DE102017214449B4 - Method and means for the diagnosis of tumors by detection and quantification of heterogeneously methylated epialleles - Google Patents

Abstract

The invention relates to methods and means for the diagnosis of tumors, in particular for early diagnosis and for distinguishing benign and malignant tumors by means of digital PCR, especially in Körperflüssigkeiten.Die invention is characterized in that not only a proof of fully methylated Epiallele, but also a proof and quantification of partially methylated epialles using sequence-specific probes in a digital PCR.

Description

The invention relates to methods and means for the diagnosis of tumors, in particular for the early diagnosis and for distinguishing benign and malignant tumors by means of digital PCR, in particular in body fluids.

For the prognosis of cancers, such as prostate and breast cancer as well as other solid tumors, the timing of the diagnosis is crucial. For example, 5- and 10-year survival rates are strongly dependent on the tumor stage in which the disease was detected. A late diagnosis of a tumor disease is often associated with metastases already occurring. For a timely diagnosis of malignant degeneration biomarkers are therefore required to indicate such degeneration with high diagnostic sensitivity and specificity early. The tumor markers hitherto known and used in clinical-chemical diagnostics have hitherto proven only for the follow-up of tumor patients after therapeutic interventions, but they do not allow, with a few exceptions, no use in the early diagnosis (screening or prevention) of tumor diseases. Here, the diagnostic sensitivities and specificities are still insufficient to distinguish with the help of these biomarkers tumor patients of healthy, especially in the early stages of development, certainly.

Tumor cells differ in sequence-dependent sequence-independent epigenetic changes in DNA, including hypermethylation. These tumor-specific changes in DNA methylation can be used as new cancer markers (eg WO2012007462A1 . WO2013064163A1 . US20130022974 . US20110301050 ).

The detection of altered patterns of heterogeneously methylated epialleles of selective DNA sections in blood, urine or other human body fluids or swabs and histological preparations is so far only qualitatively, but not quantitatively possible for routine diagnostics.

The widespread use of methyl-specific PCR for the study of gene methylation in the last decade has largely ignored the presence of heterogeneously methylated epialleles [1]. The later developed MS-HRM technique (MS-HRM stands for "methylation-sensitive high resolution melting") was able to detect heterogeneously methylated epialleles for the first time on the basis of complex melting curves of the amplified DNA [2]. However, this technique is unable to accurately identify and quantify each heterogenous methylated epiallele [1]. A further development of the MS-HRM technique relates to the digital modification of this technique [3], which provides for amplification by means of limited dilution followed by melting curve and sequencing analyzes of the amplification products in the individual reaction spaces. Disadvantage of this technique is the execution of the analysis in a limited number of available reaction spaces, eg in a 96-well format. This number is clearly too low compared to 20,000 to 10 ⁶ reaction spaces in the digital droplet PCR, which is the basis of the new technique described here, to comparable analytical sensitivity and reproducibility by the digital MS-HRM technique without substantially increased cost compared to the method described here.

New techniques such as New Generation Sequencing, Pyrosequencing, and SequenomMassArray are able to quantify heterogenous methylated epialleles, but these techniques involve high investment and investigation costs, and so far have not been included in routine diagnostic exams [1]. Also, these techniques had an analytical sensitivity of only 5-10% [4].

The recently described ligase-based method is also capable of quantifying heterogenomethylated epialleles [5, 6]. Here, however, the determination is influenced by the proportion of non-methylated alleles and leads to distorted results in the presence of high concentrations of non-methylated epialleles, as is the case in tumors, especially in the early stages [5, 6]. However, the diagnosis of tumors in early stages of development is of crucial importance, as already stated, to significantly improve the prognosis of tumors after appropriate therapies such as surgery, chemotherapy and radiotherapy. The influence of high concentrations of non-methylated epialles on the test results of ligase-based methods is therefore a great disadvantage. In contrast, the determination of heterogenously methylated epialleles using the method described here is not influenced by high concentrations of non-methylated epialleles, as no PCR bias occurs in the dPCR [7].

How important the establishment of new biomarkers and the provision of appropriate commercial test kits z. As for the diagnosis of prostate cancer (PCa), is shown by the high number (more than 150,000) in Germany alone annually performed prostate biopsies, which are indicated on the basis of PSA determinations and in which only in about 25% of investigated cases tumors are detected [8, 9]. Prostate biopsy is an invasive diagnostic procedure that involves approximately 5% of biopsies that require treatment, such as bleeding and inflammation [8, 9]. In addition, not all PCa are detected by means of a single tissue biopsy, so that repeat examinations are necessary, and a negative finding does not rule out the presence of a PCa [8, 9].

The object of the invention is to provide an improved method for the diagnosis of tumors and a kit for its implementation, which is particularly suitable for early diagnosis and for distinguishing benign and malignant tumors by means of PCR, in particular in body fluids.

Contents of the present invention is the description of a newly developed method, referred to herein as epiallele-sensitive digital PCR (EAST-dPCR) method, with the aid of tumor-specific heterogenous methylated epialleles in serum, plasma or urine, for example. After digital-rectal examination (DRU) can be identified and quantified and helps to reduce the number of unnecessary biopsies significantly. In addition, when tissue biopsy was required, the diagnostic sensitivity and specificity, and thus the accuracy, of malignant changes to the sole histopathological examinations can be significantly improved in the punch biopsies taken, thereby also reducing unnecessary repetition of tissue biopsies.

1. a) Bisulfit-Umwandlung der DNA in der Probe (Umwandlung nicht-methylierter Cytosine in Uracil),
2. b) Identifizierung und Quantifizierung heterogen methylierter Epiallele mittels digitaler PCR (dPCR), wobei ein Set an bevorzugt unterschiedlichen fluoreszenzmarkierten Sonden zum Einsatz kommen, deren Sequenzen jeweils komplementär zu den zu untersuchenden Epiallelen sind. Die Sonden können mit unterschiedlichen Fluorochromen markiert werden, die eine parallele Quantifizierung, d.h. in ein und demselben Ansatz, ermöglicht. Auf diese Weise können auch Epiallele mit 4, 5, 6 oder mehr CpG-Stellen, die in 16, 32, 64 und entsprechend noch mehr EpiallelVarianten vorliegen können, durch ein Hochdurchsatz-Verfahren (High-Throughput-Screening) kostengünstig quantifiziert werden.

The object is achieved by a method for diagnosing a tumor disease according to claim 1 in an isolated sample with the steps

1. a) bisulfite conversion of the DNA in the sample (conversion of non-methylated cytosines into uracil),
2. b) Identification and quantification of heterogeneously methylated epialleles by means of digital PCR (dPCR), using a set of preferably different fluorescently labeled probes whose sequences are each complementary to the epialleles to be investigated. The probes can be labeled with different fluorochromes, which allows a parallel quantification, ie in one and the same approach. In this way epialles with 4, 5, 6 or more CpG sites, which can be present in 16, 32, 64 and correspondingly even more epiallelic variants, can be inexpensively quantified by a high-throughput screening.

The present invention involves the use of a set of different fluorescently labeled probes in the digital PCR, whereby highly circulating tumor DNA based on methylated sequences in patient samples such as serum, plasma, urine, cerebrospinal fluid, sputum, bronchial lavage, sperm fluid or mammary secretions as so-called liquid biopsy can be selectively detected.

Heterogeneously methylated epialleles within the meaning of the invention are understood as meaning partially methylated epialleles. Not only a detection of completely methylated and unmethylated epialleles, but also multiple partially methylated epialels, which differ in their patterning of methylated and unmethylated CpG sites, is advantageously carried out in the invention. Ie. the partially methylated epialels each contain multiple CpG sites that are partially methylated and partially unmethylated, with each epiallel (i.e., each epiallelic variant) having a different methylation pattern.

An epiallel in the sense of the invention is thus an epigenetic variant of a sequence section in a genome, in particular a gene section but also a sequence section which contains regulatory elements, wherein each epiallel of a sequence section differs only in the methylation pattern as described above, d. H. otherwise has an identical sequence. The terms "target gene" (target of interest) or target sequence used synonymously below also include regulatory sequences that lie outside an ORF (open reading frame).

The basic idea of the invention is not only to construct and insert into the dPCR probes which are complementary to fully methylated and unmethylated DNA fragments, but additionally to construct and employ a set of probes complementary to heterogeneously methylated DNA fragments. Sequences are. In this way, the full spectrum of potential tumor DNA epialleles in liquid biopsy is specifically identified and quantified using dPCR. This makes it much easier to differentiate early malignant changes from benign changes, thereby improving the diagnostic Sensitivity (by identifying early forms of malignant changes) and diagnostic specificity (by quantifying only tumor-specific DNA) can be significantly increased. For example, it was found in studies that under appropriate conditions in the dPCR, the fluorescence signals that occur due to mismatching of the probes with heterogenously methylated epialleles, compared to the signals in complementary hybridization to significantly reduced signal amplitudes, a distinction between nonspecific and allowed specific signals, as shown in the following explanations.

The process according to the invention is advantageously much more sensitive than the methods known from the prior art for the quantification of heterogeneously methylated epialleles. The analytical sensitivity of the novel technique according to the invention is below 1%.

In the present sketch ( 1 ) the principle of EAST-dPCR is shown. In order to be able to identify and quantify heterogeneously methylated DNA fragments in addition to fully methylated and unmethylated epialleles, which arise in particular in early developmental stages of malignant diseases and are therefore of great relevance for the early diagnosis of tumor diseases, the construction of additional probes with complementary sequences intended for Epiallele. The number of probes required depends on the number of CpG sites to be studied. According to calculation formula 2 ⁿ , in addition to two probes for fully methylated and unmethylated epialleles, two further probes for heterogeneously methylated epialleles result in 2 CpG sites to be investigated; For 3 CpG sites, in addition to two probes for homogeneously methylated and non-methylated epialleles, 6 additional probes for heterogeneously methylated epialleles (see 1 ), for 4 CpG sites correspondingly two probes for homogeneously methylated and unmethylated epialles and 14 further probes for heterogeneously methylated epialleles (see 16 ) and so on.

Advantageously, it has been shown that in the method according to the invention the quantification of methylated sequences is also possible in samples in which a high background DNA (non-methylated DNA) is present. This does not interfere in the process according to the invention. Thus, the inventive method is also suitable for the analysis of tumor DNA (free circulating tumor DNA - fc tumor DNA) in body fluids, smears or tissue samples.

1. a) Bisulfit-Umwandlung der DNA in der Probe,
2. b) Vervielfältigung eines teilweise (heterogen) methylierten Sequenzabschnittes eines Genoms mittels digitaler PCR, wobei in der digitalen PCR ein Set an fluoreszenzmarkierten Sonden eingesetzt wird, das sowohl Sonden mit komplementären Sequenzen zu dem vollständig methylierten Epiallel und dem unmethylierten Epilallel des Sequenzabschnittes als auch Sonden mit komplementären Sequenzen zu den teilweise methylierten Epiallelen des Sequenzabschnittes enthält, wodurch eine Identifizierung und/oder Quantifizierung der vollständig methylierten und unmethylierten als auch teilweise methylierten Epiallelen des Sequenzabschnittes erfolgt.

The method for diagnosing a tumor disease in an isolated sample comprises the following steps:

1. a) bisulfite conversion of the DNA in the sample,
2. b) amplification of a partially (heterogeneously) methylated sequence portion of a genome by means of digital PCR, wherein in the digital PCR, a set of fluorescently labeled probes is used, both probes with complementary sequences to the fully methylated epiallel and the unmethylated epilallel of the sequence portion and probes with contains complementary sequences to the partially methylated epialleles of the sequence section, whereby an identification and / or quantification of fully methylated and unmethylated as well as partially methylated epialleles of the sequence section is carried out.

In step b), a quantification is carried out by means of digital PCR - also referred to below as dPCR. In the known methods of digital PCR, a limiting dilution of the DNA used is such that in a maximum number of compartments there is no or exactly one DNA molecule (Poisson distribution).

In one embodiment, the identification and quantification of heterogeneously methylated epialleles is preceded by a pre-amplification (step b ') in which either the methylated DNA sequences as well as the unmethylated DNA sequences are amplified if the original epiallelic pattern to be examined does not change Alternatively, the methylated DNA sequences (if they are tumor-specific) should preferably be amplified over non-methylated DNA sequences or the non-methylated DNA sequences (if they are tumor-specific) should be amplified more than the methylated ones DNA sequences to detect tumor DNA epitopes with high analytical and diagnostic sensitivity.

The combination of steps b) and b ') advantageously achieves significantly higher analytical and diagnostic sensitivities. On the other hand, the method according to the invention surprisingly allows a much more reliable statement as to whether a malignant tumor is present or not. This makes the procedure suitable for early detection screening (prevention). Next advantageous allows the invention Method a distinction of benign (benign) from malignant (malignant) tumors and the detection of a minimal residual disease (MRD) and thus the therapy monitoring and follow-up. The principle of the method according to the invention is based on embodiments in 1 summarized.

The higher the proportion of specific heterogeneously methylated epialleles, the higher the likelihood that a malignant tumor disease, especially at an advanced stage, will be present.

By step b) a methylation pattern (epiallelic pattern) is advantageously obtained, which provides both information about the proportion of fully methylated and unmethylated epiallelic as well as the proportion of only partially methylated epiallelic.

A comparison of the methylation data obtained in step b) (methylation pattern) with 1st data from samples of healthy tissue and / or data of benign tumors (negative comparative data) and Tumors (positive comparative data). The negative and positive comparison data are preferably obtained beforehand by performing steps a) and b). Alternatively or additionally, samples of healthy tissue and / or data of benign tumors or corresponding cells or cell lines are included as a negative control and / or samples of malignant tumors or corresponding cells or cell lines as a positive control in the method according to the invention.

The individual steps of the method and preferred embodiments are explained in more detail below:

Prior to step a), DNA isolation (step a ') is preferably carried out by known methods. The implementation of bisulfite conversion is also known to the person skilled in the art. For both steps, the expert can use commercially available kits.

The isolated sample can in principle be a tissue sample. Advantageously, however, the method of the invention is particularly suitable for the detection of methylated tumor DNA in body fluids ("liquid biopsy"), such as. Whole blood, serum, plasma, urine, cerebrospinal fluid, sputum, bronchial lavage, sperm fluid, mammary gland secretions, vaginal secretions (smears) or lymph, most preferably serum, plasma or urine.

In step b), the heterogenously methylated epiallel is quantified by means of probes which in each case hybridize in a complementary manner to the corresponding epiallele without pre-amplification or after pre-amplification (step b ').

Depending on the number of 5'-CG-3 'dinucleotides to be included in the investigations, the number of probes required is calculated according to the formula 2 ⁿ . For example, in addition to two probes for fully methylated and unmethylated epialleles, two additional probes for heterogeneously methylated epialleles are obtained at 2 CpG sites to be investigated. For 3 CpG sites, in addition to two probes for homogeneously methylated and unmethylated epialles, 6 additional probes for heterogeneously methylated epialleles (see 1 ), 4 probes for heterogeneously methylated epialleles (see 16 In addition to two probes for homogeneously methylated and unmethylated epialles, 30 additional probes for heterogeneously methylated epialleles etc. are required for 5 CpG sites.

Alternatively, the probes can be used to detect the minus strand (also complementary strand or complementary strand) in the amplified DNA double-stranded molecule. This is done separately from the probes for the plus strand (also called coding strand) For the detection of the complementary strand, the probes for the non-methylated DNA sequences have 5'-TG-3 'dinucleotides (instead of the 5'). The number of 5'-CG-3 'dinucleotides in the probes for the methylated DNA sequences preferably corresponds to the number of 5'-CA-3' dinucleotides (or 5'-TG -3 'dinucleotides) in the probes for the non-methylated DNA sequences of the same target gene.

The digital PCR is otherwise performed and quantified according to common methods. The compartments are preferably oil droplets (droplet digital PCR - ddPCR). Alternatively preferably, the compartments are arranged on a chip. In the digital PCR, however, preferably more than one amplification cycle is carried out, preferably at least 5, preferably at least 15, more preferably 20 to 50, particularly preferably 30 to 45, more preferably up to 40. For quantification, the compartments in which an amplification has taken place and with which the probes hybridize for the methylated or non-methylated DNA sequences. The primers used for the dPCR can in each case be the same primers as used for the pre-amplification before step b). Alternatively and preferably, nested primers are chosen which bind to other sites of the pre-amplified sequence. A nested PCR can further increase the specificity.

A digital PCR (dPCR) within the meaning of the invention thus means a PCR in a large number of separate compartments, preferably with a volume in the femtoliter range or in the nanoscale. The dPCR is characterized by the fact that the quantification of the compartments follows a digital result (amplification: yes or no). By counting a large number of reaction compartments (high throughput screening with preferably 10,000 to 100,000 compartments per PCR) a statistical significance is achieved. The proportion of reaction chambers with successful amplification is proportional to the amount of DNA used in the amplified DNA sequence, which is used for quantification.

The probes are preferably fluorescently labeled, with the probes for methylated and non-methylated sequences preferably being provided with different fluorescence markers. The fluorescent dye is preferably attached to one end of the probe (preferably the 5 'end). At the other end there is preferably a quencher suitable for the fluorescent dye which suppresses the fluorescence signal. By hybridization or after hybridization and subsequent polymerase effect with the amplified DNA, the quenching effect is abolished and the fluorescence signal detectable. Such fluorescent dyes and quenchers are well known to those skilled in the art and commercially available for any probe sequences.

If appropriate multi-color fluorescence detection systems are available, the digital PCR is preferably also performed as a multiplex PCR. For quantification, different colored probes are preferably used for each amplified target gene. Alternatively, the probes are inserted and evaluated in separate batches (wells) in the dPCR.

If several probes are used per target gene, these can have the same fluorescent dyes and the signal can be integrated. Alternatively, the two alternatives described above for the multiplex PCR are used.

In one embodiment of the method according to the invention, prior to step b), methylated and corresponding non-methylated DNA sequences of the same gene segment are either pre-amplified by PCR so that the methylated DNA sequences as well as the non-methylated DNA sequences (without PCR bias ) are amplified if the original Epiallelmuster to be examined is not to be changed or the methylated DNA sequences are amplified more than the non-methylated DNA sequences (by PCR bias), when the tumor DNA Epiallele are present in very low concentration and these should be detected with high analytical and diagnostic sensitivity.

Preferably, the PCR conditions (in particular primer sequences, magnesium concentration, annealing temperature and in particular also the number of cycles) are adjusted so that no bias in favor of the methylated DNA sequences in the pre-amplification occurs.

The method according to the invention is applicable in principle for each target gene to be investigated (target of interest). Preferably, in the method according to the invention, DNA sequences of three to five, preferably up to six, target genes are analyzed with regard to the methylation pattern of the heterogeneously methylated epiallelic. To this end, step b) is preferably carried out as a multiplex PCR, ie the probes are used simultaneously in the dPCR, depending on the possible fluorescence channels of the subsequent evaluation measuring devices in a set. The annealing temperature in the dPCR is chosen as a function of the MgCl ₂ concentration used so that the signals can be completely distinguished from one another by complementary hybridization of the signals by mismatching.

Depending on the number of simultaneously measurable fluorescences, the set consists of 2 probes (1 fluorescently labeled probe for methylated and 1 probe labeled with another fluorescent dye for the non-methylated epiallel) for a duplex dPCR or the set consists of several probes labeled with different fluorescent dyes for heterogeneously methylated epiallelic and simultaneously labeled with a different fluorescent dye probe for the non-methylated epiallel for a multiplex dPCR. The number of probes in each set depends on the number of simultaneously measurable fluorescent dyes. The number of sets with the different probes for heterogeneously methylated epialleles depends on the number of CpG sites and the resulting number of possible epiallelic variants to be determined.

The higher the proportion of fully and heterogenously methylated sequences (especially if they are specific for tumor DNA), the higher the likelihood of advanced malignant tumor disease. If non-methylated sequences are tumor-specific, the opposite is true.

As tumor patients, patients are preferably classified which have elevated values for fully methylated or heterogenously methylated epi-alleles for at least one, preferably two, or more epiallelic for at least one, preferably two, or even more target genes.

1. i) Primer für die dPCR, welche sowohl vollständig methylierte und nicht-methylierte als auch heterogen methylierte Epiallele amplifizieren, d. h. ein Primerpaar, welches so gewählt ist, dass es die Amplifikation des gesamten Spektrums der möglichen heterogen methylierten und vollständig methylierten und nicht-methylierten Epiallele ermöglicht,
2. ii) Sonden zur Quantifizierung der heterogen methylierten und vollständig methylierten und nicht-methylierten DNA-Sequenzen der Zielgensequenzen, bevorzugt wie oben beschrieben jeweils mit einem Fluoreszenzfarbstoff und einem Quencher.

The object is further solved by a kit for diagnosing a tumor disease in an isolated sample comprising:

1. i) primers for the dPCR which amplify both fully methylated and unmethylated as well as heterogeneously methylated epialles, ie a primer pair chosen to amplify the entire spectrum of possible heterogeneously methylated and fully methylated and unmethylated epialles allows
2. ii) probes for quantifying the heterogenously methylated and fully methylated and unmethylated DNA sequences of the target gene sequences, preferably as described above, each with a fluorescent dye and a quencher.

• iii) Nested Primer für die Prä-Amplifikation zur Amplifikation von methylierten und nicht-methylierten Zielgensequenzen, deren Epiallele in der nachfolgenden dPCR quantifiziert werden sollen,

The primers used for the digital PCR are either identical to those of the pre-amplification. Alternatively and preferably, the kit additionally contains:

• iii) nested primers for the pre-amplification for the amplification of methylated and unmethylated target gene sequences whose epialleles are to be quantified in the following dPCR,

These additional primers are preferably used as extrinsic primers when intrinsic primers (nested primers) were used in the dPCR.

• iv) Reaktionspuffer für die Bias-freie PCR-Prä-Amplifikation, bevorzugt mit einer Magnesiumchloridkonzentration von 0,5 bis 15,0 mmol/l, bevorzugt 1,5 bis 8 mmol/l, weiter bevorzugt bis 3,5 mmol/l Endkonzentration, besonders bevorzugt 2,5 bis 3,5 mmol/l, oder alternativ einen Standard PCR Puffer und eine konzentrierte Magnesiumlösung,
• v) Reaktionspuffer für die dPCR bevorzugt mit einer Magnesiumchloridkonzentration von 0,5 bis 15,0 mmol/l, bevorzugt 1,5 bis 8 mmol/l, weiter bevorzugt bis 3,5 mmol/l Endkonzentration, besonders bevorzugt 2,5 bis 3,5 mmol/l, oder alternativ einen Standard PCR Puffer und eine konzentrierte Magnesiumlösung
• vi) Desoxyribonukleotid-Mix
• vii) eine DNA-Polymerase, wie z. B. HotStarTaq Plus,
• viii) Kontroll-DNA, bevorzugt neben vollständig methylierten und nicht-methylierten Sequenzen, DNA-Fragmente mit für alle heterogen methylierten Epiallele-Varianten komplementären Sequenzen,
• ix) Gebrauchsanweisung u.a. mit der in Abhängigkeit der im Reaktionspuffer für die dPCR verwendeten MgCl₂-Konzentration entsprechend einzustellende Annealingtemperatur für die jeweiligen Sets an Sonden und ggf. Computersoftware zur Auswertung.

The kit preferably contains further constituents selected from:

• iv) Reaction buffer for the bias-free PCR pre-amplification, preferably with a magnesium chloride concentration of 0.5 to 15.0 mmol / l, preferably 1.5 to 8 mmol / l, more preferably to 3.5 mmol / l final concentration , more preferably 2.5 to 3.5 mmol / l, or alternatively a standard PCR buffer and a concentrated magnesium solution,
• v) reaction buffer for the dPCR is preferred with a magnesium chloride concentration of 0.5 to 15.0 mmol / l, preferably 1.5 to 8 mmol / l, more preferably to 3.5 mmol / l final concentration, particularly preferably 2.5 to 3 , 5 mmol / l, or alternatively a standard PCR buffer and a concentrated magnesium solution
• vi) Deoxyribonucleotide mix
• vii) a DNA polymerase, such as. HotStarTaq Plus,
• viii) control DNA, preferably in addition to fully methylated and unmethylated sequences, DNA fragments with sequences complementary to all heterogeneously methylated epiallele variants,
• ix) Instructions for use, inter alia, with the annealing temperature to be set correspondingly for the respective sets of probes and possibly computer software for evaluation, depending on the MgCl ₂ concentration used in the reaction buffer for the dPCR.

Preferably, the primers and probes, as well as the control DNA for the kit are selected as described above for the method.

Preferred by the reaction buffer for the dPCR with a magnesium chloride concentration of 0.5 to 15.0 mmol / l, preferably 1.5 to 8 mmol / l, more preferably to 3.5 mmol / l final concentration, particularly preferably 2.5 to 3 , 5 mmol / l, there is preferably a selective annealing temperature in the dPCR for maximum separation of specific and nonspecific fluorescence signals resulting from mismatching.

As control DNA for the method or kit according to the invention, DNA or artificially produced DNA fragments isolated from primary cells or cell lines (eg by oligonucleotide-directed mutagenesis or chemical oligonucleotide synthesis) are preferably used.

Preferably, the non-methylated control (negative control) used is DNA from cells or cell lines in which the target sequences are not methylated. More preferably, DNA from epithelial cells of the healthy tissue corresponding to the tumor is used as the non-methylated DNA control. For example, DNA from human prostate epithelial cells (PrEC) or human mammary epithelial cells (HMEC) is used as the unmethylated control for the diagnosis of prostate or breast cancer. As a negative control for the detection of breast cancer, DNA from MCF10A cell lines is alternatively used.

As a methylated control (positive control) for the detection of prostate carcinomas DNA for each target gene is preferably selected from a cell line such as the LNCaP, PC-3 and DU-145 cell lines in which the target sequence of the epiallel to be examined is present in high concentration.

The invention also relates to the use of the kit for carrying out the method according to the invention.

The term tumor diagnosis within the meaning of the invention includes in particular the early detection screening (screening), the prognosis, the course diagnostics, therapy control and the detection of MRD.

The detection of free-circulating tumor DNA with the aid of the EAST-dPCR can be used in principle for the diagnosis of any solid tumor, especially if in body fluids or smears ("liquid biopsies") corresponding target gene sequences (target of interest) occur.

The method and kit according to the invention is particularly suitable for the diagnosis of malignant tumors, such as prostate, mammary, renal cell and urinary tract and bladder, pancreatic, testicular, oral and pharyngeal, esophageal, laryngeal and thyroid glands -, gastric, esophageal, intestinal (especially colon and rectal), lung, ovarian, cervical and uterine carcinomas and gall bladder carcinomas, malignant melanoma of the skin, astrocytoma, glioblastoma, neuroblastoma, but also for diagnosis non-Hodgkin's and Hodgkin's lymphomas, skin, CNS, GI tract, gastric lymphoma and intestinal lymphoma and leukemia.

A preferred application of the method and kit consists in the general early detection (independent screening or prevention) of malignant diseases (which includes the above-mentioned tumor entities), but especially in combination with the PSA determination and the indication for tissue biopsy at elevated PSA levels and the diagnosis of a prostate carcinoma or in combination with the mammography and suspected findings in the diagnosis of breast cancer or in combination with the presence of a gene mutation with increased familial risk for breast and ovarian carcinoma and the decision for a prophylactic mastectomy and / or ovariectomy.

If a tumor disease has been diagnosed, the determination of tumor-specific epiallelic patterns using EAST-dPCR after surgery, chemo- or radiological therapy can control the course of the disease and diagnose or rule out the presence of a minimal residual disease (MRD). If no tumor-specific heterogenous methylated epialleles can be detected after therapy, a good response to the therapy can be assumed and MRD can be ruled out. If tumor-specific epialles are still detectable, this can be the reason for therapy optimization. If tumor DNA can be detected in the course, after this was not the case, a recurrence is expected, which is the reason for a renewed optimized therapy.

Advantageously, the method and kit according to the invention are particularly suitable for the differential diagnosis of benign diseases and malignant tumor diseases. The invention is particularly suitable for the differential diagnosis of benign prostatic hyperplasia (s), prostatitis and prostate carcinomas, in particular if, according to the prior art, a prostate tissue biopsy is indicated due to increased PSA values. By the determination according to the invention of the heterogenously methylated epialleles of the target genes to be investigated, EAST-dPCR can be used to determine to what extent fcT-DNA can be detected in serum, plasma, urine and / or seminal fluid. Based on the determined heterogenous methylated epiallelic patterns, a distinction can be made between benign prostatic hyperplasia, prostatitis and prostate cancer, so that in many cases unnecessary prostate tissue biopsy and surgery can be avoided. Can be z. B. in a serum or urine sample using the EAST dPCR no tumor DNA can find another PSA determination (eg after 3 or 6 months) is awaited. If, on the other hand, tumor DNA can be detected in the samples, the indication for tissue biopsy must be strengthened.

Advantageously, the method and kit according to the invention is also suitable for the differential diagnosis of breast carcinomas, in particular if tissue biopsy is indicated on the basis of suspicious mammographic findings according to the prior art. By the determination according to the invention of the heterogenously methylated epialleles of the target genes to be investigated, EAST-dPCR can be used to determine the extent to which fcT DNA can be detected in serum, plasma, urine and / or breast secretions. Based on the determined heterogenously methylated epiallelic patterns, a distinction can be made between a benign microcalcification or cyst and a breast carcinoma, so that tissue biopsy and surgery can be avoided in many cases in the case of false-positive mammographic screening findings. About two-thirds of all women starting annual mammography screening at the age of 40 experience false-positive results in the first ten years. At 7 percent there is an unnecessary biopsy [10]. On the other hand, false-negative findings in mammography screening can be reduced by the method according to the invention and corresponding kits.

The method and kit according to the invention is also suitable for the differential diagnosis of ovarian and cervical carcinomas, especially if the question of a benign change such as cysts or a carcinoma of the ovaries or uterus is due to suspicious ultrasound findings and according to the prior art further invasive diagnostic Procedures should be used.

Furthermore, the method and kit of the invention is in the presence of a pathological gene mutation with an increased familial risk for mammary and ovarian carcinomas, e.g. in the BRCA1 and BRCA2 gene, as a further aid to decision making for prophylactic mastectomy and / or ovariectomy, especially if the family planning of the affected patients has not yet ended.

The method according to the invention opens up new possibilities in the diagnosis of tumor diseases, in particular in the early diagnosis, since in this way individual fc tumor DNA copies corresponding to the large background of normal wild-type DNA in blood, urine or other biological samples are correspondingly (specifically) duplicated, before quantification in the dPCR follows.

Although a bias that can not be completely eliminated changes the original relationship between the target gene (methylated DNA fragments as a sign of malignant degeneration) and the normal wild-type DNA (non-methylated DNA fragments) due to pre-amplification Help from the EAST-dPCR data for a prognosis assessment (tumor burden), response to treatment (decrease or constancy of the fcT-DNA), residual disease (minimal residual disease) and the follow-up of cancer patients (relapse due to recovery of fcT-) DNA) are used. The basis for this is that in the method according to the invention the degree of bias can be made variable by the choice of annealing temperature, magnesium concentration in the reaction buffer and number of cycles and the bias under constant conditions between the individual samples to be examined the same size and thus a relative quantification is possible.

In one embodiment of the invention, the kit contains the primers according to SEQ ID Nos. 25 and 26. In the method according to the invention, these primers are used in the dPCR and preferably also for preamplification.

In a preferred embodiment of the invention, the kit contains probes selected from SEQ ID Nos. 1 to 24 (see also 1B and 16 ). In the method according to the invention, these probes are used in the dPCR for the quantification of the epialleles of PLA2R1. These probes are therefore particularly suitable for the diagnosis, differential diagnosis and early detection of prostate cancer and breast cancer. The inventors have found that not only the signal obtained with the probe PLA2R1-3 (SEQ ID NO: 7) for completely (triply) methylated PLA2R1 DNA fragment (methylation in positions 4, 5 and 6), but also the in each case with the probes PLA2R1-2a and PLA2R1-2b (SEQ ID Nos. 4 and 5) for partially (twice) methylated PLA2R1 fragments (methylation in positions 4 and 5 or 5 and 6) signal obtained in malignant tumors (in comparison to healthy or benign tumors) was significantly increased. However, the signal obtained with the PLA2R1-2c probe (SEQ ID NO: 6) for the partially (doubly) methylated PLA2R1 (methylation in positions 4 and 6) was not increased in malignant tumors. The signal obtained with probes PLA2R1-1a, 1b, 1c (SEQ ID NOs: 1 to 3) for partially (simply) methylated PLA2R1 (methylation in positions 4, 5 or 6) was as high in normal and benign cells as it was in malignant tumors.

Ie. a raised signal obtained in relation to negative comparison data or a negative control with probes selected from SEQ ID Nos. 4, 5 and 7 (PLA2R1-2a, PLA2R1-2b and PLA2R1-3) respectively indicates the presence of a malignant tumor. Negative and positive controls are preferably selected as described above for the detection of prostate cancer.

In accordance with the use of the PLA2R1 probes for 3 CpG sites to be investigated, probes PLA2R1-1 to PLA2R1-14, PLA2R1-m, PLA2R1-un (SEQ ID NO: 1) can be tested in the study of 4 CpG sites and the corresponding 16 possible epialleles 9 to 24). Thus, the studies on the probes PLA2R1-m and PLA2R1-un (SEQ ID Nos. 23 and 24) have shown that at an annealing temperature of, for example. B. 50.7 ° C, a specific FAM signal with high amplitude was generated at the same time high HEX signal amplitude. In this way, the further probes PLA2R1-1-14 (SEQ ID Nos. 9-22) can be used under the same reaction conditions as the probes PLA2R1-m and PLA2R1-un (at the same annealing temperatures and magnesium concentrations) and the signals due to Mismatching can be specifically differentiated from the specific signals due to the significantly reduced signal amplitudes even when using probes with 4 CpG sites.

As far as the term "embodiment" is used above and below in the text, it is to be understood that individual embodiments can be combined with each other.

Based on the following figures and examples, the invention is explained in more detail without limiting this.

• 1: Schematische Illustration der Lokalisation (A) und Design (B) der fluoreszenzmarkierten Sonden zur Identifikation und Quantifizierung heterogen methylierter PLA2R1-Epiallele in der epiallele-sensitive digital PCR (EAST-dPCR). Die schwarz ausgefüllten Kreise in (B) zeigen methylierte, die offenen Kreise nicht-methylierte CpG-Stellen (Position 4: -547 bp, Position 5: -549bp und Position 6: -552bp upstream vom Transkriptionsstartpunkt [TSS] in (A)). Das Prinzip der EAST-dPCR kann auf alle potentiell geeigneten Gensequenzen (targets of interest) für die Etablierung neuer Biomarker angewendet werden.
• 2 und 3: 2D-Projektion von FAM- (positiv für methylierte PLA2R1-DNA-Fragmente) und HEX-Signalen (positiv für nicht-methylierte PLA2R1-DNA-Fragmente) in DNA-Proben aus Blutleukozyten (linke Seite, E188) und Knochenmarkzellen (rechte Seite, K189) nach 40 Zyklen in der ddPCR. FAM-positive Signale (methylierte DNA) sind eingekreist. Rechts der Grafiken sind jeweils die verwendeten Sonden (PLA2R1-1a - PLA2R1-3) dargestellt, deren Sequenzen in der Tabelle in 1B gezeigt sind. Die DNA-Proben wurden jeweils in Dreifachbestimmungen analysiert und die Daten der Untersuchungen sind gemeinsam dargestellt. Schwarz eingekreist sind jeweils die FAM-positiven Signale.
• 4: 2D-Projektion von FAM-Signalen (positiv für methylierte PLA2R1-DNA-Fragmente) und HEX-Signalen (positiv für nicht-methylierte PLA2R1-DNA-Fragmente) in DNA-Proben aus normalen Epithelzellen der Prostata (PrEC) nach 40 Zyklen in der ddPCR. FAM-positive Signale (methylierte DNA) sind eingekreist. Die verwendeten Sonden (PLA2R1-1a - PLA2R1-3) sind oberhalb der Grafiken angezeigt, deren Sequenzen in der Tabelle der 1B zusammengefasst sind.
• 5: 2D-Projektion von FAM-Signalen (positiv für methylierte PLA2R1-DNA-Fragmente) und HEX-Signalen (positiv für nicht-methylierte PLA2R1-DNA-Fragmente) in DNA-Proben aus benignen Hyperplasiezellen der Prostata (BPH-1) nach 40 Zyklen in der ddPCR. FAM-positive Signale (methylierte DNA) sind eingekreist. Die verwendeten Sonden (PLA2R1-1a - PLA2R1-3) sind oberhalb der Grafiken angezeigt, deren Sequenzen in der Tabelle der 1B zusammengefasst sind.
• 6: 2D-Projektion von FAM-Signalen (positiv für methylierte PLA2R1-DNA-Fragmente) und HEX-Signalen (positiv für nicht-methylierte PLA2R1-DNA-Fragmente) in DNA-Proben aus LNCaP-Prostatatumorzellen nach 40 Zyklen in der ddPCR. FAM-positive Signale (methylierte DNA) sind eingekreist. Die verwendeten Sonden (PLA2R1-1a - PLA2R1-3) sind oberhalb der Grafiken angezeigt, deren Sequenzen in der Tabelle der 1B zusammengefasst sind.
• 7: 2D-Projektion von FAM-Signalen (positiv für methylierte PLA2R1-DNA-Fragmente) und HEX-Signalen (positiv für nicht-methylierte PLA2R1-DNA-Fragmente) in DNA-Proben aus PC-3-Prostatatumorzellen nach 40 Zyklen in der ddPCR. FAM-positive Signale (methylierte DNA) sind eingekreist. Die verwendeten Sonden (PLA2R1-1a - PLA2R1-3) sind oberhalb der Grafiken angezeigt, deren Sequenzen in der Tabelle der 1B zusammengefasst sind.
• 8: 2D-Projektion von FAM-Signalen (positiv für methylierte PLA2R1-DNA-Fragmente) und HEX-Signalen (positiv für nicht-methylierte PLA2R1-DNA-Fragmente) in DNA-Proben aus DU 145-Prostatatumorzellen nach 40 Zyklen in der ddPCR. FAM-positive Signale (methylierte DNA) sind eingekreist. Die verwendeten Sonden (PLA2R1-1a - PLA2R1-3) sind oberhalb der Grafiken angezeigt, deren Sequenzen in der Tabelle der 1B zusammengefasst sind.
• 9 und 10: Quantifizierung der FAM-Signale (positiv für methylierte PLA2R1-Epiallele) und HEX-Signale (positiv für nicht-methylierte PLA2R1-Epiallele) in DNA-Proben aus normalen Epithelzellen der Prostata (PrEC), benigner Prostatahyperplasiezelllinie (BPH) und den malignen LNCaP (LNCa), PC-3 (PC-3) und DU-145 (DU) Prostatazelllinien nach 40 Zyklen in der ddPCR. Die Grenzen (cut-off bzw. thresholds) sind eingezeichnet, die verwendeten Sonden sind jeweils oberhalb der Grafiken beschrieben. Die Pfeile zeigen FAM-Signale mit niedriger Amplitude, die Folge eines Mismatching der Sonden mit Epiallelen sind. Die Untersuchungen wurden in Doppelbestimmung durchgeführt. NTC, Negativkontrolle ohne DNA-Template.
• 11: Konstellationen, die durch Mismatching der 8 Sonden auf Grund eines einzelnen Nukleotidunterschiedes mit den Epiallelen möglich sind. (A-H) zeigen jeweils die Sonden (groß eingerahmt) und deren mögliche Mismatching-Hybridisierungspartner, die neben der Sonde (jeweils klein eingerahmt) gekennzeichnet sind. Die schwarz ausgefüllten Kreise stellen die CpG-Stellen dar, die methyliert vorliegen, die offenen Kreise stellen die CpG-Stellen in den Epiallelen dar, die nicht-methyliert vorliegen. Die Sterne weisen auf ein Mismatching hin, indem durch unterschiedliche Methylierungen an diesen CpG-Stellen nach Bisulfitumwandlung der DNA keine Komplementarität besteht. Tritt ein Mismatching mehr als an einer CpG-Stelle auf (mindestens 2 Sterne), werden durch die entsprechenden Sonden keine Fluoreszenzsignale unter den gewählten dPCR-Bedingungen generiert. In (A) ist die Sonde für das Epiallel 1a dargestellt (an oberster Stelle eingerahmt) und deren mögliche Mismatching zu den anderen 7 Epiallelen. So kann neben den spezifischen Fluoreszenzsignalen mit hoher Signalamplitude für das Epiallel 1a bei Verwendung der Sonde 1a nur durch ein einfaches Mismatching (gekennzeichnet durch nur einen Stern) mit den Epiallelen 2a, 2c und 0 (klein eingerahmt) auftreten, so dass die unspezifischen Signale mit erniedrigter Signalamplitude nur diesen Epiallelen zuzuordnen sind. Keine Signale werden gegenüber den Epiallelen 1b, 1c, 2b, und 3 bei Einsatz der Sonde 1a generiert. In (B) sind die Sonde 1b (wieder an oberster Stelle eingerahmt) und deren mögliche Hybridisierungspartner während der dPCR dargestellt. Hier können neben den spezifischen Fluoreszenzsignalen mit hoher Signalamplitude für das Epiallel 1b bei Verwendung der Sonde 1b nur durch ein einfaches Mismatching (wieder gekennzeichnet durch nur einen Stern) mit den Epiallelen 2a, 2b und 0 (klein eingerahmt) auftreten, so dass die unspezifischen Signale mit erniedrigter Signalamplitude nur diesen Epiallelen zuzuordnen sind. Keine Signale werden gegenüber den Epiallelen 1a, 1c, 2c, und 3 bei Einsatz der Sonde 1b generiert. In (C-H) sind entsprechend die weiteren Sonden (jeweils an oberster Stelle eingerahmt) und die entsprechenden Hybridisierungspartner, bei denen neben den spezifischen mit hoher Signalamplitude unspezifische Fluoreszenzsignale mit erniedrigter Amplitude gebildet werden können.
• 12 (Vergleichsdaten): Methylierung des PLA2R1-Gens in DNA-Proben aus Blutleukozyten (A) und Knochemarkzellen (B) eines 8-jährigen männlichen Patienten nach einem B-ALL-Rezidiv. MS-HRM-Analyse einer PLA2R1-amplifizierten Sequenz, die 9 CpG-Stellen umfasst (siehe schematische 1A) nach Isolierung und Bisulfitmodifizierung der genomischen DNA. Dargestellt sind die Schmelzkurven (dF/dT) von 0% und 100% methylierter Standard-DNA (unterbrochene Linien) und DNA aus Blutleukozyten (A) und Knochenmarkzellen (B). Die Pfeile in (A) zeigen 3 Subfraktionen heterogen methylierter DNA neben der Hauptfraktion der nicht-methylierten DNA, in (B) zeigt der Pfeil die Hauptfraktion an vollständig methylierter DNA neben einer kleinen Subfraktion nicht-methylierter DNA.
• 13 (Vergleichsdaten): Methylierung des PLA2R1-Gens in DNA-Proben aus normalen Epithelzellen der Prostata (PrEC, B) und benignen Prostatahyperplasiezellen (BPH-1, C) und den malignen LNCaP (D), PC-3 (E) und DU-145 (F) Prostatazelllinien. MS-HRM-Analyse einer PLA2R1-amplifizierten Sequenz, die 9 CpG-Stellen umfasst (siehe schematische 1A) nach Isolierung und Bisulfitmodifizierung der genomischen DNA. Dargestellt sind die Schmelzkurven (dF/dT) von 0% und 100% methylierter Standard-DNA (unterbrochene Linien, A) und DNA aus Prostatazellen (B-F). Die Pfeile in (B-F) zeigen den Anteil methylierter DNA.
• 14: Prozentuale Häufigkeit der heterogen methylierten Epiallele im Verhältnis zur Gesamt-DNA (fractional abundance in %) in den DNA-Proben aus peripherem Blut (E188) und Knochenmark (K189) eines 8-jährigen männlichen Patienten mit einem B-ALL-Rezidiv (A) und aus normalen Epithelzellen der Prostata (PrEC), benigner Prostatahyperplasiezelllinie (BPH-1) und den malignen LNCaP, PC-3 und DU-145 Prostatazelllinien (B). Nach Isolierung und Bisulfitmodifizierung der genomischen DNA wurden die Proben für 40 Zyklen in der ddPCR unter Einsatz von 8 fluoreszenzmarkierten Sonden PLA2R1-1a - PLA2R1-3 (1a/1b/1c/2a/2b/2c/3, deren Sequenzen in der Tabelle in 1B gezeigt sind) analysiert. Die Untersuchungen wurden in Dreifach- (A) bzw. Zweifachbestimmungen (B) durchgeführt und die Ergebnisse sind repräsentativ für jeweils zwei unabhängig voneinander durchgeführten Experimenten.
• 15: Quantifizierung der FAM-Signale (positiv für methylierte PLA2R1-Epiallele) in DNA-Proben aus normalen Epithelzellen der Prostata (PrEC), benigner Prostatahyperplasiezelllinie (BPH) und den malignen LNCaP (LNCa), PC-3 (PC-3) und DU-145 (DU) Prostatazelllinien nach 40 Zyklen in der ddPCR unter Einsatz der PLA2R1-3-Sonde. Im Gegensatz zu 10, wo die Grenze (thresholds) so eingestellt waren, dass nur die FAM-Signale mit hoher Signalamplitude berücksichtigt wurden, die auf Grund komplementärer Hybridisierung mit der PLA2R1-3-Sonde resultierten (hier als PLA2R1-3s bezeichnet), wurden die Grenze (threshold, eingezeichnet) so erniedrigt, dass alle FAM-positiven Signale (gekennzeichnet mit PLA2R1-3w), d.h. sowohl die Signale, die durch komplementäre Hybridisierung als auch durch Mismatching mit anderen Epiallelen durch die PLA2R1-3-Sonde entstanden, in die Berechnungen miteinbezogen wurden (Ergebnisse siehe Tabelle 2).
• 16: Schematische Illustration des Designs der fluoreszenzmarkierten Sonden zur Identifikation und Quantifizierung heterogen methylierter PLA2R1-Epiallele in der epiallele-sensitive digital PCR (EAST-dPCR) unter Berücksichtigung von 4 CpG-Stellen im Vergleich zu 3, wie in 1 dargestellt. Die schwarz ausgefüllten Kreise zeigen methylierte, die offenen Kreise nicht-methylierte CpG-Stellen der Position 3: -558 bp, Position 4: -547 bp, Position 5: - 549bp und Position 6: -552bp upstream vom Transkriptionsstartpunkt [TSS] (siehe 1A). Das Prinzip der EAST-dPCR unter Berücksichtigung von 4 CpG-Stellen kann auf alle potentiell geeigneten Gensequenzen (targets of interest) für die Etablierung neuer Biomarker angewendet werden, die 4 CpG-Stellen innerhalb eines Bereiches von etwa 30 bp enthalten, wie hier im Fall des PLA2R1 Promotors gezeigt.
• 17: Quantifizierung der FAM-Signale (positiv für methylierte PLA2R1-Epiallele) und HEX-Signale (positiv für nicht-methylierte PLA2R1-Epiallele) in einer 50%-igen homogen methylierten und nicht-methylierten Standard-DNA-Probe in Abhängigkeit von der Annealingtemperatur zwischen 50,0°C und 60,0°C nach 40 Zyklen in der ddPCR unter Einsatz der PLA2R1-1- (SEQ-ID 9) und PLA2R1-un-(SEQ ID 24)-Sonden mit 4 CpG-Stellen. Die Grenzen (cut-off bzw. thresholds) sind eingezeichnet.

The pictures show:

• 1 : Schematic illustration of the localization (A) and design (B) of fluorescently labeled probes for the identification and quantification of heterogeneously methylated PLA2R1 epialleles in epiallelic-sensitive digital PCR (EAST-dPCR). The black filled circles in (B) show methylated, open circles non-methylated CpG sites (position 4: -547 bp, position 5: -549bp, and position 6: -552bp upstream from the transcription start point [TSS] in (A)) , The principle of EAST-dPCR can be applied to all potentially suitable gene sequences (targets of interest) for the establishment of new biomarkers.
• 2 and 3 2D projection of FAM (positive for methylated PLA2R1 DNA fragments) and HEX (positive for unmethylated PLA2R1 DNA fragment) signals in blood leukocyte DNA samples (left side, E188) and bone marrow cells (right side , K189) after 40 cycles in the ddPCR. FAM-positive signals (methylated DNA) are circled. To the right of the graphs are shown the probes used (PLA2R1-1a - PLA2R1-3) whose sequences are shown in the table in 1B are shown. The DNA samples were each analyzed in triplicate and the data from the studies are presented together. The circled in black are the FAM-positive signals.
• 4 2D projection of FAM signals (positive for methylated PLA2R1 DNA fragments) and HEX signals (positive for unmethylated PLA2R1 DNA fragments) in DNA samples from normal prostate epithelial cells (PrEC) after 40 cycles in the ddPCR. FAM-positive signals (methylated DNA) are circled. The probes used (PLA2R1-1a - PLA2R1-3) are displayed above the graphs whose sequences are shown in the table of the 1B are summarized.
• 5 : 2D projection of FAM signals (positive for methylated PLA2R1 DNA fragments) and HEX signals (positive for unmethylated PLA2R1 DNA fragments) in DNA samples from benign prostate hyperplasia cells (BPH-1) at 40 Cycles in the ddPCR. FAM-positive signals (methylated DNA) are circled. The probes used (PLA2R1-1a - PLA2R1-3) are displayed above the graphs whose sequences are shown in the table of the 1B are summarized.
• 6 2D projection of FAM signals (positive for methylated PLA2R1 DNA fragments) and HEX signals (positive for unmethylated PLA2R1 DNA fragments) in DNA samples from LNCaP prostate tumor cells after 40 cycles in the ddPCR. FAM-positive signals (methylated DNA) are circled. The probes used (PLA2R1-1a - PLA2R1-3) are displayed above the graphs whose sequences are shown in the table of the 1B are summarized.
• 7 2D projection of FAM signals (positive for methylated PLA2R1 DNA fragments) and HEX signals (positive for unmethylated PLA2R1 DNA fragments) in DNA samples from PC-3 prostate tumor cells after 40 cycles in the ddPCR , FAM-positive signals (methylated DNA) are circled. The probes used (PLA2R1-1a - PLA2R1-3) are displayed above the graphs whose sequences are shown in the table of the 1B are summarized.
• 8th : 2D projection of FAM signals (positive for methylated PLA2R1 DNA fragments) and HEX signals (positive for unmethylated PLA2R1 DNA fragments) in DNA samples from DU 145 prostate tumor cells after 40 cycles in the ddPCR. FAM-positive signals (methylated DNA) are circled. The probes used (PLA2R1-1a - PLA2R1-3) are displayed above the graphs whose sequences are shown in the table of the 1B are summarized.
• 9 and 10 : Quantification of FAM signals (positive for methylated PLA2R1 epialleles) and HEX signals (positive for non-methylated PLA2R1 epialleles) in DNA samples from normal prostate epithelial cells (PrEC), benign prostate hyperplasia cell line (BPH) and malignant LNCaP (LNCa), PC-3 (PC-3) and DU-145 (DU) prostate cell lines after 40 cycles in the ddPCR. The boundaries (cut-offs or thresholds) are drawn, the probes used are each described above the graphics. The arrows show low amplitude FAM signals resulting from mismatching of probes with epialleles. The investigations were carried out in duplicate. NTC, negative control without DNA template.
• 11 : Constellations that are possible by mismatching the 8 probes due to a single nucleotide difference with the epialleles. (AH) each show the probes (boxed in large) and their possible mismatching hybridization partners, which are labeled next to the probe (each boxed in small). The black circles represent the CpG sites that are methylated, the open circles represent the CpG sites in the epialleles that are unmethylated. The stars indicate mismatching in that there is no complementarity due to different methylations at these CpG sites after bisulfite conversion of the DNA. If mismatching occurs more than at a CpG site (at least 2 stars), the corresponding probes will not generate fluorescence signals under the selected dPCR conditions. In (A), the probe is for the epiallel 1a (framed at the top) and their possible mismatching to the other 7 epialleles. Thus, besides the specific fluorescence signals with high signal amplitude for the epiallel 1a when using the probe 1a only by a simple mismatching (characterized by only one star) with the epialleles 2a . 2c and 0 (framed in small) occur, so that the non-specific signals with reduced signal amplitude can be assigned only to these epialleles. No signals are given to the epialleles 1b . 1c . 2 B , and 3 when using the probe 1a generated. In (B) are the probe 1b (framed again at the top) and their possible hybridization partners during the dPCR. Here, in addition to the specific fluorescence signals with high signal amplitude for the epiallel 1b when using the probe 1b only by a simple mismatching (again characterized by only one star) with the epialleles 2a . 2 B and 0 (framed in small) occur, so that the non-specific signals with reduced signal amplitude can be assigned only to these epialleles. No signals are given to the epialleles 1a . 1c . 2c , and 3 when using the probe 1b generated. In (CH), the other probes (each framed at the top) and the corresponding hybridization partners, in which in addition to the specific with high signal amplitude nonspecific fluorescence signals with reduced amplitude can be formed.
• 12 (Comparative data): Methylation of the PLA2R1 gene in DNA samples from blood leukocytes (A) and bone marrow cells (B) of an 8-year-old male patient after a B-ALL recurrence. MS-HRM analysis of a PLA2R1 amplified sequence comprising 9 CpG sites (see schematic 1A) after isolation and bisulfite modification of the genomic DNA. Shown are the melting curves (dF / dT) of 0% and 100% methylated standard DNA (broken lines) and DNA from blood leukocytes (A) and bone marrow cells (B). The arrows in (A) show 3 subfractions of heterogenous methylated DNA next to the major fraction of unmethylated DNA, in (B) the arrow shows the major fraction of fully methylated DNA next to a small subfraction of unmethylated DNA.
• 13 (Comparative data): Methylation of the PLA2R1 gene in DNA samples from normal prostate epithelial cells (PrEC, B) and benign prostatic hyperplasia cells (BPH-1, C) and the malignant LNCaP (D), PC-3 (E) and DU- 145 (F) prostate cell lines. MS-HRM analysis of a PLA2R1 amplified sequence comprising 9 CpG sites (see schematic 1A) after isolation and bisulfite modification of the genomic DNA. Shown are the melting curves (dF / dT) of 0% and 100% methylated standard DNA (broken lines, A) and DNA from prostate cells (BF). The arrows in (BF) show the proportion of methylated DNA.
• 14 : Percentage frequency of heterogenous methylated epiallele relative to total fractional abundance (%) DNA in peripheral blood (E188) and bone marrow (K189) DNA samples of an 8-year-old male patient with B-ALL recurrence (A ) and normal prostate epithelial cells (PrEC), benign prostate hyperplasia cell line (BPH-1) and the malignant LNCaP, PC-3 and DU-145 prostate cell lines (B). After isolation and bisulfite modification of the genomic DNA, the samples were incubated for 40 cycles in the ddPCR using 8 fluorescently labeled probes PLA2R1-1a - PLA2R1- 3 (1a / 1b / 1c / 2a / 2b / 2c / 3, whose sequences are given in the table in 1B shown). The studies were performed in triplicate (A) and double determinations (B), respectively, and the results are representative of two independent experiments.
• 15 : Quantification of the FAM signals (positive for methylated PLA2R1 epialleles) in DNA samples from normal prostate epithelial cells (PrEC), benign prostate hyperplasia cell line (BPH) and the malignant LNCaP (LNCa), PC-3 (PC-3) and DU -145 (DU) prostate cell lines after 40 cycles in the ddPCR using the PLA2R1-3 probe. In contrast to 10 where the thresholds were set to account for only the high signal amplitude FAM signals resulting from complementary hybridization to the PLA2R1-3 probe (referred to herein as PLA2R1-3s), the threshold (threshold , plotted) in such a way that all FAM-positive signals (labeled PLA2R1-3w), ie both the signals which were formed by complementary hybridization and by mismatching with other epialleles by the PLA2R1-3 probe, were included in the calculations (Results see Table 2).
• 16 : Schematic illustration of the design of fluorescently labeled probes for the identification and quantification of heterogeneously methylated PLA2R1 epialleles in epiallelic-sensitive digital PCR (EAST-dPCR) considering 4 CpG sites compared to 3, as in 1 shown. The black-filled circles show methylated, open circles non-methylated CpG sites at position 3: -558 bp, position 4: -547 bp, position 5: -549 bp, and position 6: -552bp upstream from the transcription start point [TSS] (see 1A) , The principle of EAST dPCR, taking into account 4 CpG sites, can be applied to all potentially suitable target of interest targets for the establishment of new biomarkers containing 4 CpG sites within a range of about 30 bp as in this case of the PLA2R1 promoter.
• 17 : Quantification of FAM signals (positive for methylated PLA2R1 epialleles) and HEX signals (positive for non-methylated PLA2R1 epialleles) in a 50% homogeneous methylated and unmethylated standard DNA sample as a function of annealing temperature between 50.0 ° C and 60.0 ° C after 40 cycles in the ddPCR using the PLA2R1-1 (SEQ ID 9) and PLA2R1-un (SEQ ID 24) probes with 4 CpG sites. The boundaries (cut-off or thresholds) are shown.

General regulations:

Isolation of free-circulating DNA (fc-DNA): First, the free-circulating DNA (fc-DNA) from the sample to be examined, such. B. from 1-5 ml serum samples using the QIAamp Circulating Nucleic Acid Kit from Qiagen GmbH (Hilden, DE) according to the test kit description isolated and eluted in each 25 ul.

Bisulfite conversion of the fc DNA: The bisulfite conversion of each 20 ul of the fc DNA was carried out using the EpiTect Fast Bisulfite Conversion Kit from Qiagen GmbH according to the test kit description. After elution, each 15 μl of bisulfite-treated fc-DNA solution is obtained.

Bias-free or bias-inducing pre-amplification of the samples: After bisulfite conversion, the DNA concentrations of the DNA samples were determined using the Quantus ™ Fluorometer (Promega). If the DNA concentration was below or above 1 ng / μl, 2 or 1 μl of the bisulfite-treated DNA were introduced into the EAST-dPCR directly or initially in a pre-amplification.

Master mix for the preamplification: reagent Vol. [Μl] Final concentration. PCR buffer [15 mM MgCl] 2.50 1.5 mmol / l dNTPs [2.5 mmol / l] 2.00 200 μmol / l forward primer (SEQ ID No. 25) 1.00 400 nmol / l reverse primer (SEQ ID No. 26) 1.00 400 nmol / l HotStarTaq Plus [5 U / μl] 0,125 0.625 U MgCl ₂ [25 mmol / l] 0 to 13.50, 0 to +13.5, preferably 1.00-2.00 preferably + 1-2 mmol / l RNase free aqua dest 2.875 to 17.375, preferably 13.50-15.50 Carrier DNA 1.00-2.00 final volume 25,00 mM = mmol / l

Master Mix for EAST-dPCR: component Batch vol [μl] final concentration 2x ddPCR Supermix (BioRad) 10.0 20x primer / probe mix 1.0 900 mmol / l / 250 nmol / l DNA 2.0 RNase-free dist. 7.0 final volume 20.0

Primer for the pre-amplification and the EAST-dPCR: Primer ID Sequences (5 '→ 3') SEQ ID PLA2R1-forward (forward) 5'-GGG GTA AGG AAG GTG GAG AT-3 ' 25 PLA2R1 reverse (reverse) 5'-ACA AAC CAC CTA AAT TCT AAT AAA CAC-3 ' 26 The primers give a product length of 168bp.

Probes: for the dPCR
All probes are labeled in the examples 5'-FAM (methylated DNA) or HEX (unmethylated DNA) and at the 3 'end with the quencher BHQ-1 (see 1B) and were each used singly as duplex EAST dPCR. If more than 2 fluorescent dyes can be measured simultaneously, the probes can each be labeled at the 5 'end with different fluorescent dyes and the corresponding quenchers at the 3' end and assembled in a set and used for a multiplex EAST dPCR.

• 20 µl Mastermix mit DNA-Probe
• + 70 µl Öl in entsprechende Cartridges (BioRad);
• Generierung von etwa 20.000 Öl/Emulsionströpfchen,
• davon 35 µl in die nachfolgende dPCR

Production of Droplets (Droplet Generator [BioRad]):

• 20 μl master mix with DNA sample
• + 70 μl of oil in corresponding cartridges (BioRad);
• Generation of about 20,000 oil / emulsion droplets,
• thereof 35 μl into the following dPCR

Temperature and time diagram for the dPCR using the T100 PCR device (BioRad): PCR temperature Time denaturation: 95 ° C 10 mins PCR cycle: 94 ° C (denaturation) 30 seconds 40x Primer and probe specific temperature (annealing and elongation, 50-72 ° C) 1 minute 98 ° C (stabilization of the droplets) 10 mins 20 ° C hold

Measurement of fluorescence in the pots:
Droplet fluorescence was measured by QY100 (BioRad) according to the manufacturer's description, which allows the simultaneous determination of multiple fluorescences or an alternative Measuring device that allows the simultaneous determination of multiple fluorescences (more than 2 different fluorescent dyes).

EMBODIMENT 1 Results of EAST dPCR Studies of Bone Marrow Cell DNA and Blood Leukocytes of an 8-Year Male Patient with a B-ALL Recurrence

DNA samples from a bone marrow aspirate of an 8-year-old boy with a B-ALL (K189 sample) were examined in a duplexed ddPCR using a FAM-PLA2R1-3 probe (complementary to fully methylated DNA) and HEX-PLA2R1 Probe (complementary to completely unmethylated DNA) revealed that the DNA was predominantly methylated ( 3 , right column, graphic PLA2R1-3). The proportion of fully methylated and unmethylated DNA was 84.8% and 13.6%, respectively, and the remaining 1.7% represented heterogeneously methylated epialles in this sample (Table 1).

By comparison, using the same probes in the DNA of the blood leukocytes of the same patient, a subfraction with a comparable signal amplitude as in the case of the DNA from the bone marrow of the patient also appeared ( 3 , left column, graphic PLA2R1-3). However, the proportion of fully methylated DNA was only 14.2% and that of non-methylated DNA was 73.6%. In addition, further subfractions showed a significantly lower signal amplitude and amounted to 12.4% (Table 1).

Examination of the samples with the PLA2R1-2a 2c probes revealed numerous FAM-positive subfractions with different fluorescence amplitudes ( 2 and 3 ). Particularly in the DNA of the blood leukocytes, distinct subfractions with high FAM signal amplitude could be detected. These subfractions could be well separated from the other subfractions, which had significantly lower signal amplitude. For this reason, the thresholds were also used in the evaluation of the data of sample K189, in which the proportion of epialleles PLA2R1-2a-2c was significantly lower compared to the E188 sample ( 2 and 3 ). In addition, a dominant subfraction was observed in both samples (E188 and K189) whose fluorescence amplitude was just above the amplitude of the negative droplets without amplificates (empty droples, 2 and 3 , left and right column, graphics PLA2R1-2a, -2b and -2c). The amount of this subfraction corresponded to the amount that was present when using the PLA2R1-3 probe, in particular in the K189 sample, but also in the E188 sample ( 3 , Graphic PLA2R1-3). It was concluded that this is the fully methylated epiallelic fraction PLA2R1-3, which resulted in a FAM signal due to mismatching with the PLA2R1-2a 2c probes, its reduced amplitude due to lower hybridization and amplification efficiencies under the chosen reaction conditions in the ddPCR (MgCl ₂ concentration and annealing temperature) compared to the complete complementarity by using the PLA2R1-3 probe ( 2 and 3 , left and right column, graphics PLA2R1-2a, -2b and -2c). When using the PLA2R1-2c probe, there was another subfraction in the DNA from the blood leukocytes (sample E188), whose signal amplitude was slightly higher than that of the subfraction assigned to the PLA2R1-3 epiallel, but still well below PLA2R1 -2c-specific FAM signals ( 2 and 3 , left column, graphic PLA2R1-2c). When using the PLA2R1-1a-1c probes, the dominant subfraction was absent in the blood leukocyte and bone marrow DNA samples, which was predominant when using the PLA2R1-3 and PLA2R1-2a 2c probes ( 2 , left and right column, graphics PLA2R1-1a, -1b and -1c). This suggested that under the ddPCR conditions chosen, no mismatching occurs with a twofold nucleotide difference and the probes PLA2R1-1a-1c do not hybridize to the fully methylated DNA fragments (PLA2R1-3 epiallelic).

Except for the DNA sample from the blood leukocytes (E188), where a small subfraction was seen just above the FAM signal amplitude of the negative droplets (empty droplets) when using the PLA2R1-1b probe ( 2 , left column, graphic PLA2R1-1b), all three PLA2R1-1a-1c probes could only detect separate fractions with high FAM signal amplitudes, in particular in the E188 DNA sample. For this reason, the thresholds used here were also used in the evaluation of the data of the K189 sample, in which the PLA2R1-1a-1c epialleles occurred only in low concentration due to the almost completely methylated DNA ( 2 , right column, graphics PLA2R1-1a, -1b and -1c), as also shown in the MS-HRM analysis ( 12 ).

Based on the observation that i) FAM signals with high signal amplitude arise when a complementary hybridization takes place and ii) a mismatching associated with a FAM signal generation at significantly reduced signal amplitude only occurs when there was a single nucleotide difference, but not a double nucleotide difference , the constellations could be deduced that in 11 are shown.

Based on these constellations, it could be concluded that, in addition to the PLA2R1-2c complementary PLA2R1-2c subfraction with high signal amplitude and the fully methylated PLA2R1-3 subfraction (signal amplitude just above that of the empty droplets) when using the PLA2R1-2c probe the third subfraction must be around the PLA2R1-1a and / or PLA2R1-1c epialleles that caused FAM signals due to mismatching with the PLA2R1-2c probe ( 2 and 3 , left column, graphic PLA2R1-2c). Since the PLA2R1-1a and PLA2R1-1c subfractions were present in the DNA of the bone marrow only to a small extent, this explains the absence of this subfraction in the 2D projection of the DNA sample of the bone marrow ( 2 and 3 , right graphic: PLA2R1-2c).

According to the possible constellations, using the PLA2R1-3 probe further results in mismatching of this probe with the PLA2R1-2a-2c epialleles, and thus in the DNA sample from the blood leukocytes of the B-ALL patient apart from the fully methylated DNA (PLA2R1-3 epiallel) two further subfractions with reduced FAM signal amplitude ( 3 , left column, graphic PLA2R1-3). The PLA2R1-3 as well as the PLA2R1-1a-1c probes did not generate an additional FAM signal when there was a double nucleotide difference as well as the calculated recovery rates when using the PLA2R1-3 probe (Table 2). For this purpose, the copies of the PLA2R1-3 epiallel were summed with those of the PLA2R1-2a-2c and the resulting copy numbers gave good agreement with the values obtained after appropriate limit settings involving all positive FAM signals (see Thresholds) 15 for PLA2R1-3w and recovery rates see Table 2). On the other hand, when the copies of PLA2R1-1a-1c were included in the calculations, this resulted in a recovery rate of 141% instead of 99% in the case of the E188 sample, which contained a high proportion of epialleles 1a-1c (Table 2). In the case of the K189 sample, the proportion of these epialleles was too small to have an effect on the recovery rate since the DNA in this sample was 98.2% as the PLA2R1-3 epiallel. It could be concluded that similar to the PLA2R1-1a-1c probes, the PLA2R1-3 probe also generates only low-amplitude signals in a mismatching due to a simple, but not twofold nucleotide difference.

Embodiment 2: Results of EAST dPCR studies on DNA samples from normal epithelial cells, benign hyperplasia cells and malignant cells of the prostate

In order to verify whether the episodes can be similarly identified and quantified in other DNA samples using the 8 probes, DNA from normal and malignant prostate cell lines was isolated and analyzed. It was found that significant FAM signals could only be detected in LNCaP cells using the PLA2R1-3 probe ( 6 ). A small number of PLA2R1-1b, -2b, and -2c probes were found, and no FAM signals were found on the PLA2R1-1a, -1c, and -0 probes. In contrast, negligible amounts of PLA2R1-3-specific signals were detected in PrEC cells, but significant amounts of PLA2R1-1c and PLA2R1-1a-specific signals ( 4 ). In comparison, the signal levels for the PLA2R1-3 epialleles were slightly higher for BPH-1 than for PrEC ( 5 ), but significantly lower compared to the malignant LNCaP, PC-3 and DU-145 prostate cell lines, which had significantly increased levels of fully methylated DNA (PLA2R1-3 epiallel) ( 6 to 8th ).

Similar to the result for the blood leukocytes of the 8-year-old patient (sample E188), the PLA2R1-2c complementary fraction (above the set thresholds) showed two additional subfractions with different signal amplitude below the thresholds limit ( 10 , right column, graphic PLA2R1-2c). The subfraction with the FAM signal amplitude could be assigned slightly above that of the negative droplets by comparing the signal amplitude in the LNCaP cells to the PLA2R1-3 epiallel, since in the LNCaP cells only this fraction was present in the corresponding amount ( 10 ). According to the established constellation possibilities ( 11 ), the only possibility for the PLA2R1-2c probe is that mismatching with the PLA2R1-1a and PLA2R1-1c epialleles can lead to a FAM signal based on a single nucleotide difference. As a result, the second subfraction in the PrEC, BPH-1, PC-3 and DU-145 cells, whose signal amplitude was slightly above the PLA2R1-3 epiallel, was the PLA2R1-1a strain. and / or PLA2R1-1c epiallelic ( 9 and 10 ). This conclusion is in line with the results and conclusion drawn previously for samples E188 and K189. The lack of these subfractions in the LNCaP cells in the study with the PLA2R1-2c probe can also be explained ( 10 , Graphic for PLA2R1-2c). In addition, the PLA2R1-2c-specific signal levels of these second subfractions were consistent with the signal levels obtained with probes PLA2R1-1a and PLA2R1-1c in the PrEC, BPH-1, PC-3, and DU-145 ( 9 and 10 ).

Furthermore, the 3 subfractions resulting from the use of the PLA2R1-3 probe could be limited by the results in the PC-3 cells. Thus, in this cell line, in addition to the fraction of the complementary and therefore completely methylated DNA ( 9 and 10 , Signal amplitude above the thresholds limit) two further subfractions ( 9 and 10 , Signal amplitudes below the thresholds limit). Because the PLA2R1-3 probe can generate low-amplitude FAM signals due to simple mismatching with the PLA2R1-2a-2c epialleles and the amount of positive FAM signals in the PC-3 cells with those of the PLA2R1-2a -Epialleles in these cells, which was determined using the PLA2R1-2a probe, the sub-fraction with a slightly higher signal amplitude could be assigned to the PLA2R1-2a epiallel (in 10 , left column, graphic PLA2R1-3 marked with the upper arrow). It was also possible to conclude from the comparable amounts of the PLA2R1-2a epiallel in the PC-3 and DU-145 cells and the increased signal quantity of this subfraction in the DU-145 cells in comparison to the PC-3 cells ( in 10 , Graphic PLA2R1-3) that in addition to the PLA2R1-2a epiallel, the epialleles PLA2R1-2b or PLA2R1-2c also mismatched signals of comparable amplitude when using the PLA2R1-3 probe. The subfraction with reduced signal amplitude (in 10 marked by the lower arrow in the graphic PLA2R1-3) was missing in the PC-3 cells. Since the levels of the PLA2R1-2b and PLA2R1-2c epialleles were very low in the PC-3 cells, this subdomain prominent in the DU-145 cells should be around the PLA2R1-2b or PLA2R1-2c -Epiallele act. This was in good agreement with the amounts of FAM signals found for the PLA2R1-2b ( 10 , left column, graphic PLA2R1-2b) and PLA2R1-2c epiallel ( 10 , right column, graphic PLA2R1-2c) and the PLA2R1-3-generated FAM signals in the PrEC, BPH-1 and DU-145 cells ( 10 , left column, graphic PLA2R1-3).

Similarly good recovery rates as in the E188 sample after summation of the PLA2R1-3 and PLA2R1-2a 2c epialleles compared to the total FAM positive signals using the PLA2R1-3 probe (PLA2R1-3w, see 15 ) were also found in the DNA samples of the PrEC, BPH-1, PC-3 and DU-145 prostate cells, in which the epithelial PLA2-1a-1c also occurred in relatively high concentration (Table 2). These results also revealed that using the PLA2R1-3 probe, no PLA2R1-1a-1c epitopes are identified and quantitated. These epialleles can therefore only be detected by using probes with complementary sequences (such as the PLA2R1-1a-1c specific probes) directly or by using probes complementary to 2 of the 3 CpG sites studied (such as PLA2R1-2a-2c Probes) can be quantified indirectly by mismatching. This surprising observation is of particular interest since these subfractions may already be present in early malignant changes [11] and their abundance is overlooked when only probes complementary to 3 fully methylated CpG sites are used (as in the present example Probe PLA2R1-3).

On the basis of the results obtained, it was possible to plausibly show that, under selective reaction conditions in the dPCR (annealing temperature depending on the MgCl 2 concentration used), the 7 possible heterogeneously methylated epialleles were analyzed by using 3 selective CpG sites of the PLA2R1 Promoters can be sensitively and specifically identified and quantified. The results for the individual epialleles in the DNA samples isolated from the blood leukocytes, bone marrow cells and prostate cell lines are in 14 and Tables 1 and 3-5.

To include 4 or 5 instead of the 3 CpG sites in methylation studies, a total of 16 or 32 individual probes are required. This would require a relatively high number of individual examinations. As new dPCR gauges are developed and made available in the future, which can simultaneously detect more than 2 fluorescence regions, e.g. Current flow cytometry instruments for investigations of cellular surface structures with 5 to 7 fluorescence channels or fluorescence microscopes with up to 20 different fluorescence channels in clinical chemistry laboratories are used in the future instead of the duplex multiplex dPCR conceivable, in addition to FAM and HEX / VIC Fluorochromes other fluorescent dyes can be used for the labeling of the probes.

In this way, several sets can be produced, for example, each consisting of 7 differently fluorescently labeled probes for the heterogenously methylated epiallelic. In this case, 4 sets of CpG sites to be studied in parallel, 2 sets of 7 probes each and a set of 2 probes, 5 sets of CpG sites would require 4 sets of 7 probes each and 1 set of 4 probes. This would significantly reduce the costs for determining the 16 or 32 epiallelic variants to 3 or 5 approaches for a sample to be examined, so that a high sample throughput would also be possible here. In this way, the complex composition of heterogenous methylated epiallele can be determined at a reasonable cost and laboratory effort. As a result, in addition to an improved understanding of the mechanisms of Emergence of heterogenous methylated epialles find new biomarkers for improved differential diagnosis of benign and malignant diseases, which may be the basis for improved diagnostic test development.

Embodiment 3 Results of MS-HRM Studies of DNA Samples from Blood Leukocytes, Bone Marrow Cells and Normal Epithelial Cells, Benign Hyperplasia Cells and Malignant Cell Lines of the Prostate

The MS-HRM studies (conventional method of the prior art) of the DNA samples isolated from the bone marrow of an 8-year-old patient with B-ALL (sample K189) showed almost completely methylated DNA and only a small proportion on non-methylated DNA ( 12B) , In contrast, the DNA isolated from the patient's blood leukocytes (sample E188) contained, in addition to a high proportion of unmethylated DNA fragments, at least 3 differently methylated subfractions (arrows which mark the heterogenously methylated epiallelic subfractions, 12A) , In contrast to the EAST-dPCR, the MS-HRM technique was unable to assign the 3 subfractions detected in the melting curve to individual heterogenously methylated epialleles, nor could this technique quantify the proportion of individual methylated epialleles.

The same applies to the study of the prostate cell lines by means of the MS-HRM technique (conventional method according to the prior art). Thus, the DNA of the normal PrEC cells showed a congruent melting curve profile with the 0% DNA standard ( 13 ). It was concluded that the DNA of the PrEC cells was unmethylated. In fact, however, the DNA contained 0.4% of fully methylated DNA, which was not apparent in the MS-HRM melting curve ( 13 ). In the MS-HRM of the DNA from BPH-1 cells, on the other hand, a small fraction was found whose melting point was slightly shifted to the right, ie that a small proportion of the amplified DNA was heterogeneously methylated. Again, no single heterogeneously methylated epiallele could be assigned to this fraction, nor could they be quantified.

In the invention, heterogeneously methylated epialleles of the PLA2R1 promoter taking into account a further CpG site (eg position 3: -558 bp from the TSS of the PLA2R1 promoter, 1A) In addition to the three sites already analyzed, two FAM and HEX-labeled PLA2R1 probes for fully methylated and unmethylated epialles with four CpG sites were constructed and analyzed in the ddPCR (PLA2R1-m and PLA2R1-un, 16 ). Here, it was shown that both probes generated a correspondingly high fluorescence signal amplitude after 40 cycles in the ddPCR at an annealing temperature of 50.7 ° ( 17 ). In this way, by means of another 14 different fluorescently labeled probes (PLA2R1-1 to PLA2R1-14 probes, 16 ) under the same reaction conditions (annealing temperature as a function of the MgCl2 concentration used) the complete PLA2R1 epiallelic pattern can be investigated in a more differentiated manner. Assuming that 16 different fluorescences can be determined simultaneously, a set of 16 different fluorescently labeled probes could be prepared and used to determine the complete epiallelic pattern in one run. In this way, new heterogenous methylated epialles could be identified, which allow an improved differentiation between benign and malignant diseases.

Table 1: Percentage distribution of the heterogenously methylated epialleles (abs./%: relative proportion of all 8 epialleles; rel./%: relative proportion of the 7 methylated epialleles) in the peripheral blood (E188) and bone marrow (K189) DNA samples 8-year-old male patient with a B-ALL recurrence. After isolation and bisulfite modification of the genomic DNA, the samples were incubated for 40 cycles in the ddPCR using 8 fluorescently labeled probes PLA2R1-1a - PLA2R1-3 (their sequences are given in the table in FIG 1B are shown) examined. The analyzes were performed in triplicate and the results are presented as mean and standard deviation. E188 K189 Probe ID Section./% rel./% Section./% rel./% PLA2R1-1a 2.2 ± 0.3 8.5 ± 1.0 0.1 ± 0.0 0.1 ± 0.0 PLA2R1-1b 1.2 ± 0.1 4.4 ± 0.3 0.1 ± 0.0 0.1 ± 0.0 PLA2R1-1c 4.5 ± 0.3 17.0 ± 1.2 0.1 ± 0.0 0.1 ± 0.0 PLA2R1-2a 0.8 ± 0.2 2.9 ± 0.9 0.9 ± 0.1 1.0 ± 0.1 PLA2R1-2b 1.8 ± 0.3 6.6 ± 1.0 0.2 ± 0.1 0.2 ± 0.1 PLA2R1-2c 1.9 ± 0.1 7.1 ± 0.4 0.3 ± 0.0 0.3 ± 0.1 PLA2R1-3 14.2 ± 0.5 53.6 ± 1.8 84.8 ± 4.1 98.2 ± 4.7 PLA2R1-0 73.6 ± 0.7 13.6 ± 1.2

Table 2: Recovery rates (mean ± standard errors in%) after totaling of copies for fully methylated PLA2R1-3 epiallelic with copies at epialleles PLA2R1-2a-2c alone and in combination with copies of epiallelic PLA2R1-1a-1c in ratio to the copies made with the help of the PLA2R1-3 probe and the corresponding cut-off or thresholds setting for all FAM-positive signals (as for PLA2R1-3w in 15 shown) were determined. rehearse PLA2R1-epialleles PLA2R1-epialleles 3+ (2a + 2b + 2c) 3+ (2a + 2b + 2c) + (1 a + 1 b + 1 c) E188 99.0 ± 5.0 140.7 ± 4.0 K189 91.7 ± 5.0 92.0 ± 5.0 PrEC 103.0 ± 5.5 449.6 ± 9.0 BPH-1 97.5 ± 8.0 423.8 ± 2.0 LNCaP 93.9 ± 67.0 96.7 ± 66.0 PC-3 95.5 ± 20.0 136.3 ± 15.0 DU-145 103.3 ± 5.5 172.6 ± 7.0

Table 3: Percentage frequency of heterogenous methylated epiallele relative to the total fractional abundance (%) DNA in the peripheral blood (E188) and bone marrow (K189) DNA samples of an 8-year-old male patient with a B-ALL recurrence , After isolation and bisulfite modification of the genomic DNA, the samples were incubated for 40 cycles in the ddPCR using 8 fluorescently labeled probes PLA2R1-1a-3 (their sequences are given in the table in FIG 1B are shown) examined. The analyzes were performed in triplicate and the results are shown as mean and standard deviation. Probe ID E188 K189 PLA2R1-1a 3.0 ± 0.2 0.6 ± 0.2 PLA2R1-1b 1.5 ± 0.1 0.3 ± 0.2 PLA2R1-1c 5.7 ± 0.3 0.9 ± 0.1 PLA2R1-2a 0.8 ± 0.1 1.7 ± 0.5 PLA2R1-2b 2.3 ± 0.3 0.2 ± 0.1 PLA2R1-2c 2.4 ± 0.1 0.3 ± 0.0 PLA2R1-3 14.6 ± 0.5 84.8 ± 4.1

Table 4: Percentage distribution of the heterogenously methylated epialleles (abs./%: relative proportion of all epialleles relative to the total number of 8 epialleles; rel./%: relative proportion of the methylated epialleles relative to the sum of all methylated epialleles) in the DNA samples from normal epithelial cells of the prostate (PrEC), benign prostate hyperplasia cell line (BPH-1) and the malignant LNCaP, PC-3 and DU-145 prostate cell lines. After isolation and bisulfite modification of the genomic DNA, the samples were incubated for 40 cycles in the ddPCR using 8 fluorescently labeled probes PLA2R1-1a - PLA2R1-3 (their sequences are given in the table in FIG 1B are shown) examined. The analyzes were performed in triplicate and the results are presented as mean and standard deviation. Probe ID PrEC BPH-1 LNCaP PC-3 DU-145 Section./% rel./% Section./% rel./% Section./% rel./% Section./% rel./% Section./% rel./% PLA2R1-1a 3.6 ± 1.1 20.1 ± 6.0 8.0 ± 1.0 27.1 ± 3.3 0.8 ± 1.1 0.8 ± 1.1 8.6 ± 0.8 17.3 ± 1.6 7.4 ± 0.5 17.4 ± 1.2 PLA2R1-1 b 1.3 ± 0.2 7.3 ± 0.9 2.6 ± 0.2 8.7 ± 0.8 2.1 ± 1.9 2.1 ± 1.9 2.1 ± 0.2 4.1 ± 0.4 3.7 ± 0.1 8.7 ± 0.1 PLA2R1-1c 8.9 ± 0.4 49.7 ± 2.2 12.2 ± 1.1 41.2 ± 3.6 0 0 4.3 ± 1.6 8.6 ± 3.1 6.7 ± 0.4 15.8 ± 1.0 PLA2R1-2a 0.5 ± 0.2 2.6 ± 1.3 1.0 ± 0.2 3.5 ± 0.5 8.8 ± 2.3 8.9 ± 2.3 15.5 ± 1.7 31.2 ± 3.4 4.0 ± 0.8 9.3 ± 1.9 PLA2R1-2b 1.3 ± 0.4 7.0 ± 2.2 1.7 ± 0.3 5.8 ± 1.1 2.2 ± 1.1 2.2 ± 1.1 2.3 ± 1.0 4.6 ± 2.0 6.3 ± 0.6 14.9 ± 1.5 PLA2R1-2c 2.0 ± 0.8 11.0 ± 4.3 2.6 ± 0.1 8.7 ± 0.3 2.0 ± 1.1 2.0 ± 1.1 1.7 ± 0.7 3.5 ± 1.3 4.1 ± 0.3 9.6 ± 0.6 PLA2R1-3 0.4 ± 0.2 2.4 ± 1.1 1.5 ± 0.2 5.0 ± 0.5 82.9 ± 29.3 84.0 ± 29.7 15.3 ± 1.8 30.7 ± 3.6 10.3 ± 1.2 24.3 ± 2.9 PLA2R1-0 82.0 ± 7.0 70.4 ± 8.0 1.3 ± 1.1 50.2 ± 4.1 57.4 ± 4.1

Table 5: Percentage frequency of heterogenously methylated epiallele relative to the total fractional abundance (%) DNA in DNA samples from normal epithelial cells of the prostate (PrEC), benign prostatic hyperplasia cell line (BPH-1), and malignant LNCaP, PC-3 and DU-145 prostate cell lines. After isolation and bisulfite modification of the genomic DNA, the samples were incubated for 40 cycles in the ddPCR using 8 fluorescently labeled probes PLA2R1-1a - PLA2R1-3 (their sequences are given in the table in FIG 1B are shown) examined. The analyzes were performed in triplicate and the results are shown as mean and standard deviation. nd, not determined, since due to the low copy numbers of non-methylated DNA, the calculation of the fractional frequency is not meaningful. Probe ID PrEC BPH-1 LNCaP PC-3 DU-145 PLA2R1-1a 3.3 ± 0.6 8.5 ± 0.9 nd 11.7 ± 0.5 11.2 ± 0.3 PLA2R1-1b 1.2 ± 0.1 3.0 ± 0.3 nd 3.5 ± 0.1 5.5 ± 0.1 PLA2R1-1c 7.8 ± 0.4 12.6 ± 1.0 nd 6.3 ± 1.0 9.8 ± 0.3 PLA2R1-2a 0.4 ± 0.2 1.4 ± 0.2 nd 23.1 ± 0.4 6.4 ± 0.7 PLA2R1-2b 1.3 ± 0.3 2.1 ± 0.3 nd 5.0 3 ± 0.7 10.1 ± 0.7 PLA2R1-2c 2.0 ± 0.4 3.3 ± 0.5 nd 3.1 ± 0.8 6.8 ± 0.1 PLA2R1-3 0.5 ± 0.1 2.1 ± 0.4 98.6 ± 0.5 23.4 ± 2.4 15.3 ± 0.5

Cited non-patent literature

1. [1] Mikeska T, Candiloro IL, Dobrovica. Epigenomics. 2010; 2: 561-73 ,
2. [2] Candiloro II, Mikeska T, Dobrovic A. Methods Mol Biol. 2011; 791: 55-71 ,
3. [3] Candiloro II, Mikeska T, Hokland P, Dobrovic A., Epigenetics & Chromatin 2008; 1: 7 ,
4. [4] Ehrich M, Nelson MR, Stanssens P, et al. Proc Natl Acad Sci USA. 2005; 102: 15785-90 ,
5. [5] Palanisamy R, Connolly AR, Trau M. Anal Chem. 2011; 83: 2631-7 ,
6. [6] Wee EJ, Rauf S, Shiddiky MJ, Dobrovic A, Trau M. Clin Chem. 2015; 61: 163-71 ,
7. [7] Sykes PJ, Neoh SH, Brisco MJ, High E, Condon J, Morley AA. Biotechniques. 1992; 13: 444-9 ,
8. [8th] Tombal B. EurUrol. 2012; 62: 997-8 ,
9. [9] Tombal B. Eur Urology Suppl. 2006; 5: 511-513 ,
10. [10] Hubbard RA. Annals of Internal Medicine. 2011; 155: 481-92 ,
11. [11] Szyf M. Epigenomics. 2012; 4: 13-4 ,

Claims (15)

Method for diagnosing a tumor disease in an isolated sample with the steps a) bisulfite conversion of the DNA in the sample, b) amplification of a heterogeneously methylated sequence portion of a genome by means of digital PCR, wherein in the digital PCR, a set of fluorescently labeled probes is used, which includes both probes with complementary sequences to the fully methylated epiallel and the unmethylated epilallel of the sequence section as well as probes with complementary sequences contains the partially methylated epialleles of the sequence section, whereby an identification and / or quantification of fully methylated and unmethylated and partially methylated epialleles of the sequence section is carried out. Method according to Claim 1 , characterized in that before step b) a pre-amplification of the DNA to be examined is carried out, wherein preferably the methylated epialleles are amplified more than the non-methylated epiallel. Method according to Claim 1 or 2 , characterized in that the isolated sample is a body fluid. Method according to one of Claims 1 to 3 , characterized in that in step b) a primer pair is used which amplifies both the fully methylated and the unmethylated and partially methylated epiallelic. Method according to one of Claims 1 to 4 , characterized in that in step b) depending on the number n of CpG sites to be examined in the gene sections to be analyzed according to the resulting number 2 ⁿ of possible Epiallelvarianten 2 ⁿ probes with sequences complementary to the individual epialleles on the plus Strand or the minus strand are used. Method according to one of Claims 1 to 5 , characterized in that in step b) the probes for complete and partially methylated epilales are labeled with a fluorescent dye and the unmethylated epiallel probe is labeled with another fluorescent dye such that the probes for the methylated and non-methylated epiallel are each in one duplex dPCR. Method according to one of Claims 1 to 6 , characterized in that in step b) all probes are labeled with different fluorescent dyes and a multiplexed dPCR is performed. Method according to Claim 7 , characterized in that in step b) for each possible epiallel a complementary probe is contained in the set and all probes of the set are used together in a multiplexed dPCR. Kit for the diagnosis of a tumor disease in an isolated sample comprising: i) a primer pair which amplifies both the fully methylated and the unmethylated as well as partially methylated epialleles of a sequence section of a genome, ii) a set of fluorescently labeled probes, the kit containing probes with complementary sequences to the fully methylated and unmethylated epialleles and also probes with complementary sequences to the partially methylated epialleles. iii) reaction buffer for the digital PCR. Kit after Claim 9 , characterized in that all probes are labeled with different fluorescent dyes. Kit after Claim 9 or 10 , characterized in that the probes for quantifying the partially methylated epiallelic have at least two 5'-CG-3 'dinucleotides. Kit after one of Claims 9 to 11 , characterized in that the probes for the quantification of the non-methylated epilule has a total of at least two 5'-CA-3 'dinucleotides or at least two 5'-TG-3' dinucleotides. Kit after one of Claims 9 to 12 characterized in that the probes contain nucleic acids which are selected from SEQ ID no. 1 to 24 and / or the primers contain nucleic acids which are selected from SEQ ID no. 25 and 26. Using a kit according to one of Claims 1 to 13 for the diagnosis of a tumor disease in an isolated sample, preferably with a method according to one of Claims 1 to 8th , Use of a method according to one of Claims 1 to 8th or kits according to one of Claims 9 to 13 or a set of probes like in one of the Claims 9 to 13 defines i.) for the differential diagnosis of benign prostatic hyperplasia, prostatitis and prostate cancer in an isolated specimen, especially at elevated PSA levels or otherwise suspected prostate cancer, ii.) for the diagnosis of mammalian, ovarian or cervical carcinoma iii) for the diagnosis of breast and ovarian carcinoma in an isolated specimen, in particular in the presence of a pathological gene mutation associated with an increased familial risk for mammary and ovarian carcinoma for risk assessment (in particular in case of ambiguous findings in mammography or sonography, iii) Time) and decision for a prophylactic mastectomy and / or ovariectomy, especially if the family planning of the affected patient is not yet completed, or iv.) For the follow-up of a tumor disease, in particular to rule out a minimal residual disease (MRD).

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