any of the methods and systems herein within 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hrs, 6 hrs, 3 hrs, 2 hrs, or 1 hr from the time the sample is obtained or longer if the sample has been pre-frozen.

In some embodiments, a blood sample can be combined with an agent that selectively lyses one or more cells or components in a blood sample. For example platelets and/or enucleated red blood cells are selectively lysed to generate a sample enriched in nucleated cells. The cells of interest can subsequently be separated from the sample using methods known in the art. When obtaining a sample from a subject (e.g., blood sample), the amount can vary depending upon subject size and the condition being screened. In some embodiments, up to 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ml. of a sample is obtained. In some embodiments, 1 -50, 2-40, 3-30, or 4-20 ml. of sample is obtained. In some embodiments, more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ml of a sample is obtained.

Nucleic acids

Nucleic acids from samples that can be analyzed by the methods herein include double-stranded DNA or single-stranded DNA. In some embodiments, less than 1 pg, 5pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 200 pg, 500 pg, 1 ng , 5ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 200 ng, 500 ng, 1 ug, 5ug, 10 ug, 20 ug, 30 ug, 40 ug, 50 ug, 100 ug, 200 ug, 500 ug or 1 mg of nucleic acids are obtained from the sample for further genetic analysis. In some cases, about 1 -5 pg, 5-10 pg, 10- 100 pg, 100 pg- 1 ng, 1 -5 ng, 5 - 10 ng, 10 - 100 ng, 100 ng- 1 ug of nucleic acids are obtained from the sample for PCR analysis.

As described herein, cell-free DNA comprises single or double-stranded DNA fragments that are relatively short typically 130 to 180 base-pairs) and are normally at low concentration (e.g. 1 -100 ng/ml in plasma).

The methods of the present disclosure preferably detect deletion/insertion polymorphisms (DIPs) of less than 100bp, less than 50bp, or between 10 and 50bp, between 10 and 30 bp, or between 10 and 15bp. A polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs (referred to as the "DIP locus" herein). Preferred markers have at least two alleles, each occurring at a frequency of preferably greater than 1 %, and more preferably greater than 10% or 20% of a selected population.

Circulating nucleic acids and transplant rejection

During cellular turnover, cell-free DNA derived from the donor organ is shed into the circulation of the recipient as dscfDNA. The release of cell-free DNA into the circulation of the recipient is thought to arise following cellular apoptosis, necrosis and active secretion (Jahr S et al (2001 ) Cancer Research 61 (4):1659-65). During episodes of increased cell death following graft injury (i.e. ischemic injury, immunity- mediated rejection and sepsis), an increased level of dscfDNA is quantifiable in the circulation of the transplant recipient.

Without wishing to be bound by theory, the relative amount of donor-specific DNA sequences in circulating nucleic acids should provide a predictive measure of oncoming organ failure in the transplant recipient for many types of solid organ transplantation (e.g. as exemplified for liver transplantation herein).

The methods of the present disclosure provide for quantifying circulating dscfDNA either free in plasma or serum or other biological sample, for the diagnosis, prognosis, detection and/or treatment of a transplant status or outcome.

The methods of the present disclosure also provide a useful adjunct to clinical methods of detecting organ transplant rejection, organ dysfunction or organ failure, such as pathology, facilitating the interpretation of pathology results and clinical observations.

Monitoring organ health following transplantation

Surveillance of organ health after solid transplantation remains a fundamental aspect of post-transplantation care. The estimation of graft function is essential for the surveillance of post-transplant complications and the adjustment of immunosuppression therapy.

Depending on the type of solid-organ transplantation, the definitive evaluation of graft health is typically based on the histological assessment of tissue biopsies. These carry significant risks of pain, bleeding and sepsis (Grant A and Neuberger J (199) Gut 45(Supplement 4):pp iv1 -iv1 1 ). Furthermore, the accuracy of tissue biopsies is often challenged by sampling errors and inter-observer variation (Regev A et al (2002) The American Journal of Gastroenterology 97(10):2614-8). Clearly, methods with are non-invasive, rapid and accurate for surveillance of allograft health are desirable.

By performing the methods of the present disclosure at a single time point, the methods of the present disclosure can be used to detect the status of a transplant in a transplant recipient. For example, the inventors have demonstrated the levels of dscfDNA in transplant recipients with non-rejection at different time points. Comparing a level of dscfDNA in a recipient with those of recipients with non-rejection allows determining the status of the transplant in the recipient. For example, if the level of dscfDNA in the recipient are elevated relative to levels in recipients with non- rejection, an acute rejection can be detected.

Figure 13 shows the change in dscfDNA levels in recipients with non-rejection. In one embodiment non-rejection is characterised by one or more of the following: less than about 3500 dscfDNA copies/ml in the biological sample by three days post-transplant; less than about 1500 dscfDNA copies/ml in the biological sample by seven days post- transplant; less than 1000 dscfDNA copies/ml in the biological sample by two weeks post-transplant; and less than 500 dscfDNA copies/ml in the biological sample by four weeks post-transplant.

By performing the methods of the present disclosure over at least two time points, the methods of the present disclosure can be used to monitor or predict transplant survival in a recipient over time.

The level of dscfDNA in the recipient can be monitored at a number of time points post-transplant to look for increases or decreases in dscfDNA copies/ml recipient plasma. In some embodiments, temporal differences in the quantity of dscfDNA from the transplant donor are indicative of a transplant status or outcome. For instance, a transplant recipient can be monitored over time to determine the quantity of dscfDNA from the transplant donor. A temporary increase in the quantity of dscfDNA from the transplant donor, which subsequently return to normal values, may indicate less serious pathology rather than transplant rejection. On the other hand, a sustained increase (over several days) in the quantity of dscfDNA from the transplant donor can indicate significant pathology such as transplant rejection. Preferably, monitoring is performed at least two, three, four, five, six, eight, ten, twelve, fourteen or fifteen times within the first twelve months post-transplantation. Monitoring may also be performed six monthly or yearly after the first twelve months or as required at the discretion of the clinician.

The term "temporal" as used herein denotes enduring for a certain period of time. More particularly, it denotes an increase in dscfDNA copies/ml that is detected compared to a reference value or which is detected between two time-points in the recipient samples.

In one example, the methods of the present disclosure can be performed daily. In an example, the methods of the present disclosure can be performed weekly. In another example, the methods of the present disclosure can be performed monthly. In another example, the methods of the present disclosure can be performed bi-monthly. In another example, the methods of the present disclosure can be performed every three months, every four months, every six months. In another example, the methods of the present disclosure can be performed yearly.

In another example, the methods of the present disclosure can be used to predict treatment failure.

In one embodiment, the disclosure provide a method for monitoring or predicting transplant survival in a recipient who has received an organ transplant (allograft). In another embodiment, the disclosure provides methods of diagnosing or predicting whether a transplant (allograft) in a recipient will survive post-transplant. In certain embodiments, the disclosure provides methods of diagnosing or predicting the presence of long-term graft survival. By "long-term" graft survival is meant graft survival for at least about 5 years beyond current sampling, despite the occurrence of one or more prior episodes of acute rejection. In certain examples, transplant survival is determined for recipients in which at least one episode of acute rejection has occurred. As such, these embodiments provide methods of determining or predicting transplant survival following acute rejection.

In another embodiment, the disclosure provides methods of predicting or monitoring transplant survival in a recipient who has received an organ transplant comprising: quantifying circulating dscfDNA in a transplant recipient using probe-free quantitative PCR comprising the use of a forward or reverse primer which fully hybridises to at least one donor-specific deletion/insertion polymorphism (DIP) allelic sequence present in the donor but which is not present in the recipient, said donor- specific DIP allelic sequence comprising a) one or more allelic breakpoints corresponding to either an insertion or deletion junction of the polymorphism in the donor, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to either an insertion or deletion junction for the same polymorphism in the recipient; or b) donor-specific insertion sequence, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to a deletion junction for the same polymorphism in the recipient; and

(iii) less than 500 dscfDNA copies/ml in the biological sample by four weeks post-transplant; and

(iv) less than 500 dscfDNA copies/ml in the biological sample at the stable phase.

The present inventors have demonstrated that immediately following transplantation there is an initial "post-transplant surge" in levels of dscfDNA in many transplant recipients immediately following transplantation, and which peaks within the first three days following transplantation. Accordingly, in one embodiment the continuous decline in dscfDNA copies/ml in the biological sample over time following transplantation is a continuous decline in dscfDNA copies/ml in the biological sample over time following the initial post-transplantation surge.

The term "stable phase" as used herein is intended to refer to the period of time post- transplant in which the quantity of dscfDNA copies/ml does not substantially decrease further in the recipient. This may be recipient dependent. For example, a recipient may reach the stable phase by day 28, another recipient may reach the stable phase by day 35, or day 42.

In one embodiment non-rejection is characterised by one or more of the following: a reduction from less than about 3500 dscfDNA copies/ml in the biological sample at three days post-transplant; to less than about 1500 dscfDNA copies/ml in the biological sample at seven days post-transplant; to about less than about 1000 dscfDNA copies/ml in the biological sample at two weeks post-transplant; to less than about 500 dscfDNA copies/ml in the biological sample by four weeks post-transplant. In another embodiment non-rejection is characterised by one or more of the following: a reduction from less than about 3500 dscfDNA copies/ml in the biological sample at three days post-transplant; to less than about 1400 dscfDNA copies/ml in the biological sample at seven days post-transplant; to about less than about 800 dscfDNA copies/ml in the biological sample at two weeks post-transplant; to less than about 400 dscfDNA copies/ml in the biological sample by four weeks post-transplant.

In another embodiment acute rejection is characterised by one or more of the following: more than about 3500 dscfDNA copies/ml in the biological sample at three days post-transplant; more than about 1500 dscfDNA copies/ml in the biological sample at seven days post-transplant; more than about 1000 dscfDNA copies/ml in the biological sample at two weeks post-transplant; more than about 500 dscfDNA copies/ml in the biological sample at four weeks post-transplant; any temporal increase in dscfDNA copies/ml in the biological sample following the initial post- transplant surge; an increase in dscfDNA copies/ml in the biological sample of at least 50% between two or more time points between days 3 and 28 post-transplant; an at least two-fold increase, or at least three-fold increase, or greater than three-fold increase in dscfDNA copies/ml in the biological sample between days 3 and 28 post- transplant compared to the corresponding dscfDNA copies/ml in the biological sample from post-transplant recipients with no evidence of transplant rejection or other pathologies.

In another embodiment, quantification of dscfDNA is used to determine whether the recipient is displaying graft tolerance.

Non-rejection transplant injury or organ dysfunction

In some embodiments, the disclosure provides methods for diagnosis or prediction of non-rejection based transplant injury or organ dysfunction. Examples of non-rejection based graft injury include, but are not limited to, ischemic injury, sepsis, virus infection, peri-operative ischemia, reperfusion injury, hypertension, physiological stress, injuries due to reactive oxygen species and injuries caused by pharmaceutical agents. By performing the methods of the present disclosure at a single time point, the methods of the present disclosure can be used to detect non-rejection based transplant injury or organ dysfunction in a recipient.

By performing the methods of the present disclosure over at least two time points, the methods of the present disclosure can be used to monitor or predict non-rejection based transplant injury or organ dysfunction in a recipient over time.

For example, Figure 8 shows detection of ischemic reperfusion injury following liver transplantation.

Monitoring organ transplant rejection or organ failure

In some embodiments, the methods described herein are used for diagnosing or predicting transplant status or outcome (e.g. organ transplant rejection). As used herein, the terms "organ transplant rejection", "transplant rejection", and "organ rejection", are used interchangeably. The term "transplant rejection" as used herein encompasses both acute and chronic rejection. Furthermore, as used herein, the terms "acute rejection" and "acute cellular rejection", are used interchangeably. "Acute rejection" or "Acute Cellular Rejection" is the rejection by the immune system of a tissue transplant recipient when the transplanted tissue is immunologically foreign. Acute cellular rejection is characterized by infiltration of the transplanted tissue by immune cells of the recipient, which carry out their effector function and destroy the transplanted tissue. The onset of acute rejection is rapid and generally occurs in humans within a few weeks following transplant surgery.

Accordingly, in one embodiment the predicting transplant rejection includes prognosis of a response to particular treatment with immunosuppressive drugs such as rapamycin, cyclosporin A, anti-CD40L monoclonal antibody and the like.

The term "organ dysfunction" as used herein includes an impairment of organ function.

The term "organ failure" as used herein included the loss of function and/or necrosis of one or more cells comprising the organ and may affect one or more of the lungs, heart, kidneys, liver, pancreas, brain, stomach, intestine (small and/or large) and/or genitor-urinary or haematopoietic systems.

As used herein the term "diagnose" or "diagnosis" of a transplant status or outcome includes predicting or diagnosing the transplant status or outcome, determining predisposition to a transplant status or outcome, monitoring treatment of transplant patient, diagnosing a therapeutic response of transplant patient, and prognosis of transplant status or outcome, transplant progression, and response to particular treatment (e.g. immunosuppression therapy), or determining the risk of a subject rejecting having a transplant status or transplant outcome at any point following the transplant.

The term "transplant status or outcome" as used herein refers to the status of the transplanted organ, tissue or cells, and may comprise rejection, tolerance, non- rejection based transplant injury, transplant function, transplant survival, chronic transplant rejection or pharmacological immunosuppression. The term "transplant survival" includes when the subject does not reject a graft organ, tissue or cell(s) that has been introduced into the subject. In other words, the subject tolerates or maintains the organ, tissue or cell(s) that has been transplanted.

In one embodiment, organ transplant rejection, organ dysfunction or organ failure is characterised by a temporal increase in the quantity of dscfDNA in the recipient within 1 year post-transplant. In some examples, the increase will be detected within days 3, 4, 5, 6, 7, 8, 9, 10, 14, 20, 30, 2 months, 3 months, 5 months, 8 months, 10 months or 12 months post-transplant.

In another example, the increase in quantity of dscfDNA is observed between days 3 and 10 post-transplant. In another example, the increase in quantity of dscfDNA is observed between days 4 and 10 post-transplant.

In another embodiment, organ transplant rejection, organ dysfunction or organ failure is characterised by a continuously higher quantity of dscfDNA in the recipient plasma compared to the corresponding dscfDNA copies/ml recipient plasma from stable posttransplantation recipients with no evidence of transplant rejection or other pathologies.

In another embodiment, organ transplant rejection, organ dysfunction or organ failure is characterised by greater than 50% increase in quantity of dscfDNA in the recipient post-transplant.

In another embodiment, organ transplant engraftment, is characterised by less than 50% increase in quantity of dscfDNA in the recipient post-transplant.

In another example, the level of dscfDNA and/or the level recipient DNA are compared with the total level of DNA to provide a ratio of organ transplant rejection, organ dysfunction or organ failure. However, the high dynamic range and discrimination power of the methods of present invention within the dynamic range do not require recipient DNA to be measured.

In another embodiment, methods of diagnosing or predicting transplant status or outcome comprising the steps of: (i) providing a sample from a recipient who has received a transplant from a donor; (ii) quantifying circulating dscfDNA in the recipient using probe-free quantitative PCR comprising the use of a forward or reverse primer which fully hybridises to at least one donor-specific deletion/insertion polymorphism (DIP) allelic sequence present in the donor but which is not present in the recipient, said donor-specific DIP allelic sequence comprising a) one or more allelic breakpoints corresponding to either an insertion or deletion junction of the polymorphism in the donor, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to either an insertion or deletion junction for the same polymorphism in the recipient; or b) donor-specific insertion sequence, and wherein said forward or reverse primer does not fully hybridise to the one or more allelic breakpoints corresponding to a deletion junction for the same polymorphism in the recipient; and (iii) diagnosing or predicting transplant status or outcome based on the quantity of dscfDNA in the recipient.

In some embodiments, the quantity of dscfDNA can be determined as a concentration.

Various mathematical software models can be used to derive a standardised "quantity of dscfDNA" in the recipient. In one example, quantification is determined using QuantaSoft (BioRad) or other equivalent software. For droplet digital PCR typically, quantification is standardised based on the following criteria (i) total droplets in each reaction is greater than 10, 000 and ii) the presence of negative droplets. In one example, the number of copies of dscfDNA per μΙ of assembled PCR reaction is calculated using Poisson distribution as shown in the examples. The skilled person will appreciate that as the gene dosage per cell for a homozygote marker is double that for a heterozygous marker, homozygous markers used for quantification of dscfDNA will need to be adjusted by half.

(ii) an increase in dscfDNA copies/ml in the biological sample of at least 50% between two or more time points between days 3 and 28 post-transplant; (iii) an at least two-fold increase, or at least three-fold increase, or greater than three-fold increase in dscfDNA copies/ml in the biological sample between days 3 and 28 post-transplant compared to the corresponding dscfDNA copies/ml in the biological sample from post-transplant recipients with no evidence of transplant rejection or other pathologies;

In some embodiments, the quantity of dscfDNA above a pre-determined threshold is indicative of a transplant status or outcome. For example, the normative values for clinically stable post-transplantation recipients with no evidence of graft rejection or other pathologies can be determined. An increase in the quantity of dscfDNA in the recipient above the normative values for clinically stable post-transplantation recipients could indicate a change in transplant status or outcome such as transplant rejection or transplant injury. On the other hand, a quantity of dscfDNA below or at the normative values for clinically stable post-transplantation recipients could indicate graft tolerance or graft survival.

In one example, the threshold value is 200, 300, 500, 800 or 1 ,000 dscfDNA copies/ml recipient biological sample (e.g. plasma) post-transplant. In another example, the threshold value is the quantity of dscfDNA copies/ml at two weeks, at three weeks or at four weeks onward post-transplant.

The disclosure provides methods that are sensitive and specific. In some embodiments, the methods described herein for diagnosing or predicting transplant status or outcome have at least 56 %, 60%, 70%, 80%, 90%, 95% or 100% sensitivity. In some embodiments, the methods described herein have at least 56 % sensitivity. In some embodiments, the methods described herein have at least 78 % sensitivity. In some embodiments, the methods described herein have a specificity of about 70% to about 100%. In some embodiments, the methods described herein have a specificity of about 80% to about 100%. In some embodiments, the methods described herein have a specificity of about 90% to about 100%. In some embodiments, the methods described herein have a specificity of about 100%

Treatment of organ rejection

In another embodiment, the disclosure provides methods for determining an immunosuppressive regimen for a transplant (e.g. allograft) recipient. In some embodiments, the invention further includes methods for determining the effectiveness of an immunosuppressive regimen for a subject who has received a transplant.

In some embodiments, temporal differences in the quantity of dscfDNA can be used to monitor effectiveness of an immunosuppressant treatment or to select an immunosuppressant treatment. For instance, the quantity of dscfDNA in the recipient can be determined before and after an immunosuppressant treatment. A decrease dscfDNA in the recipient after treatment may indicate that the treatment was successful in preventing transplant rejection. Additionally, the quantity of dscfDNA in the recipient can be used to choose between immunosuppressant treatments, for examples, immunosuppressant treatments of different strengths. For example, a higher quantity of dscfDNA in the recipient may indicate that there is a need of a very potent immunosuppressant, whereas a lower quantity of dscfDNA in the recipient may indicate that a less potent immunosuppressant may be used.

Examples of suitable immunosuppressants include calcineurin inhibitors such as cycolsporin and tacrolimus, corticosteroids such as methylprednisolone, dexamethasone, prednisolone, cytotoxic immunosuppressants such as azathioprine, chlorambucil, cyclophosphamide, methotrexate, immunosuppressant antibodies such as antithymocyte globulins, basiliximab, infliximab, sirolimus derivatives such as everolimus, rapamycin, and others such as mycophenolate.

Kits

Also provided are reagents and kits thereof for practicing one or more of the above- described methods. The subject reagents and kits thereof may vary greatly. Reagents of interest include reagents specifically designed for use in production of the above- described: (i) genotyping of a transplant donor and a transplant recipient and quantification of dscfDNA in a sample obtained from a transplant recipient. The kits may include one or more of the forward and reverse primer sets described herein in Tables 2 and 4.

The kits of the present disclosure may additionally comprise one or more therapeutic agents. The kit may further comprise a software package for data analysis, which may include reference profiles for comparison with the test profile.

The kit may further comprise other reagents, including DNA intercalating agents, buffers etc, required to perform an amplification reaction such as a buffer, nucleotides and/or a polymerase, as well as reagents for extracting nucleic acids from a biological sample.

Such kits may also include instructions to access a database. The kits may also include protocols for performing the methods described herein.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

EXAMPLES

Example 1 : Assessment of high resolution melting analyses for genotyping of small deletion and insertion polymorphisms

The inventors evaluated the genotyping of deletion/insertion polymorphisms (DIPs) by high resolution melting analysis (HRMA).

Methods

DNA samples

Genomic DNA was extracted from the leukocyte-rich component of blood from 39 individual blood donors using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. Genomic DNA was genotyped for nine bi-allelic small deletion/insertion polymorphisms (DIPs). Consent was obtained specifically for this project.

Selection of deletion/insertion polymorphisms (DIPs)

Nine DIPs were selected using the Marshfield Clinic database (Weber et al. (2002) Am J Hum Genet 71 :854-62; Marshfield Clinic. Mammalian Genotyping Service [Internet]. Natl. Hear. Lung Blood Inst. Available from: https://www3.marshfieldclinic.org/mqs/). DIPs were selected based on the following criteria (a) high heterozygosity >0.35, (b) small deletion and insertion lengths between 10-50bp, (c) absence of repetitive sequences, (d) absence of polymorphisms >1 % minor allele frequency flanking 50bp of the locus. Table 1 summarises the characteristics of these polymorphisms. Polymorphism identifications were retrieved from the Database of Single Nucleotide Polymorphisms (dbSNP) build ID: 142 (Bethesda, 2015 Bethesda (MD). Database of Single Nucleotide Polymorphisms (dbSNP) [Internet]. Natl. Cent. Biotechnol. Information, Natl. Libr. Med. (dbSNP Build ID 142). Available from: http://www.ncbi.nlm.nih.gov/SNP/). Sequence coordinates were retrieved from the University of California Santa Cruz (UCSC) human genome browser assembly hg38 (Kent WJ et al. (2002) Genome Res 12:996-1006).

Design of genotyping primer sets and establishing genotype-specific melting profiles

Genotyping primer sets were designed based on the following criteria (a) flanking the bi-allelic DIP locus to produce three effective genotypes [insertion/insertion (LONG), insertion/deletion (HET) or deletion/deletion (SHORT)], (b) short amplicons to minimize inclusion of unknown variants and enhance differentiation of the alleles (Do H, et al. (2008) BMC Cancer 8:142), and (c) a melting temperature of 65°C. The details of the genotyping primer sets are summarized in Table 1 .

Genotyping by HRMA

Genomic DNA from 39 individuals were genotyped by HRMA against each DIPs to (a) establish unique genotype-specific melting profiles for each DIP and (b) to determine the likely diverse coverage of our panel of 9 DIPs.

PCR for HRMA analysis was performed in 0.1 mL tubes on a rotor-based platform, Rotor-Gene 6000 (Corbett Life Science) in the presence of the fluorescent DNA intercalating dye, SYTO 9 (Invitrogen) as previously described (Kristensen et al. 2008 above). In brief, the reaction mixture in a 20 μΙ final volume contained: 1 χ PCR buffer, 2.5 mM MgCI₂, 200 nM forward primer, 200 nM reverse primer, 20 ng of genomic DNA, 200 μΜ of dNTPs, 5 μΜ of SYTO 9, 0.5 U of HotStarTaq (Qiagen) DNA polymerase and PCR-grade water. The cycling and melting conditions were as follows: one cycle of 95°C for 15 min; 55 cycles of 95°C for 10 s, 60°C for 20 s, 72°C for 30 s; one cycle of 97°C for 1 min and a melt fr om 65°C to 95°C rising 0.2°C per second.

All samples were tested in duplicate. Analyses of data were performed using Rotor- Gene 6000 software (Corbett Life Science). Melting curves were determined and normalized by setting temperature windows before and after major fluorescence changes. Genotypes were determined by direct visualization of normalized curve overlays. Genotypes for each DIP were further validated by fragment length analysis using microfluidics capillary electrophoresis.

Results

HRMA effectively genotyped all 39 individuals. All three genotypes for each DIP were detected (Figure 1 ). Furthermore, normalized melting curves for each genotype were distinct and readily distinguished (Figure 1 ). The genotypes were also confirmed by microfluidics capillary electrophoresis.

The coverage diversity of the panel of 9 DIPs was also evaluated using the genotypes of 39 individuals. The individuals were cross-matched against each other to generate a potential combination of 1482 unique donor-recipient pairs. Using our panel of 9 DIPs, at least 1 informative locus in 98.2% (n=1455) of the unique donor-recipient pairs. Remarks

Effective genotyping of small DIPs is essential to identify donor alleles that can be used for quantification of genomic chimerism. The amplification of bi-allelic small DIPs using respective primer sets described in Table 1 will yield three different genotypes: insertion/insertion (LONG) with a long amplicon, deletion/deletion (SHORT) with a short amplicon and insertion/deletion (HET) with both long and short amplicons. The genotypes can be rapidly and effectively assessed by HRMA.

The difference in amplicon lengths generated easily distinguishable homozygous melting profiles for each locus. Furthermore, all heterozygote melting curves for each locus were clearly identified.

HRMA was determined to be the preferred platform owing to the rapidity, simple workflows, cost-effective and closed-tube methodology. It also proved to be the most rapid with the turnaround time to genotype 48-96 samples being just under 3 hours with less than 1 hour hand-on-time.

The panel of DIPs was demonstrated to be have a diverse coverage and this will facilitate the identification of informative alleles that will differentiate donor-specific DNA from recipient-specific DNA.

Example 2: Analysis of dscfDNA as a marker of organ health after liver transplantation

Liver transplantation is the only effective treatment with excellent long-term outcome for patients with irreversible liver diseases. Surveillance of organ health after transplantation remains a fundamental aspect of post-transplantation care. The estimation of graft function is essential for the surveillance of post-transplant complications, in particular organ rejection, a common and well recognized complication. Current approaches used to organ diagnose rejection are inaccurate or highly invasive. However, these major limitations may be circumvented by the use of novel molecular biomarkers such as dscfDNA.

Adopting a two-step workflow, dscfDNA was readily quantified using an innovative probe-free digital PCR approach in a cohort of 8 liver transplant recipients. Method

A two-step workflow was developed to successfully quantify dscfDNA (Figure 2). In Step 1 , donor and recipient genomic DNA are genotyped against a panel of DIPs to identify informative allelic sequences that are present in the donor and absent in the recipient. These informative sequences can be subsequently used as markers to quantify chimerism. In Step 2, selective amplification of donor-specific alleles by primers that hybridizes to the allelic breakpoints (allele-specific priming) enables the absolute quantification of dscfDNA by a novel probe-free ddPCR methodology. By using small DIPs coupled with allele-specific primers, this allowed us to maintain the specificity of amplification of donor alleles, simplify the genotyping, allow for the use of cheaper cost-effective agents in place of probes and eliminate the complexity of optimization that is associated with the use of probes.

Study Approval

Institutional human research ethics committee approval for this study was obtained. Written informed consent was obtained from the individual donors and recipients prior to the collection of blood samples for analyses.

Processing of blood sampling

Blood samples from eight transplant recipients were collected and processed within 3 hours at the following time points: pre-transplant and post-transplant days 3, 7, 14, 28 and 42 using previously described protocols (Tsao, S.C.-H. et al., (2015). Scientific reports, 5, p.1 1 198). In brief, each blood sample was centrifuged at 800 g for 10 mins. The plasma fraction of the blood sample was transferred into a collection tube for a second centrifugation step at 1600 g for 10 mins to minimize contamination by cellular DNA. Subsequently, the plasma was carefully aspirated and transferred into cryovials (Corning) for storage at -80°C.

Plasma DNA Extraction

4ml_ of plasma (2ml_ when plasma volumes were inadequate) from the transplant recipients was thawed to ambient temperature and plasma DNA was batch extracted using the QIA Circulating Nucleic Acid Kit (Qiagen) DNA using our standardized laboratory protocols. All samples were eluted in 100μΙ_ of AVE buffer and stored at 4°C.

Design of allele-specific quantification primer sets

The design of either a forward or reverse primer that hybridizes to up to 2 allelic breakpoints (i.e. across two junctions) in combination with a common primer (downstream or upstream respectively) will selectively amplify the allele of interest in a deletion-insertion polymorphism (Figure 2).

Such primer sets facilitate the accurate amplification of the target of interest (i.e. donor-specific fraction of the cell-free DNA in the plasma of the transplant recipient) eliminating the need for fluorescent probes for allelic discrimination.

Allele-specific primer sets were designed to produce small amplicons between 50- 130bp to minimize the underrepresentation of fragmented dscfDNA. The details of 18 primer sets that were designed to specifically amplify the long and short alleles of each DIP locus are summarized in Table 2. The specificity of these primer sets were further confirmed by in-silico PCR available online on UCSC Genome Bioinformatics (Kent WJ et al. (2002) Genome Research 12(6):996-1006).

Step 1 of Workflow - Genotyping

This step is only performed once per donor-recipient pair. Blood samples from eight de-identified deceased organ donors and matched transplant recipients were obtained prior to organ procurement and prior to liver transplantation respectively. The samples were processed and genotyped as described in Example 1 . DIPs were considered informative if allelic sequences for the polymorphisms are present in the donor and absent in the recipient or vice versa (for e.g. see Figure 4).

Step 2 of Workflow - Quantification

This step allows quantification of dscfDNA using informative allelic sequences established in Step 1 . The same informative locus can be used to monitor dscfDNA levels over time and this is demonstrated in Figure 3. Quantitative analysis of DNA is performed and presented in accordance to the digital minimum information for publication of quantitative digital PCR experiments (Huggett JF et al. (2013) Clinical Chemistry 59(6):892-902).

(i) Determining informative donor-specific alleles

A donor allele that is not present in the recipient is considered informative and the allele-specific primer set will be selected for the quantification of dscfDNA (Table 2). Based on the genotyping information derived from a panel of 9 DIP loci, up to two informative donor alleles (where there was more than a single unique donor allele) were selected to serially quantify dscfDNA levels for each transplant recipient.

(ii) Probe-free ddPCR methodology for quantification of dscfDNA

Conditions were standardized across all assays. In brief, 22 μΙ_ reaction comprising 1 x QX200 ddPCR EvaGreen Supermix (Bio-Rad Laboratories), 100 nM of forward and reverse primers (allele-specific primer set - Table 2), 2 μΙ_ of plasma DNA and PCR-grade water was assembled. 20 μΙ_ from each ddPCR mix was loaded onto the DG8 droplet generator cartridge (Bio-Rad) for droplet generation. After partitioning, the reactions were thermocycled using the following conditions: one cycle of 95°C for 5 min; 40 cycles of 95°C for 30 s and 61 °C for 60 s ; one cycle of for 5 mins; one cycle of 90°C for 5 min and a brief hold at 4°C. Af ter the endpoint PCR, the 96 well plate was transferred onto a QX200 Droplet Reader (Bio-Rad) for analyses.

(iii) Data analyses

Using the supplied software, QuantaSoft (Bio-Rad), rare event detection mode using the EvaGreen chemistry was selected. The following criteria are observed to standardize quantification; i) total droplets in each reaction is greater than 10,000 and ii) the presence of negative droplets. The number of copies of dscfDNA per μΙ_ of the assembled PCR reaction is calculated using the Poisson distribution. The derived concentration is further converted into copies of dscfDNA per mL of recipient plasma by the following formula:

As the gene dosage per cell for a homozygote marker is double that for a heterozygote marker, homozygote markers utilized for the quantification of dscfDNA were adjusted by half to normalize the interpretation of data in this study. The levels of dscfDNA were also correlated with the clinical progress, serum liver function tests and histological findings (if available) for each transplant recipient.

64

Results

Recipients who did not develop any complications after transplantation

Donor and recipient pairs were genotyped by HRMA to identify informative DIP loci dscfDNA was selectively amplified using informative alleles.

As shown in Figure 3, positive droplets (containing template, in this case dscfDNA) clustered at a higher fluorescent intensity compared to the negative droplets (containing no template). The lack of dscfDNA in the pre-transplant sample confirmed the specificity of the allele-specific primer utilized in this methodology.

Figure 5 shows box plot of dscfDNA levels in six recipients who underwent uneventful liver transplantation. The recovery of dscfDNA levels was stereotypical. Consistently, dscfDNA levels were significantly elevated at day 3 after transplantation. This is expected from the traumatic process of organ procurement, preservation and reperfusion. There were significant reductions in the dscfDNA levels with subsequent time points. The dscfDNA levels reached steady state 4 weeks after transplantation. The mean baseline dscfDNA levels for recipients with healthy grafts was 95 (+/-88) dscfDNA copies/mL of recipient plasma.

Figure 13 shows box plot of dscfDNA levels in the six recipients who underwent uneventful liver transplantation, combined with 7 further successful recipients. The recovery of dscfDNA levels was again stereotypical. The dscfDNA levels reached steady state 4 weeks after transplantation. The mean baseline dscfDNA levels for recipients with healthy grafts was 97 (+/-87) dscfDNA copies/mL of recipient plasma at 42 days.

One recipient who developed an episode of acute cellular rejection (ACR) after transplantation

One recipient developed an episode of ACR at day 7. The persistent elevation of serum liver function tests prompted a liver biopsy that confirmed the diagnosis. Following the adjustment of immunosuppression, the serum liver function tests improved. In this recipient, allele BTR16-L0NG (using SEQ ID NO:52 and SEQ ID NO:53) was informative and was selected to quantify dscfDNA. The marked elevation of dscfDNA levels coincided with the diagnosis of ACR and improved with further treatment (Figure 6).

Figure 7 shows the surveillance of dscfDNA levels in a recipient with ACR following the transplantation compared to 6 other recipients who underwent liver transplantation without any complications. Interestingly, the level of dscfDNA remained above the level compared to the normal (i.e. uncomplicated) transplant recipients.

One recipient who developed ischemic reperfusion injury after transplantation

Figure 8 shows the surveillance of dscfDNA levels in a recipient with ischemic reperfusion injury following transplantation compared with the average of six uncomplicated recipients. Following abnormalities in serum liver function tests, a liver biopsy was performed on day 7. The results of the liver biopsy confirmed ischemic reperfusion injury.

In this recipient, allele BTR09-LONG (using primers SEQ ID NO:44 and SEQ ID NO:45) was informative and was selected to quantify dscfDNA. It was interesting to observe in this recipient that there was a four-fold increase in dscfDNA copies/ml at day 3 after transplantation. Importantly, the rapid decline in dscfDNA levels at day 7 to "near-normal" levels" of the corresponding recipients who did not develop any complications at day 7. The significant elevation at day 3 after transplantation and marked reduction in dscfDNA may be characteristic of recipients who develop ischemic reperfusion injury.

Remarks

Increasing number of studies are evaluating dscfDNA as a non-invasive biomarker of organ health and organ rejection. The methodologies utilized to date have intrinsic limitations (such as being costly, laborious and/or have long turnaround times to generate results) that may preclude their implementation into clinical practice. The methodology described herein can overcome some of these limitations and facilitate its translation into the diagnostic laboratories of specialized transplantation centers. Our methodology is based on a straightforward two-step workflow. In Step 1 , genotyping of DIPs is performed on pre-transplantation blood samples by HRMA.

Droplet digital PCR has enabled the absolute quantification of low abundance DNA. This technology is therefore attractive for the monitoring of dscfDNA after transplantation. Our methodology described in Step 2 harnesses the novelty of allele- specific primers, the cost-effectiveness of EvaGreen (a DNA intercalating dye) and the precision of the ddPCR to quantify dscfDNA.

The specificity of our assays is based on the design of allele-specific primer sets, one of which hybridizes across the allelic breakpoints at the insertion and deletion junction (unique to DIPs) to selectively amplify donor alleles. As such, DIPs were selected in this study to facilitate the use allele-specific primers for the detection and quantification of dscfDNA. The utilization of EvaGreen chemistry on a ddPCR platform has several key benefits pertinent to the quantification of dscfDNA. First, ddPCR offers the technical advantages of reproducibility and absolute quantification without the need of standard-curve calibration of real-time quantitative PCR (Bruno DL et al. (2014) Clinical Chemistry 60(8):1 105-1 1 14). Second, the use of EvaGreen in ddPCR eliminates the need for costly fluorescent probes. As probe-based assays are often challenged by meticulous optimizations, co-hydrolysis (resulting in significant rain that can confound downstream analysis) and costly redesign, a probe- free approach was more economical and straightforward in our experience.

More importantly, the methodology employed in this study to quantify dscfDNA does not require a PCR pre-amplification step. The PCR pre-amplification is considered to be diagnostically unfavorable, requires arduous manual handling and carry a significant risk of contamination. Despite the lack of pre-amplification, detection and quantification of dscfDNA was still feasible in this study.

The lack of standardization in the measurement of dscfDNA is accurately acknowledged in a review by Gielis et al (Gielis et al. (2015) American Journal of Transplantation 15(10):2541 -51 ). dscfDNA is frequently measured based on the fractional abundance of donor DNA (percentage of donor DNA = donor DNA divided by sum of donor and recipient DNA). However, calculations that include the concentration of recipient DNA have inherent limitations. Studies have shown that the improper handling of blood samples as well as infection and exercise can falsely elevate recipient DNA (van der Vaart M & Pretorius PJ (2008) Annals of the New York Academy of Sciences 1 137:18-26), and these factors are likely to confound the measurement of fractional abundance. As such, absolute measurements of dscfDNA related to the initial plasma volume are presented in this study.

Our methodology to assess dscfDNA levels was demonstrated to be feasible as depicted by the monitoring of recipients who underwent liver transplantation. The graft health of the 8 recipients who underwent liver transplantation was well reflected by the levels of dscfDNA measured using our two-step workflow. A role for well- designed clinical trials to qualify the clinical utility of dscfDNA as a biomarker of organ rejection and potential role as a biomarker to individualize immune suppression is proposed.

The results of the pilot study demonstrated that the methodology to genotype donor- recipient pairs using deletion/insertion polymorphisms was readily performed and reproducible. Further, the methodology for quantifying dscfDNA was highly sensitive and specific. The methodology provides a convenient and cost effective method of monitoring recipients long-term and tailoring effective treatment.

Example 3: Quantification of dscfDNA using allele-specific primers

The present inventors have demonstrated the quantification of dscfDNA using insertion allele-specific primers. In Figure 9 (a), (b), (d) and (e) a forward or reverse primer which fully hybridises to a donor-specific insertion polymorphism allelic sequence present in the donor but which is not present in the recipient is used. The allelic sequence includes one allelic breakpoint corresponding to an insertion junction of the polymorphism in the donor. In Figure 9 (c) and (f), a forward or reverse primer which fully hybridises to a donor-specific insertion polymorphism allelic sequence present in the donor but which is not present in the recipient is used. The allelic sequence includes allelic breakpoints corresponding to two insertion junctions of the polymorphism in the donor. In Figure 9 (g) and (h), deletion allele specific primers are used; a forward or reverse primer which fully hybridises to a donor-specific deletion polymorphism allelic sequence present in the donor but which is not present in the recipient is used. The allelic sequence includes one allelic breakpoint corresponding to a deletion junction of the polymorphism in the donor. The design of either a forward or reverse primer that hybridizes to donor-specific insertion sequence in combination with a common primer (downstream or upstream respectively) allows selective amplification of the allele of interest in a DIP (Figures 10 and 1 1 ).

The inventors designed allele-specific primer sets to specifically amplify the long and short alleles of a DIP locus BTR20, as summarized in Tables 3 and 4.

Example 4: Association of elevated dscfDNA in recipients with organ rejection but not in recipients with hepatitis without organ rejection

Twelve recipients who underwent liver transplantation were recruited and levels of dscfDNA were quantified using the methodology described in Example 1 and Example 2. Recipients U001 , U002, U003 and U004 were four clinically stable and healthy recipients with no complications who are greater than 12 months posttransplantation. Blood samples were collected from these recipients for the quantification of dscfDNA levels. Recipients S001 to S008 were recipients who had clinical suspicions of acute rejection. Blood samples were collected from these recipients prior to their liver biopsies. DscfDNA levels were measured and correlated with the liver biopsy outcomes.

Figure 12 (a) shows 10 clinically stable recipients. Day 14 of six of the recipients (recipients T01 1 , T012, T002, T006, T008 and T009; also included in Figure 5) demonstrated low levels of dscfDNA. The remaining four recipients were healthy recipients who are greater than 12 months after transplantation. This data shows that in stable recipients, levels of dscfDNA were consistently low.

Figure 12 (b) shows 8 recipients with abnormal serum liver biochemistry undergoing liver biopsy at time points greater than 3 months post-transplantation. The liver biopsies of recipients S007 and S006 showed hepatitis with no evidence of organ rejection and the dscfDNA levels in these recipients remain low. In recipients with liver biopsies confirming organ rejection (e.g. S005, S002, S008, S004, S003 and S001 ), dscfDNA levels were demonstrably higher. For example, Figure 12 (b) shows recipients with organ rejection have 500 or more copies dscfDNA per ml of recipient plasma.

This data demonstrates dscfDNA can be used as a marker of organ rejection following liver transplantation.

Example 5: LONG and SHORT allele specific amplification

Primers were selected to examine specificity of ddPCR assays using allele specific primer sets. Figures 14, 15 and 16 show ddPCR using primer sets specific for either the LONG allele or the SHORT allele as described in Figure 2. This data demonstrates that the use of a primer which fully hybridises to at least one donor- specific deletion/insertion polymorphism (DIP) allelic sequence comprising one or more allelic breakpoints corresponding to an insertion junction of the polymorphism, is able to specifically amplify the LONG allele, but does not amplify the SHORT allele.

This data also demonstrates a primer which fully hybridises to at least one donor- specific deletion/insertion polymorphism (DIP) allelic sequence comprising one or more allelic breakpoints corresponding a deletion junction of the polymorphism is able to specifically amplify the SHORT allele, but does not amplify the LONG allele.

Example 6: Acute cellular rejection following transplantation

Further recipients were recruited, and following genotyping as described in Example 1 , dscfDNA in the recruited transplant recipients was examined as described in Example 2.

One recipient (T015) developed an episode of acute cellular rejection (ACR) at day 9, immediately following which immunosuppressive therapy was adjusted. For example, Figure 17 shows the surveillance of dscfDNA in the plasma of recipient T015 with ACR after liver transplantation compared to the mean of thirteen recipients who underwent liver transplantation without any complications. The elevation in dscfDNA levels (determined using primers specific for alleles of BTR08, BTR03 and BTR17) at day 9 coincided with the diagnosis of ACR on liver biopsy. Immunosuppression was adjusted and a subsequent liver biopsy performed on day 14 confirmed mild improvement in organ health. The elevation of dscfDNA levels coincided with the diagnosis of ACR and improved with adjustment of immunosuppressive treatment.

One recipient (T038) developed an episode of ACR at day 14, immediately following which immunosuppressive therapy was adjusted. For example, Figure 18 shows the surveillance of dscfDNA in the plasma of recipient T038 with ACR after liver transplantation compared to the mean of thirteen recipients who underwent liver transplantation without any complications. The elevation in dscfDNA levels (determined using primers specific for the BTR12-LONG) at day 14 coincided with the diagnosis of ACR on liver biopsy and improved with adjustment of immunosuppressive treatment Example 7: Biliary complications do not alter dscfDNA

One recipient (T022) of the cohort of further recipients referred to at Example 6 developed a significant biliary anastomotic stricture after liver transplantation. For example Figure 19 shows the surveillance of dscfDNA in the plasma of recipient T022 who developed cholestasis secondary to biliary complications compared to the mean of thirteen recipients who underwent liver transplantation without any complications. Continued improvement of dscfDNA levels (mean determined using primers specific for the BTR02-LONG; BTR03-LONG, BTR08-LONG and BT18-SHORT alleles) indicates that dscfDNA is independent of cholestasis.

Example 8: Normal transplant recipients do not have elevated levels of dscfDNA

Many of the cohort of further recipients referred to at Example 6 underwent uneventful liver transplantation. Figure 13 shows a box plot of dscfDNA levels in 13 recipients who underwent uneventful liver transplantation. The recovery of dscfDNA levels was stereotypical. Consistently, dscfDNA levels were frequently elevated at day 3 after transplantation. There were significant reductions in the dscfDNA levels with subsequent time points. The dscfDNA levels reached steady state 4 weeks after transplantation.

Example 9: Analysis of dscfDNA as a marker of organ health after liver transplantation

DscfDNA was examined in a liver transplant recipient as described in Example 2. Figure 20 shows a representative 1 D plot derived from the probe-free ddPCR methodology evaluating the dscfDNA levels of the recipient. Positive droplets (containing template, in this case dscfDNA) clustered at a higher fluorescent intensity compared to the negative droplets (containing no template). The lack of dscfDNA in the pre-transplant sample confirmed the specificity of the allele-specific primer utilized in this methodology.

Claims

CLAIMS:

1 . A method for detecting organ transplant rejection, organ dysfunction or organ failure, the method comprising:

2. The method according to claim 1 , further comprising:

genotyping a donor and recipient pair against a panel of multi-allelic deletion/insertion polymorphisms (DIPs) to identify one or more donor-specific DIP alleles which are present in the donor but absent in the recipient.

3. The method according to claim 1 or claim 2, further wherein the multi-allelic DIPs include bi-allelic small deletion/insertion polymorphisms (DIPs).

4. The method according to claim 1 , further comprising a corresponding primer which is a common primer located either downstream or upstream or the forward or reverse primer respectively wherein the common primer hybridises to allelic sequences common to both the donor and the recipient.

5. The method according to any one of claims 1 or 4, wherein:

(i) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the insertion junction of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient;

(ii) the forward primer is a common primer and the reverse primer hybridises to allelic sequence comprising the insertion of the DIP in the donor but does not fully hybridise to allelic sequence of the deletion junction of the DIP in the recipient;

6. The method according to any preceding claim, wherein an allelic breakpoint corresponds to each insertion junction of the insertion sequence of the DIP in the allelic sequence.

7. The method according to any preceding claim, wherein an allelic breakpoint corresponds to a DIP deletion in the allelic sequence.

8. The method according to any preceding claim, wherein the organ transplant is selected from the group consisting of liver, lung, intestine, bowel, pancreas, kidney, liver or heart or a combination of any of these.

9. The method according to any preceding claim, wherein the quantitative PCR is digital PCR (dPCR).

10. The method according to any preceding claim, wherein digital PCR is droplet digital (ddPCR).

1 1 . The method according to any one of claims 3 to 7, wherein the identification of donor-specific DIP alleles comprises performing high resolution melting analysis (HRMA).

12. The method according to any preceding claim wherein one, two, three, four or five donor-specific DIP alleles are quantified.

13. The method according to any preceding claim, wherein the insertion or deletion sequence of the DIP consists of a length of between 10 and 50 bases.

14. The method according to any preceding claim, wherein the insertion or deletion sequence of the DIP consists of a length of between 10 and 100 bases.

15. The method according to any preceding claim, wherein the DIP is characterised by (i) small deletion and insertion lengths between 10-50bases, (ii) absence of repetitive sequences and (iii) absence of polymorphism >1 % minor allele frequency flanking 100 bases of the locus.

16. The method according to any preceding claim, wherein the panel comprises a sufficient number of DIPs to identify at least one donor-specific DIP allele in at least 98% of donor-recipient pairs.

17. The method according to any preceding claim, wherein the panel comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more DIPs.

18. The method according to any one of claims 13 to 17, wherein the panel comprises at least fifteen or at least twenty small DIPs.

19. The method according to any one of claims 12 to 16, wherein the DIPs is selected from at least one set forth in Table 1 or Table 3.

20. The method according to any preceding claim, wherein the forward and reverse primers are selected from at least one primer set for a given DIP locus set forth in Table 2 or Table 4.

21 . The method according to any preceding claim, wherein the method is performed in a biological sample obtained from the recipient or a biological sample obtained from the donor and a biological sample obtained from the recipient.

22. The method according to claim 21 , wherein the biological sample is selected from smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, plasma, lymph fluid, saliva and urine.

23. The method according to any preceding claim, wherein a temporal increase in the quantity of dscfDNA is indicative of transplant rejection, organ dysfunction or organ failure.

24. The method according to any preceding claim, wherein organ transplant rejection, organ dysfunction or organ failure is characterised by one or more of the following in the recipient:

25. The method according to any preceding claim, wherein the transplant rejection is selected from the group consisting of acute cellular rejection (ACR), chronic cellular rejection or antibody mediated rejection.

26. The method according to any preceding claim, further comprising performing a biopsy.

27. The method according to any preceding claim, further comprising analysing one or more serum biochemistry markers in the recipient.

28. A method of predicting or monitoring transplant survival in a recipient who has received an organ transplant comprising:

29. The method according to any preceding claim, wherein the donor is human or a non-human donor.

30. The method according to any preceding claim, wherein the recipient is a human.

31 . The method according to any preceding claim, wherein the quantity of dscfDNA in the recipient is determined over multiple time points post-transplant.

32. The method according to any preceding claim wherein the diagnosing, predicting or monitoring transplant status or outcome comprises treating organ transplant rejection in the recipient.

33. The method according to claim 32, wherein the diagnosing, predicting or monitoring transplant status or outcome comprises determining, modifying or maintaining an immunosuppressive regimen.

34. The method according to any one of claims 1 to 31 , wherein a temporal increase in the quantity of dscfDNA between the at least two time points is indicative of transplant rejection, graft dysfunction or organ failure.

35. The method according to any one of claims 1 to 31 , wherein the quantity of dscfDNA above a predetermined threshold is indicative of a transplant status or outcome.

36. The method according to claim 35, wherein the threshold is a normative value for clinically stable post-transplantation recipients with no evidence of transplant rejection or other pathologies.

37. The method according to claim 35 or 36, wherein the threshold value is greater than 500 dscfDNA copies/ml.

38. A rejection therapy for use in treating a transplant rejection in a recipient thereof, wherein the recipient is assessed according to the method of any one of claims 1 to 27.

39. A kit comprising one or more of the forward and reverse primers set forth in Table 2 or Table 4 for amplifying one or more of the DIPs set forth in Table 1 or Table 3, together with suitable reagents and instructions for performing the quantification method according to claim 1 .

40. The steps, features, integers, compositions and/or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps or features.