WO2023187744A1

WO2023187744A1 - Non-invasive diagnosis of acute rejection in solid organ transplantation

Info

Publication number: WO2023187744A1
Application number: PCT/IB2023/053264
Authority: WO
Inventors: Silvia DEAGLIO; Tiziana VAISITTI; Monica SORBINI
Original assignee: Universita' Degli Studi Di Torino
Priority date: 2022-04-01
Filing date: 2023-03-31
Publication date: 2023-10-05
Also published as: IT202200006512A1

Abstract

The invention concerns a method for diagnosing acute rejection in solid organ transplantation in a patient. Kits for diagnosing acute rejection in solid organ transplantation in a patient comprising specific primers and probes and uses of the kit are also described.

Description

Non-invasive diagnosis of acute rejection in solid organ transplantation

DESCRIPTION

FIELD OF THE INVENTION

STATE OF THE ART

The transplant patient is followed up throughout the post-transplant period by means of a series of clinical, instrumental and laboratory tests, repeated on a regular basis, established by the schedule of medical check-ups, which together constitute the transplant monitoring. Despite advances in immunosuppressive therapies, acute rejection is the leading cause of graft failure and death in solid organ transplant recipients. In fact, about 30% of patients experience at least one episode of acute rejection in the first year after transplantation, hence the need for accurate, early, and rapid monitoring. The method of choice for the evaluation of acute rejection is tissue biopsy of the transplanted organ. However, in addition to being very invasive and consequently causing discomfort and stress to the patient, this approach is also characterised by an important degree of inter-operator variability for the evaluation of the degree of rejection. This can result in inappropriate treatments with immunosuppressant drugs that increase the risk of infections, a further potentially fatal complication in recipient subjects.

Recently, research has focused on the identification of new molecular markers and diagnostic methods that are less invasive than tissue biopsy and more effective for early detection of rejection. A potential biomarker is dd-cfDNA (Donor-derived cell-free DNA), consisting of DNA fragments released from the cells of the transplanted organ into the recipient’s blood in conjunction with cell death events that characterise tissue damage caused by acute rejection. Several researches have shown how the dd-cfDNA values correlate with the state of the transplanted organ (heart, lung, liver and kidney) and can therefore be predictive of the occurrence of an episode of acute rejection. One of the advantages of this biomarker is related to the ease of sampling: in fact, the dd-cfDNA is obtained through a venous blood sample.

Currently, several analytical approaches for dd-cfDNA quantification, mainly based on the next-generation sequencing (NGS) technology and the identification of polymorphisms in different regions of the genome, have been developed. However, this method experience limitations to its application in the public hospital routine, represented by long preparation and analysis times with consequent delays in the generation of a report, and unsustainable high costs for post-transplant monitoring, which requires repetition of the analysis multiple times over time (e.g., in the case of heart transplantation, approximately 14 routine sequential biopsies are required during the first year of patient follow-up). ddPCR (Droplet Digital™ PCR) is a method that allows to obtain quantitative and accurate results with great sensitivity and specificity. This technique makes it possible to detect and quantify the presence of the genomic region of interest in a sample thanks to the use of specific probes, designed ad hoc to unambiguously recognise the target DNA sequence. The probes are conjugated with fluorophores of different colours, which allow the detection of the target molecule through the fluorescent signal released upon excitation with a laser at a specific wavelength. From the analysis of this signal, it is possible to trace the amount of target DNA present in the sample.

Recently, it has been demonstrated that it is possible to obtain a precise and sensitive quantification of dd-cfDNA in plasma samples from heart or lung transplant recipients, through the use of the ddPCR Expert Design Assay probe panel, (Bio-rad Laboratories, Inc.), specific for 8 out of 12 alleles of the HLA-DRB1 gene (Sorbini M, et al. “HLA-DRB1 mismatch-based identification of donor-derived cell free DNA (dd-cfDNA) as a marker of rejection in heart transplant recipients: A single-institution pilot study.” J Heart Lung Transplant 2021.). HLA genes are routinely typed by transplant centres to assess the match of donors and recipients. Most of the donor-recipient pairs (>80%) have at least one allele of the HLA-DRB1 genes which is different and therefore detectable through a molecular test. However, the test thus performed has some limitations, as it does not allow for the evaluation of all donor-recipients; in fact, i) patients with rarer alleles (less frequent in the population), not included in the BioRad panel (13-15% of cases), and ii) transplant recipients who share the same alleles with their donor (5-7%) are excluded from the analysis.

The particular, non-negligible, frequency that characterises rejection in solid organ transplantation, together with the difficulties in postoperative control and follow-up for the patient who has to undergo a tissue biopsy of the transplanted organ, are the basis of the substantial need to find an alternative way to avoid episodes of acute posttransplant rejection.

The object of the present invention is therefore to provide a method suitable for a rapid and early diagnosis of rejection in solid organ transplantation in a non-invasive way, and useful for offering indications in the various subsequent phases and the follow-up of the first years after the operation, which does not present the disadvantages noted above of known methods for the same application.

SUMMARY OF THE INVENTION

The invention concerns the clinical application of a system for monitoring acute rejection in solid organ transplantation through quantification of the donor circulating free DNA (dd-cfDNA) in the plasma of the recipient.

The method according to the present invention makes it possible to evaluate all the donor-recipients, even those excluded from the analysis in the methods known up to now, such as patients presenting rare alleles, those not included in the BioRad panel and those transplant recipients who share the same alleles with their donor.

To overcome these limitations and potentially include all donor-recipient pairs in the monitoring, the invention exploits the genetic differences in the HLA-DRB1 and HLA- DQB1 genes. For this purpose, 16 probes were designed, 12 for the DRB1 gene and 4 for DQB1, divided into two groups, each conjugated with a different fluorophore, FAM and HEX, so that they can be used in combined reactions.

The invention therefore concerns a method for diagnosing acute rejection in solid organ transplantation in a patient, comprising the steps of: a. determining the phenotype of the HLA-DRB1 and HLA-DQB1 genes in a biological sample obtained from said patient and in a biological sample obtained from a donor to obtain the typing of the patient and the donor; b. pre-amplifying the circulating DNA (cfDNA) of the biological sample obtained from said patient, by using a first pair of primers specific for said patient and a first pair of primers specific for said donor, to obtain a pre-circulating amplified DNA; c. amplifying by digital droplet PCR (ddPCR) the pre-amplified circulating DNA sample of step b., by using a second pair of primers specific for said patient and a second pair of primers specific for said donor, wherein said second pairs of primers are labelled with a different coloured probe, to obtain a labelled amplified DNA; d. analysing the signal of the labelled primers of said patient and said donor, to assess whether acute rejection of the transplanted organ is in progress.

In a second aspect, the present invention discloses a kit for diagnosing acute rejection in solid organ transplantation in a patient, comprising primer pairs selected from the group consisting of:

SEQ ID NO I and SEQ ID NO: 2;

SEQ ID NO 3 and SEQ ID NO: 4;

SEQ ID NO 5 and SEQ ID NO: 6;

SEQ ID NO 7 and SEQ ID NO: 8;

SEQ ID NO 9 and SEQ ID NO: 10;

SEQ ID NO I I and SEQ ID NO: 12;

SEQ ID NO 13 and SEQ ID NO: 14;

SEQ ID NO 15 and SEQ ID NO: 16;

SEQ ID NO 17 and SEQ ID NO: 18;

SEQ ID NO 19 and SEQ ID NO: 20;

SEQ ID NO 21 and SEQ ID NO: 22;

SEQ ID NO 23 and SEQ ID NO: 24;

SEQ ID NO 25 and SEQ ID NO: 26;

SEQ ID NO 27 and SEQ ID NO: 28;

SEQ ID NO 29 and SEQ ID NO: 30;

SEQ ID NO 31 and SEQ ID NO: 32;

SEQ ID NO 33 and SEQ ID NO: 34;

SEQ ID NO 35 and SEQ ID NO: 36;

SEQ ID NO 37 and SEQ ID NO: 38;

SEQ ID NO 39 and SEQ ID NO: 40; and SEQ ID NO: 41 and SEQ ID NO: 42; the probes selected from the group consisting of:

SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 67, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, and instructions for use in the method according to the invention.

In a third aspect the invention provides the use of the kit for diagnosing acute rejection in solid organ transplantation in a patient.

The dependent claims describe particular embodiments of the invention.

DESCRIPTION OF THE FIGURES

The invention will now be described in detail and with reference to the attached Figures. Figure 1 shows the graph of the analysis results of a ddPCR profile. Points above the threshold of 2356 represent positive droplets, points below the threshold are the negative background (Wells G and H: cfDNA samples; wells B10, B1 1 , B12: positive controls; wells C10, C1 1 , C12, H12: negative controls).

Figure 2 shows the graph of the specificity results of the probes. The probes were tested on various samples of genomic and circulating DNA and positive droplets were obtained only in the presence of the allele against which the probe is directed. Figure 2A: specificity of the HLADRB1_01 probe. Figure 2B: specificity of the HLADRB1_03 probe. Figure 2C: Specificity of the HLADRB1_04 probe. Figure 2D: Specificity of the HLADRB1_07 probe. Figure 2E: Specificity of the HLADRB1_08 probe. Figure 2F: specificity of the HLADRB1_09 probe. Figure 2G: Specificity of the HLADRB1_10 probe. Figure 2H: Specificity of the HLADRB1_1 1 probe. Figure 2I: Specificity of the HLADRB1_12 probe. Figure 2J: Specificity of the HLADRB1_13 probe. Figure 2K: Specificity of the HLADRB1_14 probe. Figure 2L: specificity of the HLADRB1_15/16 probe. Figure 2M: specificity of the HLADQB1_02 probe. Figure 2N: specificity of the HLADQB1_03 probe. Figure 20: Specificity of the HLADQB1_04 probe. Figure 2P: specificity of the HLADQB1_05 probe.

Figure 3 shows the plots of the sensitivity results of DR probes. Serial dilutions of genomic DNA starting from 1 ng up to 7pg were used. The probes proved to be sensitive even in the presence of a small amount of targets. Figure 2A: sensitivity of the HLADRB1_01 probe. Figure 2B: sensitivity of the HLADRB1_03 probe. Figure 2C: sensitivity of the HLADRB1_04 probe. Figure 2D: sensitivity of the HLADRB1_07 probe. Figure 2E: sensitivity of the HLADRB1_08 probe. Figure 2F: sensitivity of the HLADRB1_09 probe. Figure 2G: sensitivity of the HLADRB1_10 probe. Figure 2H: Sensitivity of the HLADRB1_1 1 probe. Figure 2I: Sensitivity of the HLADRB1_12 probe. Figure 2J: sensitivity of the HLADRB1_13 probe. Figure 2K: sensitivity of the HLADRB1_14 probe. Figure 2L: Sensitivity of the HLADRB1_15/16 probe.

Figure 4 shows the plots of the sensitivity results of DQ probes. Figure 4A shows the results with the DQ2 probe, Figure 4B shows the results with the DQ3 probe, Figure 4C shows the results with the DQ4 probe, and Figure 4D shows the results with the DQ5 probe.

DETAILED DESCRIPTION OF THE INVENTION

The invention concerns the clinical application of a system for monitoring acute rejection in solid organ transplantation through the quantification of the donor circulating free DNA (dd-cfDNA) in the plasma of the recipient.

The invention therefore concerns a method for diagnosing acute rejection in solid organ transplantation in a patient, comprising the steps of: a. determining the phenotype of the HLA-DRB1 and HLA-DQB1 genes in a biological sample obtained from said patient and in a biological sample obtained from a donor to obtain the typing of the patient and the donor; b. pre-amplifying the circulating DNA (cfDNA) of a biological sample obtained from said patient, by using a first pair of primers specific for said patient and a first pair of primers specific for said donor, to obtain a pre-circulating amplified DNA; c. amplifying by digital droplet PCR (ddPCR) the pre-amplified circulating DNA sample of step b., by using a second pair of primers specific for said patient and a second pair of primers specific for said donor, wherein said second pairs of primers are labelled with a different coloured probe, to obtain a labelled amplified DNA; d. analysing the signal of the labelled primers of said patient and said donor, to assess whether acute rejection of the transplanted organ is in progress. The presence of organ damage and acute rejection is suspected when the dd-cfDNA values are high compared to baseline values, calculated by analysing samples from patients in stable conditions. In the present invention, when it is employed the definition:

- “rejection”, “acute rejection” or “acute transplant rejection” is intended to encompass the reaction of the recipient organism against the organ that is not recognised as its own. The acute form occurs in about a quarter of patients in the early stages, often immediately or after surgery, usually in the first two weeks. After this critical period, the possibility of having new rejections is reduced. The rejection can be of various degrees, from mild to severe, in most cases it is mild and in 80% of cases curable if treated promptly.

The inventors have observed that through this system of monitoring acute rejection based on ddPCR and on a panel of probes specific for the alleles of the HLA-DRB1 and HLA-DQB1 genes, it is possible to quantify in a precise and accurate way the percentage of dd-cfDNA present in the recipient plasma, and consequently have a rapid estimate of the state of the transplanted organ without having to resort to invasive techniques such as tissue biopsy.

Further advantages are related to the possibility of performing the test at any time (potentially immediately after taking the venous sample) and the relatively low cost of the test. The latter aspect makes it possible to repeatedly perform dd-cfDNA analysis in the post-transplant clinical follow-up period as part of the hospital patient monitoring routine. Furthermore, it is not necessary to carry out further investigations on the recipient and donor genomes before performing the test, because it exploits the HLA typing routinely performed before the transplant for the identification of the alleles that will be used later in the dd-cfDNA quantification.

Advantageously, the method of the present invention allows acute rejection to be monitored in a non-invasive manner, since it requires a peripheral blood sample as starting material, as opposed to tissue biopsy, which is a very invasive procedure.

In one embodiment of the method according to the invention, the first pair of primers specific for the patient and the first pair of primers specific for the donor of step b. of the method are different from each other and selected from the group consisting of:

SEQ ID NO: 1 and SEQ ID NO: 2;

SEQ ID NO: 3 and SEQ ID NO: 4;

SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8;

SEQ ID NO: 9 and SEQ ID NO: 10;

SEQ ID NO: 1 1 and SEQ ID NO: 12;

SEQ ID NO: 13 and SEQ ID NO: 14;

SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; and

SEQ ID NO: 19 and SEQ ID NO: 20.

Table 1 shows the sequences and characteristics of the first pairs of primers.

Table 1 :

In a further embodiment of the method according to the invention, the second pair of primers specific for the patient and the second pair of primers specific for the donor according to step c. of the method, are different from each other and selected from the group consisting of:

SEQ ID NO: 21 and SEQ ID NO: 22;

SEQ ID NO: 21 and SEQ ID NO: 23;

SEQ ID NO: 24 and SEQ ID NO: 25; SEQ ID NO: 26 and SEQ ID NO: 27;

SEQ ID NO: 28 and SEQ ID NO: 29;

SEQ ID NO: 30 and SEQ ID NO: 31 ;

SEQ ID NO: 32 and SEQ ID NO: 33;

SEQ ID NO: 34 and SEQ ID NO: 35; SEQ ID NO: 36 and SEQ ID NO: 37;

SEQ ID NO: 38 and SEQ ID NO: 39;

SEQ ID NO: 40 and SEQ ID NO: 41 ; and

SEQ ID NO: 42 and SEQ ID NO: 43.

Table 2 reports the sequences and characteristics of the second pairs of primers. Table 2:

In a further preferred embodiment of the method according to the invention, the primer pairs of the patient and the donor of step c. are labelled with fluorophores of different colours, preferably said fluorophores are HEX and FAM. Preferably the probes are selected from the group consisting of:

SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75.

Table 3 shows the sequences and characteristics of the probes present in the panel.

Table 3:

As will be better described in Example 4, these probes contain chemical modifications called LNAs denoted in Table 3 by a “+” symbol next to the nucleotide involved in the modification.

The use of specific probes for the HLA-DRB1 and HLA-DQB1 genes makes it possible to potentially include in the analysis any donor-recipient pair: in case donor and recipient share the same HLA-DRB1 alleles (5-7% of cases), it is possible to proceed with the test using the probes for the HLA-DQB1 alleles, and vice versa.

In a further preferred embodiment, negative controls and positive controls are used in the method of the invention. Negative controls consist of sterile deionized water in which no DNA molecule is present, resulting expected values equal to zero. They are used as proof of the analysis correctness.

Positive controls consist of synthetic DNA, not from biological samples, each containing the specific sequence for each probe included in the panel. When used in the analysis, the expected values are fixed and indicate the correct technical success of the quantification itself.

Table 4 shows the sequences of the positive controls.

Table 4:

In a further preferred embodiment of the method according to the invention, probespecific positive controls are used, said positive controls are selected from the group consisting of: SEQ ID NO: 76; SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81 ; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90 and SEQ ID NO: 91. In a second aspect, the present invention discloses a kit for diagnosing acute rejection in solid organ transplantation in a patient, comprising pairs of primers selected from the group consisting of:

SEQ ID NO 1 and SEQ ID NO: 2;

SEQ ID NO 3 and SEQ ID NO: 4;

SEQ ID NO 5 and SEQ ID NO: 6;

SEQ ID NO 7 and SEQ ID NO: 8;

SEQ ID NO 9 and SEQ ID NO: 10;

SEQ ID NO 1 1 and SEQ ID NO 12;

SEQ ID NO 13 and SEQ ID NO 14;

SEQ ID NO 15 and SEQ ID NO 16;

SEQ ID NO 17 and SEQ ID NO 18; and

SEQ ID NO 19 and SEQ ID NO 20

SEQ ID NO 21 and SEQ ID NO 22;

SEQ ID NO 21 and SEQ ID NO 23;

SEQ ID NO 24 and SEQ ID NO 25;

SEQ ID NO 26 and SEQ ID NO 27;

SEQ ID NO 28 and SEQ ID NO 29;

SEQ ID NO 30 and SEQ ID NO 31 ;

SEQ ID NO 32 and SEQ ID NO 33;

SEQ ID NO 34 and SEQ ID NO 35;

SEQ ID NO 36 and SEQ ID NO 37;

SEQ ID NO 38 and SEQ ID NO 39;

SEQ ID NO 40 and SEQ ID NO 41 ; and

SEQ ID NO 42 and SEQ ID NO 43; the probes selected from the group consisting of:

SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, and instructions for use in the method according to the invention.

Preferably the kit may further comprise the probe-specific positive controls selected from the group consisting of:

SEQ ID NO: 76; SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81 ; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90 and SEQ ID NO: 91

Such a kit, by exploiting the ddPCR technique, allows to accurately evaluate the percentage of dd-cfDNA present in the recipient blood with performances comparable to those obtained with other technical-analytical approaches.

The method requires reduced analysis times compared to the NGS (Next Generation Sequencing) technology: only one day is required to obtain dd-cfDNA quantification as opposed to at least three days required for sequencing. Furthermore, the test can be performed immediately after the collection of the sample with the possibility of generating the report at the latest the day following the collection.

The costs of the analyses are contained and lower compared to NGS and allow the implementation thereof not only in a research context but above all in a hospital, where the patient must be monitored numerous times throughout the post-transplant period, according to the scheduling of established clinical controls.

Table 5: Comparison between NGS and the invention for the main technical characteristics, analysis times and costs:

¹ CareDX (Brisbane, CA 94005), data from the leading market competitor in posttransplant rejection monitoring. In this calculation, the time required to recruit the number of samples required to perform the library preparation and carry out the sequencing is not considered. In the case of ddPCR, the samples can also be analysed individually as soon as the blood has been drawn and the dd-cfDNA has been obtained.

In a third aspect the invention provides the use of the kit for diagnosing acute rejection in solid organ transplantation in a patient, for clinical application of the method according to the invention for monitoring post-transplant solid organ rejection.

Examples of embodiments of the present invention are given below for illustrative purposes.

EXAMPLES

Example 1

The first phase of the project involved the design of primers and probes (the first are sequences of nucleotides that serve to amplify specific DNA regions (primer sequences in Tables 1 and 2), while the second are sequences of nucleotides that were chemically modified to recognize genetic polymorphisms within the HLA-DRB1 and HLA-DQB1 regions, thus allowing differences to be highlighted (probe sequences in Table 3).

Specifically, the functioning of the primers was evaluated and confirmed by tests performed on different DNA samples, optimizing the different reaction conditions in order to improve their amplification capacity. Their specificity and sensitivity were tested using first genomic DNA, and then circulating DNA samples from patients who received an organ transplantation (Figures 2, 3 and 4). The reaction conditions (temperature, times, and reaction volumes) were optimised to reach a common protocol to be applied to all probes. At the end of the validation of primers and probes, we proceeded with the technical optimisation of the different steps of the molecular test, performing the analysis on serial samples from transplant patients. In this phase, the entire workflow was verified and optimised, from blood sampling to purification of circulating DNA to execution of the assay in ddPCR with consequent reporting (validation of assay performance and method diagnostic capabilities).

Table 6 shows the list of alleles of the HLA-DRB1 and HLA-DQB1 genes for which specific probes were designed which can be analysed by the diagnostic method according to the present invention. Table 6:

2: Extraction of total circulating DNA

and Tvoino of

cfDNA is extracted from plasma samples collected from patients receiving solid organ transplantation, after signing an informed consent. Sampling is carried out in conjunction with the execution of clinical and histological analyses carried out during the routine monitoring of the state of the transplant.

The donor and recipient HLA locus genes are typed during the normal pre-transplant diagnostic workup. The information relating to the alleles of the HLA-DRB1 and HLA- DQB1 genes of the two subjects is therefore available, which will be subsequently exploited in the dd-cfDNA quantification assay.

Example 3: Pre-amplification of total circulating DNA (cfDNA)

2 ng of cfDNA are pre-amplified using pairs of primers (forward and reverse) specifically selected according to the typing of the HLA-DRB1 and HLA-DQB1 alleles of the recipient and the donor. In detail, the first allele will be specific and indicative of the donor cfDNA quota, while the second allele will be indicative of the recipient cfDNA quota.

The primers used in this phase (the first pair of primers of step b. of the method according to the invention) are called external primers. The complete list of primers with their sequences and genetic targets is shown in Table 1 . The pre-amplification reaction consists of:

• 2 ng of cfDNA

• 100nM Forward primer

• 100nM Reverse primer

• 25 ul of PCR mix (containing polymerase enzyme, dNTPs and salts)

In a total of final 50uL

PCR amplification conditions are as follows: per Step 1 95° 3’

12

cycles

Step 4 4° Hold

Example 4: Preparation of Droplet digital PCR (ddPCR) reaction

1 ul of the previous pre-amplification reaction is used in the subsequent ddPCR amplification. In this phase, pairs of primers (forward and reverse) called internal primers are used (they correspond to the second pairs of primers of step c. of the method of the invention), specific for the alleles enriched in the previous reaction. Pairs of probes labelled with different coloured fluorophores (FAM and HEX) are also used, associated so as to be specific for donor and recipient. In each reaction, it will therefore be possible to distinguish the cfDNAs from the two subjects and their percentage contribution.

The sequences relating to the internal primers (second pair) and to the labelled probes, together with the indication of their specific targets, are set out in Tables 2 and 3. The probes contain chemical modifications called LNAs that allow for better stability and efficiency at high temperatures, thus ensuring greater specificity of each probe for its target. These modifications are indicated in Table 3 by a “+” symbol next to the nucleotide involved in the modification.

The ddPCR reaction consists of:

• 1 ul of pre-amplified cfDNA

• 250nM FAM probe (donor-specific)

• 250nM HEX probe (recipient-specific)

• 500nM recipient forward primer

• 500nM recipient reverse primer • 500nM donor forward primer

• 500nM donor reverse primer

• 10ul of ddPCR mix (containing polymerase enzyme, dNTPs and salts)

In a final volume of 21 ul.

The reactions containing the positive and negative controls are prepared following the same protocol. Positive controls consist of nucleotide sequences containing the genetic target, specific for each allele. The sequences relating to each control are detailed in Table 4. For each of the donor and recipient alleles amplified in the analysis reactions, amplification reactions of the positive controls containing 1 fg/ul of control DNA are prepared. Negative controls are prepared for each probe by adding 1 ul of water, instead of DNA, to the reaction volume.

Example 5: Generation of droplets

20ul of the reaction mix containing cfDNA, probes and primers are used to generate the oil droplets together with 70ul of oil for droplets (according to the droplet generation protocol using the Biorad instrumentation https://www.bio-rad.com/it-it/sku/17005227- qx200-droplet-generator?ID=17005227). 40ul of droplets are dispensed in a 96-well plate (https://www.bio-rad.com/it-it/sku/17005224-ddpcr-plates--96--well-semi- skirted?ID=17005224) and amplified according to the following amplification protocol: per Step 1 95° 3’

40 cycles

Step 5 72° 5’

Step 6 98° 10’

Step 7 4° 30’

Step 8 4° Hold

The amplification protocol is common for all probes and primers, except for the specific primers for the HLADRB1_14 allele, amplified at 59°C instead of 58°C (Step 4).

Data analysis and quantification of dd-cfDNA The plate reading is performed using a Droplet reader (https://www.bio--rad.com/it- i L/sku/17005228-qx200-droplet-reader?ID-17005228) following the Biorad instrument user manual.

The number of cfDNA copies relating to the donor and the recipient is identified through the analysis of the droplet profiles. The positivity threshold is positioned based on the signal of the wells containing the positive and negative controls (Figure 1 ). From the analysis of the fluorescence of each well, it is possible to obtain the number of copies/ul of cfDNA from the donor and the recipient present in the sample.

The cfDNA contribution of the donor is obtained through the formula:

Donor pairs

Receiver pairs after normalising the signal of each of the probes used versus the signal of its own positive control.

The specificity and sensitivity of each probe was tested using genomic and circulating DNA specific for each of the DR and DQ alleles.

From the detailed description and the Examples reported above, the advantages achieved by the method of the present invention are apparent. In particular, this method has proved to be surprisingly and advantageously suitable for the non-invasive diagnosis of acute rejection in solid organ transplantation through the quantification of circulating free DNA of the donor (dd-cfDNA) in the plasma of the recipient. At the same time, being fast and extremely easy to perform, this method can be conveniently implemented in any type of laboratory.

Claims

1 . A method for diagnosing acute rejection in solid organ transplantation in a patient, comprising the steps of: a. determining the phenotype of the HLA-DRB1 and HLA-DQB1 genes in a biological sample obtained from said patient and in a biological sample obtained from a donor to obtain the typing of the patient and the donor; b. pre-amplifying the circulating DNA (cfDNA) of the biological sample obtained from said patient, by using a first pair of primers specific for said patient and a first pair of primers specific for said donor, to obtain a pre-circulating amplified DNA; c. amplifying by digital droplet PCR (ddPCR) the pre-amplified circulating DNA sample of step b., by using a second pair of primers specific for said patient and a second pair of primers specific for said donor, wherein said second pairs of primers are labelled with a different coloured probe, to obtain a labelled amplified DNA; d. analysing the signal of the labelled primers of said patient and said donor, to assess whether acute rejection of the transplanted organ is in progress.

2. The method according to claim 1 , wherein the patient and donor primer pairs of step c. are labelled with fluorophores of different colours, preferably said fluorophores are HEX and FAM.

3. The method according to any one of claims 1 or 2, wherein said first pair of primers specific for said patient and said first pair of primers specific for said donor are different from each other and selected from the group consisting of:

SEQ ID NO: 1 and SEQ ID NO: 2;

SEQ ID NO: 3 and SEQ ID NO: 4;

SEQ ID NO: 5 and SEQ ID NO: 6;

SEQ ID NO: 7 and SEQ ID NO: 8;

SEQ ID NO: 9 and SEQ ID NO: 10;

SEQ ID NO: 1 1 and SEQ ID NO: 12;

SEQ ID NO: 13 and SEQ ID NO: 14;

SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; and

SEQ ID NO: 19 and SEQ ID NO: 20.

4. The method according to any one of claims 1 to 3, wherein said second pair of primers specific for said patient and said second pair of primers specific for said donor are different from each other and selected from the group consisting of:

SEQ ID NO: 21 and SEQ ID NO: 22;

SEQ ID NO: 21 and SEQ ID NO: 23;

SEQ ID NO: 24 and SEQ ID NO: 25;

SEQ ID NO: 26 and SEQ ID NO: 27;

SEQ ID NO: 28 and SEQ ID NO: 29;

SEQ ID NO: 30 and SEQ ID NO: 31 ;

SEQ ID NO: 32 and SEQ ID NO: 33;

SEQ ID NO: 34 and SEQ ID NO: 35;

SEQ ID NO: 36 and SEQ ID NO: 37;

SEQ ID NO: 38 and SEQ ID NO: 39;

SEQ ID NO: 40 and SEQ ID NO: 41 ; and

SEQ ID NO: 42 and SEQ ID NO: 43.

5. The method according to any one of claims 1 to 4, wherein said probes are selected from the group consisting of:

6. The method according to any one of claims 1 to 5, wherein specific positive controls for the probes are used, said positive controls are selected from the group consisting of: SEQ ID NO: 76; SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81 ; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90 and SEQ ID NO: 91.

7. A kit for diagnosing acute rejection in solid organ transplantation in a patient, comprising the pairs of primers selected from the group consisting of:

SEQ ID NO: 1 and SEQ ID NO: 2;

SEQ ID NO 3 and SEQ ID NO: 4;

SEQ ID NO 5 and SEQ ID NO: 6;

SEQ ID NO 7 and SEQ ID NO: 8;

SEQ ID NO 9 and SEQ ID NO: 10;

SEQ ID NO 1 1 and SEQ ID NO: 12;

SEQ ID NO 13 and SEQ ID NO: 14;

SEQ ID NO 15 and SEQ ID NO: 16;

SEQ ID NO 17 and SEQ ID NO: 18;

SEQ ID NO 19 and SEQ ID NO: 20;

SEQ ID NO 21 and SEQ ID NO: 22;

SEQ ID NO 21 and SEQ ID NO: 23;

SEQ ID NO 24 and SEQ ID NO: 25;

SEQ ID NO 26 and SEQ ID NO: 27;

SEQ ID NO 28 and SEQ ID NO: 29;

SEQ ID NO 30 and SEQ ID NO: 31 ;

SEQ ID NO 32 and SEQ ID NO: 33;

SEQ ID NO 34 and SEQ ID NO: 35;

SEQ ID NO 36 and SEQ ID NO: 37;

SEQ ID NO 38 and SEQ ID NO: 39;

SEQ ID NO 40 and SEQ ID NO: 41 ; and

SEQ ID NO 42 and SEQ ID NO: 43, the probes selected from the group consisting of:

SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 , SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQ

ID NO: 75, and instructions for use in the method according to any one of claims 1 to 6.

8. The kit according to claim 7, further comprising the specific positive controls for the probes selected from the group consisting of:

SEQ ID NO: 76; SEQ ID NO: 77; SEQ ID NO: 78; SEQ ID NO: 79; SEQ ID NO: 80; SEQ ID NO: 81 ; SEQ ID NO: 82; SEQ ID NO: 83; SEQ ID NO: 84; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90 and SEQ ID NO: 91.

9. Use of the kit according to any one of claims 7 or 8, for diagnosing acute rejection in solid organ transplantation in a patient.