RU2830545C1 - Test system and method for detecting a, b, d mutations of npm1 gene for quantitative determination of minimal residual disease - Google Patents

Abstract

FIELD: medicine; molecular biology.

SUBSTANCE: described is a method for detecting A, B, D type NPM1 gene mutations in a test sample and determining MRD, involving: performing RT-PCR for primary analysis for detection of A, B, D type NPM1 gene mutations; setting of RT-PCR for MRD testing; determining MRD in percent, in the case of a sample containing DNA, according to the formula for calculating the allelic load (AL) of mutant allele 100*AO/(1+AO), where AO is the result of calculating the allelic ratio, wherein the allelic ratio (AO) is calculated by formula 5/2∆Ct, where ΔCt is the difference in amplification cycles for the mutant and wild allele, in the case of the sample containing RNA, according to the formula for calculating the relative expression of mutant allele 100/2ΔCt, where ΔCt is the difference in amplification cycles for the mutant and wild allele, wherein a set of reaction mixtures is used for RT-PCR, which includes four mixtures: mixture of oligonucleotides NPM1 W with sequences SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 5; mixture of oligonucleotides NPM1 A with sequences SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 5; mixture of oligonucleotides NPM1 B with sequences SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 5; mixture of oligonucleotides NPM1 D with sequences SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 5, intended for amplification of the analyzed DNA or RNA, which are specific for the wild-type NPM1 gene and for each type of NPM1 gene mutations A, B, D, after detecting the NPM1 mutation type A, B, D. Corresponding sets are disclosed.

EFFECT: invention enables molecular genetic diagnosis of NPM1 gene mutations by a highly sensitive quantitative method based on allele-specific real-time PCR; analytical sensitivity of this method is 10⁻⁴–10⁻⁵ and allows detecting minimal residual disease (MRD) in biological material (bone marrow puncture and blood) of patients diagnosed with acute myeloid leukemia.

32 cl, 15 dwg, 15 tbl

Description

The invention relates to the field of medicine and molecular biology, namely to molecular genetic diagnostics of NPM1 gene mutations by a highly sensitive quantitative method based on allele-specific real-time PCR. The analytical sensitivity of this method is 10 ^-4 -10 ^-5 and allows for detection of minimal residual disease (MRD) in biological material (bone marrow and blood aspirate) of patients diagnosed with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a rather rare disease and accounts for only 1.1% of all cases of malignant neoplasms [1], but it is the most common acute leukemia among the adult population. The incidence in Russia is 1.3 cases per 100 thousand people [2]. Despite all the latest advances in therapy, new targeted drugs, and widespread use of allogeneic stem cell transplantation, AML remains an extremely unfavorable prognostically disease: five-year survival in AML does not exceed 25-30% [1-2]. Acute myeloid leukemia develops due to the accumulation of chromosomal aberrations and gene mutations. Mutations in the nucleophosmin 1 ( NPM1 ) gene are among the most common genetic defects in AML in adults, accounting for 25-30% of all cases and 50-60% of AML cases with a normal karyotype [3-4]. The NPM1 gene mutation is a 4-nucleotide insertion at position c.956-959. Up to 75-80% of mutations have a “type A” insertion - TCtG, and another 10-14% have a “type D and B” insertion - CCtG and CATG [4].

The main methods for diagnosing NPM1 mutations include sequencing, fragment analysis, and allele-specific PCR. However, only the last method can provide the sensitivity of detection required for monitoring minimal residual disease. Modern molecular methods for detecting minimal residual disease should detect tumor cells in an amount of 10 ^-4 -10 ^-5 , i.e. one tumor cell per 10,000-100,000 normal cells. Regarding the methods for determining MRD, the following should be noted. Methods for determining MRD are of significant interest to clinicians, since they allow them to assess the “cure” of the patient, the depth of the achieved remission, make a more balanced therapeutic decision and timely switch to another line of therapy or perform allogeneic stem cell transplantation [5-8]. Numerous groups of scientists have shown the clinical significance of quantitative monitoring of NPM1 mutations in AML [9-16]. These and other studies formed the basis of the recommendations of the European MRD Monitoring Working Group in 2018 and 2021, according to which MRD monitoring of NPM1 mutations in AML should be carried out using quantitative PCR, the analytical sensitivity of which should be 10 ^-4 -10 ^-5 [17-18]. To identify patients with a high risk of relapse, MRD assessment should be performed in the bone marrow after 2 courses of polychemotherapy [18]. At the end of therapy, detection of NPM1 mutations in the bone marrow is inevitably accompanied by a relapse, on average, within several months [16-18].

Currently, there are several methods for determining NPM1 mutations described in scientific articles, as well as commercial kits, the most famous of which is the reagent kit for determining mutations in the NPM1 A gene "ipsogen NPM1 mut A MutaQuant" and the reagent kit for determining mutations in the NPM1 gene "ipsogen NPM1 mut B&D MutaQuant" by QIAGEN Group, USA. Most studies devoted to determining MRD by mutations in the NPM1 gene by real-time PCR [13-17] use the method of Gorello P. et al., 2005 and Chou WC et al., 2007 [20-21]. These methods are based on allele-specific PCR with specific primers to the mutation region, and their high analytical sensitivity is achieved through the use of a TaqMan MGB probe. This type of probes provides stronger binding to the target due to the fragment at the 3’ end of the probe (probe) that binds to the minor groove binder of DNA (MGB), which increases the Tm (melting temperature) and stability of the probe. Such probes provide better discrimination of targets. They have an integrated NFQ quencher, which allows them to better absorb (quench) the signal from fluorescent dyes at the other end of the probe. All these properties, compared to other probes, lead to an increase in the sensitivity of the technique and the accuracy of the results. At the same time, in the technique of Gorello P. et al., 2005, the forward common primer is located in exon 11, and the reverse primers specific to the mutation region and the wild-type primer are located in exon 12, which suggests that analysis is possible only on RNA (cDNA) material, since introns 11-12 of the NPM1 gene contain more than 1000 nucleotides. On the other hand, Chou WC et al., 2007, located the direct common primer in intron 11-12, which makes it possible to analyze only using genomic DNA, but not RNA (cDNA). In the reagent kits for determining mutations in the NPM1 gene “ipsogen NPM1 mut A, B&D MutaQuant Kits” by QIAGEN Group, USA, the technical approach is not disclosed, but the location of the primers is similar to Gorello P. et al., 2005, thus, RNA (cDNA) is used as the material for analysis in this reagent kit. The specified methods for determining MRD by mutation of the NPM1 gene using the PCR method are the closest analogues of the proposed invention. These analogues do not allow using two biological materials at once to detect mutations: RNA and DNA. At the same time, it is obvious that DNA is a more stable molecule than RNA, and its use as a material provides undeniable advantages, for example, the possibility of studying archival material from frozen cells. On the other hand, some laboratories also use RNA (cDNA) for primary testing in AML, in particular for the detection of chimeric transcripts.

The main task, which the present invention is aimed at solving, is the development of specific synthetic oligonucleotides (specific primer sets) and fluorescent probes (tests) that amplify a specific region of the NPM1 gene and mutations of the NPM1 gene of type A, B, D in the RT-PCR reaction, and the development of a method for quantitative determination of the relative proportion of cells with a mutation in both DNA and RNA, a method of allele-specific polymerase chain reaction in real time for detecting mutations of the NPM1 gene of type A, B, D, determining MRD, having an analytical sensitivity of 10 ^-4 -10 ^-5 . Within the framework of the set task, specific primers and tests were designed, which were located inside exon 12, which made it possible to use DNA and RNA (cDNA) as material. In addition, LNA (locked nucleotide acid) modified nucleotides were integrated into the sequence of allele-specific primers, which increased their specificity and sensitivity of mutant allele detection. LNA-modified nucleotide has a greater binding capacity during complementary interaction of nucleotides compared to a normal nucleotide. Under certain conditions, this leads to stronger binding of the primer to the target, which in turn increases the specificity and sensitivity of detection [22-26].

The stated task is solved by the presented group of inventions. Within the framework of the presented invention, specific sets of primers) and fluorescent probes (tests) have been designed, amplifying the nucleotide sequences of the gene in the PCR-RV reactionNPM1And gene mutationsNPM1A, B, D type and a reliable method of multiplex polymerase chain reaction in real time for detection of gene mutations has been developedNPM1A, B, D types, definitions of IBD.

This group of inventions is a cheaper alternative method for determining mutations of the NPM1 A, B, D type gene and determining MRD, which guarantees a reliable analysis result with a sensitivity of 10 ^-4 -10 ^-5 and the ability to use both DNA and RNA isolated from bone marrow cells or patient blood as material, including the ability to study archival material from frozen bone marrow cells or patient blood.

When describing the implementation of the claimed invention, the following abbreviations and definitions are used, which have the indicated meanings in this description, unless otherwise specified:

NPM1 - nucleophosmin gene;

AML - acute myeloid leukemia;

PCR-RT - real-time polymerase chain reaction;

MRD is a well-established term in hematology and oncology, meaning the minimum number of tumor cells after therapy, which is determined in the amount of 10 ^-4 -10 ^-5

BM - bone marrow;

DNA - deoxyribonucleic acid;

cDNA - complementary DNA;

RNA - ribonucleic acid;

AC - allele specific;

MOB-IF - determination of MOB by flow cytofluorimetry;

PCT - polychemotherapy;

Specific (amplification) primers are nucleic acids that bind to one or more target sequences, allowing its amplification to occur.

A test tube is an individual vessel that has the shape and size of a typical laboratory test tube (for example, 4.0 ml vacuum plastic Vacuette tubes for collecting blood, bone marrow, or 2, 1.5, 0.5 ml Eppendorf tubes for working with DNA/RNA, preparing PCR mixtures, or 0.2, 0.1 ml tubes for performing PCR).

An oligonucleotide is a short fragment of DNA obtained by chemical synthesis and used as a primer in the context of the present invention.

A primer is an oligonucleotide complementary to the target DNA that serves as a primer for the synthesis of a complementary chain using DNA polymerase.

Negative control sample - a DNA sample from a healthy donor that is used as a negative control.

Positive control sample - DNA sample with NPM1 type A mutation which is used as a positive control for mutations A, B and D.

Diagnostic specificity is the percentage of correct negative test results in relation to the total number of the control group.

An exon is a section of DNA that codes for proteins.

The probe is a fluorescent probe used in nucleotide sequencing.

Fluorescent probe - is an oligonucleotide with a fluorescent label and a quencher that can bind to the DNA strand during amplification and provides fluorescence during the amplification process.

Detection sensitivity is the minimum copy number that can be detected. "Higher or improved sensitivity" in this context means that the minimum detectable copy number of the mutant allele is reduced.

LNA (locked nucleotide acid) is a modified RNA nucleotide in which the ribose fragment is modified with an additional bridge connecting the 2'-oxygen group and the 4' carbon, which ensures the optimal arrangement of heterocyclic bases in the duplex and leads to a more effective (strong) Watson-Crick interaction between complementary bases. When the conditions described in the implementation of the method as one of the aspects of the present invention are met, it leads to a stronger binding of the primer to the target, which in turn increases the specificity and sensitivity of the determination.

FAM (carboxyfluorescein) - It is a fluorescent dye that is added to the 5' end of an oligonucleotide to synthesize labeled probes for real-time PCR.

BHQ1 is a quencher that is added to the 3' end of an oligonucleotide to synthesize labeled probes for real-time PCR.

Target sequences are the sequences that need to be detected.

Biological material is DNA and RNA isolated from bone marrow cells, patient's blood, including archival material from frozen cells.

Allelic load is the percentage ratio of mutant allele to the total number of alleles (mutant plus wild).

Disclosure of invention

The solution to the problem corresponding to the first aspect of the present invention is based on the list of nucleotide sequences SEQ ID No. 1-6, forming from them groups of sequences complementary to NPM1 and mutations of NPM1 type A, B, D and their use as primers for amplification of NPM1 and mutations of NPM1 type A, B, D, namely:

SEQ ID NO: 1 Homo sapiens DNA nucleotide sequence, wild-type forward primer, consisting of 22 nucleotides, containing an LNA-modified nucleotide at positions 19 and 21;

SEQ ID NO: 2 Homo sapiens DNA nucleotide sequence, forward primer mutant type A, consisting of 23 nucleotides, containing an LNA-modified nucleotide at positions 16 and 20;

SEQ ID NO: 3 Homo sapiens DNA nucleotide sequence, forward primer mutant type B, consisting of 23 nucleotides, containing an LNA-modified nucleotide at positions 17 and 20;

SEQ ID NO: 4 Homo sapiens DNA nucleotide sequence, forward primer mutant type D, consisting of 23 nucleotides, containing an LNA-modified nucleotide at position 20;

SEQ ID NO: 5 Homo sapiens DNA nucleotide sequence, TaqMan probe, consisting of 28 nucleotides, containing BHQ1 at the 3’ end and FAM at the 5’ end;

SEQ ID NO: 6 Homo sapiens DNA nucleotide sequence, reverse primer consisting of 22 nucleotides.

A detailed description of the nucleotide sequences included in the nucleotide sequence listing SEQ ID No. 1-6 is given in Table 1:

No. Name
primers Description Sequence (5'→3') Size,
(nucleotides) SEQ ID NO:1 NPM W Wild type primer TTCCAGGCtATTCAAGAT(C)T(C)T 22 SEQ ID NO:2 NPM A NPM1 gene mutation type A CtATTCAAGATCtCt(G)TCt(G)GCA 23 SEQ ID NO:3 NPM B NPM1 gene mutation type B CtATTCAAGATCtCtG(C)AT(G)GCA 23 SEQ ID NO:4 NPM D NPM1 gene mutation type D CtATTCAAGATCtCtGCCt(G)GCA 23 SEQ ID NO:5 NPM Sample FAM-TGCAGAGTGAGAACtTTCCCCtACCGTGT-BHQ1 28 SEQ ID NO:6 NPMR Reverse primer ACCATTTCCATGTCtGAGCACC 22

The description of the nucleotide sequences and fluorescent probes are presented separately in XML format, which is an appendix to this description.

A schematic representation of the arrangement of primers is shown in Figure 3.

Preferably, within the framework of the implementation of this aspect of the invention, the nucleotide sequences form the following groups:

- nucleotide sequences including the sequences of SEQ ID No. 1, 5 and 6 constitute a group of sequences intended to identify cells without mutations;

- nucleotide sequences including the sequences of SEQ ID No. 2, 5 and 6 constitute a group of sequences intended to identify cells with mutation A;

- nucleotide sequences including the sequences of SEQ ID No. 3, 5 and 6 constitute a group of sequences intended for identifying cells with mutation B;

- nucleotide sequences including the sequences of SEQ ID No. 4, 5 and 6 constitute a group of sequences intended to identify cells with the D mutation.

In relation to this aspect of the invention, the designed specific sets of primers and probes were located within exon 12 of the NPM1 gene, which made it possible to use DNA and RNA (cDNA) as material.

The applicant's data on the occurrence of type A, B, D insertions and non-standard insertions in Russian patients with AML are presented in Figure 2.

A schematic representation of the location of primers within exon 12 is shown in Figure 3.

The solution to the problem corresponding to the second aspect of the present invention is based on the creation of a set of reaction mixtures, which includes 4 mixtures of oligonucleotides for amplification, namely:

1. NPM1 W, for detection of cells without mutation (forward primer SEQ ID NO: 1; reverse primer SEQ ID NO: 6, TaqMan probe SEQ ID NO: 5, labeled with FAM and BHQ1);

2. NPM1 A, for detection of cells with mutation A (forward primer SEQ ID NO: 2; reverse primer SEQ ID NO: 6, TaqMan probe, SEQ ID NO: 5, labeled with FAM and BHQ1);

3. NPM1 B, for detection of cells with mutation B (forward primer SEQ ID NO: 3; reverse primer SEQ ID NO: 6, TaqMan probe SEQ ID NO: 5, labeled with FAM and BHQ1);

4. NPM1 D, for detection of cells with mutation D (forward primer SEQ ID NO: 4; reverse primer SEQ ID NO: 6, TaqMan probe SEQ ID NO: 5, labeled with FAM and BHQ1);

Each oligonucleotide mixture corresponds to one test tube, which is prepared by diluting the oligonucleotides in deionized water to a final concentration of 5 pmol/µl primer and 3.5 pmol/µl sample.

Each oligonucleotide amplification mixture contains the following:

Table No. 2. Composition of the NPM1 W nucleotide sequence mixture for detection of cells without mutation. Final volume of the mixture is 200 µl (100 reactions).

Component Volume, µl. Final primer/sample concentration (pmol/µl) Primer SEQ ID NO: 1 (stock solution concentration 100 pmol/µl) 10 5 Primer SEQ ID NO: 6
(stock solution concentration 100 pmol/µl) 10 5 Fluorescent probe SEQ ID NO: 5
(stock solution concentration 100 pmol/µl) 7 3.5 Deionized water (H2O) 183 µl

Table No. 3. Composition of the mixture of NPM1 A nucleotide sequences for determining cells with mutation A. The final volume of the mixture is 200 µl (100 reactions).

Component Volume, µl. Final primer/sample concentration (pmol/µl) Primer SEQ ID NO: 2 (stock solution concentration 100 pmol/µl) 10 5 Primer SEQ ID NO: 6
(stock solution concentration 100 pmol/µl) 10 5 Fluorescent probe SEQ ID NO: 5
(stock solution concentration 100 pmol/µl) 7 3.5 Deionized water (H2O) 183 µl

Table No. 4. Composition of the mixture of NPM1 B nucleotide sequences for the detection of cells with mutation B. The final volume of the mixture is 200 µl (100 reactions).

Component Volume, µl. Final primer/sample concentration (pmol/µl) Primer SEQ ID NO: 1 (stock solution concentration 100 pmol/µl) 10 5 Primer SEQ ID NO: 6
(stock solution concentration 100 pmol/µl) 10 5 Fluorescent probe SEQ ID NO: 5
(stock solution concentration 100 pmol/µl) 7 3.5 Deionized water (H2O) 183 µl

Table No. 5. Composition of the mixture of NPM1 D nucleotide sequences for the detection of cells with the D mutation. The final volume of the mixture is 200 µl (100 reactions).

Component Volume, µl. Final primer/sample concentration (pmol/µl) Primer SEQ ID NO: 1 (stock solution concentration 100 pmol/µl) 10 5 Primer SEQ ID NO: 6
(stock solution concentration 100 pmol/µl) 10 5 Fluorescent probe SEQ ID NO: 5
(stock solution concentration 100 pmol/µl) 7 3.5 Deionized water (H2O) 183 µl

Packaging information - a test tube with markings. The test tube with the prepared composition can be stored at t-20°C for 30 days.

The solution to the problem corresponding to the third aspect of the present invention is based on the creation of a method for detecting mutations of the NPM1 gene of type A, B, D in a test sample and a method for determining MRD, comprising the following steps:

1. Preparation of materials and components.

2. Conducting RT-PCR for primary testing (conducted in the presence of a mutation (insertion +4bp) of the NPM1 gene, proven by other methods , for example, by fragment analysis).

3. Conducting PCR for MRD testing.

1. Preparation of materials and components:

To implement the method within the framework of the present invention, at least a bone marrow punctate is taken into vacuum plastic tubes with ethylenediaminetetraacetate (EDTA) salt added as an anticoagulant at a final concentration of 2.0 mg/ml. Sodium citrate can also be used as an anticoagulant. To mix the bone marrow punctate with the anticoagulant after taking the material, it is necessary to invert the tube 2-3 times. To isolate DNA and RNA from bone marrow leukocytes or peripheral blood, any method that ensures a sufficient yield of DNA or RNA in quantity and quality is used. Pre-processing and storage of the material are carried out in accordance with the instructions for kits/reagent sets for DNA/RNA isolation from the corresponding biological material. When implementing the claimed invention, we isolated genomic DNA from bone marrow cells using the salt extraction method [29]. DNA concentration was measured spectrophotometrically. DNA can be stored at temperatures below -16 degrees for an indefinite period. RNA (cDNA) was isolated using the standard guanidine/phenol/chloroform extraction method according to Chomczynski [30]. RNA concentration was measured spectrophotometrically. RNA can be stored at temperatures below -40 degrees for a year. 1-2 μg of total RNA were used in the reverse transcription reaction; cDNA was obtained using the Reverta-L kit (Amplisens, Russia) according to the manufacturer's instructions.

When implementing the declared method, the following is not permitted:

1) the presence of PCR reaction inhibitors, for example, heparin;

2) the number of DNA copies in one reaction is less than 500 ng, and the amount of cDNA is less than 10,000 copies of ABL1.

2. Preparation and implementation of RT-PCR for primary research.

For each test sample, for the negative control sample and for the positive control sample, 8 tubes compatible with the real-time PCR instrument are prepared.

In 0.5 or 1.5 ml test tubes, prepare a mixture for PCR based on the following calculation:

• 2.5 x (8N+1) µl PCR buffer

• 2.0 x (8N+1) µl MgCL2

• 2.0 x (8N+1) µl dNTP

• 0.5 x (8N+1) µl Taq Pol

• 13 x (8N+1) µl deionized water,

where N is the number of samples tested, taking into account negative and positive control samples.

The tubes with the PCR mixture are shaken for 3-5 s and centrifuged for 1-3 s in a microcentrifuge/vortex (8000-14000g).

In each of the 8 tubes for real-time PCR, 2 µl of the corresponding oligonucleotide mixture is added, namely:

- 2 separate tubes for primer to NPM1W;

- 2 separate tubes for primer to NPM1A;

- 2 separate tubes for primer to NPM1B;

- 2 separate tubes for primer to NPM1D;

To each tube of 8 tubes, add 20 μl of the PCR mixture and 3 μl of the DNA of the test control sample at a concentration of 20-80 ng/μl (pre-dilute DNA at a concentration of 100-400 ng/μl 5 times: 5 μl of DNA + 20 μl of deionized H2O) or 3 μl of cDNA pre-diluted 5 times (5 μl of cDNA + 20 μl of deionized H2O). The tubes are centrifuged for 1-3 seconds (8000-14000g) in a microcentrifuge.

Then all the tubes are installed in the device block for real-time PCR. The device for real-time PCR is understood as a multifunctional device that combines the functions of a standard amplifier and a fluorescent detector - Real-Time amplifiers. Within the framework of the claimed invention, quantitative TaqMan RT-PCR was carried out on Step One Real-Time PCR (Applied Biosystems, USA) or Real-time CFX96 Touch (Bio-Rad Laboratories, USA) amplifiers.

The PCR reaction (amplification) is carried out in compliance with the following temperature conditions:

- denaturation 95° 5 min;

- then 50 amplification cycles, including: denaturation at 95° - 30 sec; annealing at 65° - 30 sec; synthesis at 72° - 20 sec.

During the initial testing of the biological material, the NPM1 mutation type is detected: A, B, D, or a non-standard insertion. The result is evaluated according to the general principles of real-time PCR. The higher the concentration of the target in the sample, the earlier the rise of the amplification (fluorescence) curve begins and the lower the threshold cycle Ct, i.e. the amplification cycle at which the rise begins. The mutation type in the biological material is determined by which rise of the RT-PCR fluorescence curve began earlier: for the amplification reaction of NPM1 gene mutations A or NPM1 gene mutations B or NPM1 gene mutations type D. The results are displayed as numerical values of the threshold cycle Ct on the device. The allelic load of the mutant allele (for DNA) or the relative expression of the mutant allele (for RNA) is calculated using the formula presented in the section Accounting for RT-PCR Results.

The obtained results are then interpreted. If there is no amplification in the primary study or if the relative amount of the mutant allele is less than 10%, the sample is considered unsuitable for MRD testing by this method due to the presence of a non-standard insertion. A non-standard insertion differs in nucleotide composition or location from type A, B, D insertions (shown in Figure 4 - "non-standard insertion"). When interpreting the results, the number of blasts in the bone marrow material according to the morphological study or the allelic load of the NPM1 mutation obtained during the study by another method are taken into account; fragment analysis is usually used for the primary verification of the NPM1 insertion. The allelic load of the mutant allele, as a rule, is 30-50% of the number of blasts in the sample. If the number of blasts is less than 20-30% in the sample, an allelic load of less than 10% can be obtained with a standard insertion. In complex cases, additional methods can be used to confirm whether a patient has a non-standard NPM1 insertion, such as sequencing, including NGS sequencing.

Thus, in the initial study, two types of answers were given:

- MRD testing is possible with an indication of the type of mutation detected, for example - a mutation of the NPM1 gene type A (B or D) was detected.

- MRD testing by this method is impossible - non-standard (not A, B, D) type of NPM1 gene mutation. Monitoring of MRD by flow cytofluorimetry is recommended.

Illustration of the primary RT-PCR study of NPM1 gene mutations. Amplification curves and interpretation options for the analysis results for standard inserts of types A, B, D and a non-standard insert are shown in Figure 4.

2. Preparation and implementation of RT-PCR for MRD testing.

In patients with NPM1 type A (B or D) mutations detected at the initial study stage, MRD is further determined during therapy. According to recommendations, MRD assessment is performed after the 2nd course of induction, before maintenance therapy, at the end of therapy or before allo-HSCT, every 3 months after the end of therapy, and also at any other points in therapy and time periods as indicated [19].

For each test sample, for the negative control sample and for the positive control sample, 6 tubes compatible with the real-time PCR instrument are prepared.

RT-PCR for determining MRD is carried out in triplicates with a wild-type primer and an already known mutant type (3W+3M).

In 0.5 or 1.5 ml test tubes, prepare a mixture for PCR based on the following calculation:

2.5 x (6N+1) µl PCR buffer;

2.0 x (6N+1) µl MgCL2;

2.0 x (6N+1) µl dNTP;

2.0 x (6N+1) µl oligonucleotide mixture

0.5 x (6N+1) µl Taq Pol

13 x (6N+1) µl deionized water

where N is the number of samples to be tested, taking into account negative and positive control samples. DNA from healthy donors is used as negative controls. A positive sample with a mutation of NPM1 type A, or type B, or type D is used as a positive control.

Next, the tube with the PCR mixture is shaken for 3-5 seconds on a vortex and centrifuged for 1-3 seconds in a microcentrifuge (8000-14000g).

Each of the 6 real-time PCR tubes is loaded with 2 µl of the corresponding oligonucleotide mixture as follows:

For the test sample in which the NPM1 type A gene mutation was detected at stage 1, for the negative control sample and for the positive control sample:

- 3 separate tubes for primer to NPM1W;

- 3 separate tubes for primer to NPM1A;

For the test sample in which the NPM1 type B gene mutation was detected at stage 1, for the negative control sample and for the positive control sample:

- 3 separate tubes for primer to NPM1W;

- 3 separate tubes for primer to NPM1B;

For the test sample in which the NPM1 type D gene mutation was detected at stage 1, for the negative control sample and for the positive control sample:

- 3 separate tubes for primer to NPM1W;

- 3 separate tubes for primer to NPM1D;

Next, 20 µl of the PCR mixture is added to these tubes. 3 µl of DNA of the test or control sample at a concentration of 200-300 ng per µl or 3 µl of cDNA are added to each of the 6 tubes.

The amplification process is carried out, and the amount of DNA in one reaction should be more than 500 ng, and the amount of cDNA should exceed 10,000 copies of ABL1 (separately tested with ABL1 standards), which allows achieving high sensitivity of the claimed method. With a lower amount of DNA (cDNA) or poor quality of DNA (cDNA), a false negative result may be obtained.

Next, the tube is centrifuged for 1-3 s in a microcentrifuge (8000-14000g).

Next, all the tubes are placed in the device block for real-time PCR amplification.

The PCR reaction (amplification) is carried out in compliance with the following temperature conditions:

- denaturation 95° 5 min;

- then 50 amplification cycles, including: denaturation at 95° - 30 sec; annealing at 65° - 30 sec; synthesis at 72° - 20 sec.

Accounting for PCR results in RV.

The difference in the Ct amplification cycles between the mutant and wild-type primers for NPM1 A gene mutations or NPM1 B gene mutations or NPM1 D gene mutations is used to determine the quantitative value of the mutant allele .

For DNA, the allelic load (AL) of the mutant allele was calculated in percent using the formula 100*AO/(1+AO), where AO is the result of calculating the allelic ratio. The allelic ratio (AR) w/m is calculated using the formula 5/2 ^Δ Ct; where ΔCt is the difference in amplification cycles between the mutant and wild primers.

For RNA (cDNA), the relative expression (%) of the mutant allele is calculated using the formula 100/2ΔCt; where ΔCt is the difference in amplification cycles between the mutant and wild-type primers.

In the case of NPM1 positive AML, each tumor cell contains this mutation, therefore, by determining the allele load or relative expression of the mutant allele, the diagnostic process actually determines the % of residual tumor cells (RTC).

Interpretation of results:

In accordance with the general principles of RT-PCR evaluation, van der Velden (2003) [31] distinguishes several types of responses: negative, positive (with quantitative evaluation and without quantitative evaluation). A positive non-quantitative response means the presence of a mutation in such a small amount that it makes its quantitative calculation impossible.

The following interpretation of the results of MRD determination by the RT-PCR method is made:

Criteria for a negative response regarding the absence of mutations of type A, B, D:

1) the start of amplification of the wild primer no later than the 21st cycle, this corresponds to a DNA quantity of more than 500 ng and indicates its good quality; for cDNA, it is necessary to focus on the copy number of ABL1, which should correspond to more than 10,000 copies ;

2) amplification with a mutant primer does not occur; and/or when calculating the MRD, the number of % tumor cells is outside the sensitivity of the method, i.e. is less than 0.001%. In the case of DNA, this corresponds to a difference in the amplification cycles (ΔCt) of the wild-type and mutant primers of more than 19 cycles, and in the case of RNA (cDNA), it corresponds to ΔCt > 16.5 and/or amplification with a mutant primer occurs after the 39th cycle, which also corresponds to the maximum sensitivity of the determination, see the graph for determining the analytical sensitivity (Figure 6).

Table 6 presents the results of the study illustrating examples that meet the criteria for a negative response. It follows from this table that all cases meet the criteria for a negative response: Δ Ct≥19 or Ct NPM1 M > 39; while Ct NPM1 W <21 cycles. The allelic load is not calculated in this case. In this example, the AN figure (%) is given only to show that it is below the declared sensitivity detection limit of <0.001%. *on - no amplification.

Table 6. Examples of negative results in determining MRD in bone marrow DNA samples.

Patient No. Insert type Ct NPM1 W (average) Ct NPM1 M (average) Δ Ct AN (%) 162-352 A 20.3 39.67 19.37 0,000736 162-268 A 18.65 42.25 23.6 3.96E-05 162-81 D 19.52 43.69 24.17 2,64E-05 162-206 A 19.45 42.29 22.84 6,67E-05 161-849 B 20.03 41.87 21.84 0,000134 160-157 A 19.2 *on - - 161-272 A 19.39 39,49 20.1 0.00045 161-105 A 161-20 A 17.36 43.85 26.49 5.3E-06

Criteria for a positive quantitative response (presence of a mutation with the possibility of its quantitative determination):

1) there is specific amplification with mutant primers in 2 or 3 repeats and the beginning of the rise no later than the 39th cycle;

2) When calculating the MRD, the amount determined is more than 0.001%, which corresponds to a ΔCt of the wild and mutant primer of less than 19 cycles and less than 16.5 cycles for RNA (cDNA);

3) 3 out of 3 repetitions have a Ct difference of less than 1.5 cycles or 2 out of 3 repetitions have a Ct difference of less than 1 cycle;

4) the amplification cycle (Ct) should be at least 3 units less than the lowest Ct of the polyclonal control. Usually, amplification of the polyclonal control either does not occur or occurs later than the 40th cycle.

Tables 7 and 8 show the results of the study confirming the fact of specific amplification with mutant primers and satisfying the criteria for a positive quantitative response in the primary DNA testing (Table 6) and in determining MRD (listed in Table 8). All cases meet the criteria for a positive quantitative response and have an allelic load (AL) of more than 10%, except for patients marked with *. With AL < 10%, it is necessary to estimate the level of blast cells in the material or AL by another method, in case of an obvious discrepancy, the insertion is non-standard (N). In this table, patients marked with * had a low percentage of allelic load, but only patients 146-167 and 146-544 had an obvious discrepancy between AL and the number of blast cells, subsequently, the non-standard NPM1 insertion was confirmed in these two patients by NGS.

Table 7. Examples of primary DNA testing to determine the mutation type in patients for whom the NPM1 +4bp mutation has been confirmed by fragment analysis.

Patient No. Insert type Ct NPM1 W (average) Ct NPM1 M (average) Δ Ct AN (%) Comment 142-544 B 24.9 30.2 5.30 11,26212 142-545 A 22.41 25,27 2.86 40,78252 142-892 A 22.48 27.12 4.64 16,70382 143-20* D 21.7 27.8 6.1 6,794078 Blast cells - 8.0% 143-525 A 23.4 27.08 3.68 28,0629 143-650 A 23.2 26.76 3.56 29,77227 143-267 A 21.94 25.56 3.62 28,91009 143-679 A 21.5 25.8 4.30 20,2443 143-900 B 22.4 26.7 4.30 20,2443 146-641 A 24.14 27.9 3.76 26,95719 146-254 D 21.7 26.45 4.75 15,66971 146-167* N 19.5 30 10.50 0.344079 Blast cells - 45%.
Non-standard insertion confirmed by NGS - “GCtG” 146-544* N 22.9 30 7.10 3,516496 Blast cells - 67%.
Non-standard insertion confirmed by NGS - “CtGG” 147-51 A 24.5 28.05 3.55 29,91739 147-374 A 24.9 28 3.10 36,83462 147-657 A 24 26.3 2.30 50,37998 147-863 A 23.48 28.9 5.42 10,45726 148-57 A 22.61 25.35 2.74 42,80564 148-798 A 22.05 25.74 3.69 27,92318 149-630 B 24.6 29.6 5 13,51351 149-595 A 22.94 28.2 5.26 11,54219 149-859 A 21.08 26.02 4.94 14,00699 151-271 A 22.1 25.8 3.7 27,78389 151-678 IN 23.64 28 4.36 19,58111 152-434 A 21.5 26 4.5 18,09796 152-130 B 22.3 27.6 5.3 11,26212 152-602 A 22.5 25.5 3 38,46154 152-601 A 24 28 4 23,80952 152-679 A 21.5 26 4.5 18,09796 153-80 A 22 26.9 4.9 14,3443 154-126 A 23.25 25.9 2.65 44,33932 154-363* A 23.41 29.66 6.25 6,164525 According to fragment analysis 8% 154-690* A 27.1 32.8 5.7 8,774369 According to fragment analysis 13% 154-476 A 22.5 25 2.5 46,91816 154-623 A 23.5 27.1 3.6 29,19583 154-625 A 22 25.8 3.8 26,41476 155-219 A 27.1 30.6 3.5 30,64907 155-687 A 19.4 24.4 5 13,51351 156-40 A 22.2 24.2 2 55,55556 156-107 A 21 22.75 1.75 59,78297 157-962 A 24.6 27.65 3.05 37,64459 159-262 A 21 25 4 23,80952 156-767 A 20 23 3 38,46154 160-265 A 22.3 27.1 4.8 15,21717 156-296 IN 19.35 24.3 4.95 13,92371 160-561 A 23.9 27 3.1 36,83462 160-961 D 22.4 27 4.6 17,09316 161-88 A 21.4 25.1 3.7 27,78389 160-994* A 25.7 32,36 6.66 4,711412 According to fragment analysis 4% 158-383 A 21.94 26.1 4.16 21,8564 158-629 B 22.4 25.6 3.2 35,23711 161-996 D 21.6 25.8 4.2 21,38656 162-244 A 21.3 24.6 3.3 33,67195 161-880 A 20.86 24.2 3.34 33,05555

Examples of positive quantitative responses in MRD determination in bone marrow DNA samples from patients with AML and a known insertion type in the NPM1 gene detected during the initial study are shown in Table 8. All cases meet the criteria for a positive quantitative response: Δ Ct<19; Ct NPM1 M <39; three Ct NPM1 M repeats have a difference of less than 1.5 cycles or two Ct NPM1 M repeats have a difference of less than 1 cycle and are amplified three cycles earlier than the polyclonal control.

Table 8. Examples of positive quantitative responses in MRD determination in bone marrow DNA samples from patients with AML and a known insertion type in the NPM1 gene identified during the initial study.

Patient No. Insert type Ct NPM1 W (average) Ct NPM1 M (average) Δ Ct AN (%) 144-688 A 17.92 33.17 15.25 0.012829 144-688 A 17.92 33.17 15.25 0.012829 144-746 A 19.3 29.8 10.50 0.344079 145-170 A 19.3 33.75 14.45 0.022335 142-693 A 20.6 38 17.40 0.002891 143-120 A 18.03 31.04 13.01 0,060577 146-526 A 19.1 35.77 16.67 0.004795 147-887 A 22.47 37.5 15.03 0.014943 148-767 A 22.17 37.7 15.53 0.010566 148-797 A 20.45 28.8 8.35 1,509263 148-816 B 20 38 18 0,001907 149-27 A 20.2 35.4 15.2 0.013282 146-838 A 21.9 35.2 13.3 0.049551 150-269 A 20.14 38 17.86 0.002102 147-504 B 18.62 28.19 9.57 0.65353 151-18 A 18.7 31.6 12.9 0.065373 150-910 IN 20.3 37.3 17 0.003815 151-597 A 22.2 35.3 13.1 0.056915 152-982 A 21.8 35.6 13.8 0.035043 152-862 A 19 35.6 16.6 0.005033 152-677 A 19.24 35.28 16.04 0,00742 153-186 A 19.5 33.6 14.1 0.028466 153-297 A 19.4 35 15.6 0.010066 153-916 A 21.91 30.44 8.53 1,334594 154-124 A 21.56 36.7 15,14 0,013846 154-576 A 21 34.8 13.8 0.035043 154-625 B 21.1 36.6 15.5 0.010788 155-454 A 17.92 33.8 15.88 0,00829 156-285 A 21 35 14 0.030508 155-864 A 20.2 37.75 17.55 0.002605 156-265 A 20.3 30.8 10.5 0.344079 156-651 A 18.2 36.96 18.76 0.001126 156-386 A 18.4 35.8 17.4 0.002891 156-704 A 20.57 33.56 12.99 0,061422 157-366 A 18.2 31,36 13.16 0.054598 158-21 A 18.55 35.24 16.69 0.004729 158-224 IN 17.4 33,44 16.04 0,00742 158-285 A 19 26 8 1,915709 160-669 A 16.8 29.5 12.7 0.075087 161-687 A 20 33.4 13.4 0.046235 161-793 A 18 35.7 17.7 0.002348 162-11 A 18.2 31.88 13.68 0.038082 162-463 A 21.46 37.8 16.34 0.006027 162-386 A 19.7 31.7 12 0.121921 162-123 D 18.8 36.5 17.7 0.002348

Criteria for a positive non-quantitative response (presence of a mutation in an amount below the counting sensitivity): all criteria for a positive quantitative response are met except for the spread between repeats: the spread between two or three repeats is more than 1 or 1.5 cycles, respectively.

Table 9 shows the results of the study confirming the fact of specific amplification with mutant primers and satisfying the criteria for a positive non-quantitative response.

Examples of a positive response to MRD determination in bone marrow DNA samples without quantification (non-quantitative) are shown in Table 9. These cases only partially satisfy the criteria for a positive quantitative response, except for the point about the coincidence of 2-3 repeats in cycles, which makes AN calculation impossible. In example No. 1, three Ct NPM1 M repeats satisfy the criteria for a positive response, but have a large spread. In example No. 2, two Ct NPM1 M repeats satisfy the criteria for a positive response, but have a difference of more than 1 cycle.

Table 9. Examples of a positive response to the determination of MRD in bone marrow DNA samples without determination of quantity (non-quantitative).

Patient No. Insert type Ct NPM1 W
average Ct NPM1 M
average
(repetitions in brackets) ΔCt Comment 161-793 A 18.71 35.39 (37.65; 32.53; 35.98) 18.95; 13.82; 17.27 AN calculation is not possible because the spread between NPM1 A repeats is 5.13- 161-760 A 18,16 37.34 (33.77; 36.11) 15.61;
17.95 AN calculation is not possible because one of the repeats has no amplification (na), and the spread between the two NPM1 A repeats is 3.6

If the criteria for a negative response are met, but the amount of DNA in the study is insufficient to achieve a sensitivity of 10 ^-5 , i.e. when Ct W (average)> 21 cycles, or the copy number in the case of RNA (cDNA) is less than 10,000 copies, the appropriate response must be given: “Insufficient sensitivity, repeat study required.”

There is a small group of results, the so-called “gray zone” or results suspicious for the presence of MRD. Such results are considered negative according to the criteria, but the criteria for a positive sample are partially met. For example, amplification meets the positive criteria only in one repeat, or amplification in 2 or 3 repeats partially meets the positive criteria, for example, it occurred a little later than cycle 39, but three cycles earlier than the polyclonal control. This situation is possible if a patient with MRD+ did not have enough cells (DNA) for a full study or the percentage of tumor cells in the sample being studied is extremely low. In any case, such cases suspicious for the presence of MRD should be noted in the form of comments to the analysis, or in the form of a response with a special wording that “the analysis is suspicious for the presence of MRD, active monitoring of the patient is recommended, including repeating the study in a month.” Examples of “gray zone” results, i.e. suspicious for the presence of MRD. There is partial compliance with positive criteria, examples are given in Table 10. From the given examples it is clear that there is partial compliance with positive criteria (see comments). Amplification took place earlier than 3 cycles from the polyclonal control, i.e. it is in the positive region. In such cases, when the researcher suspects that this case is not negative, we suggest entering the answer “suspected of having MRD”. Allelic load is not calculated in this case. In this example, the AN figure (%) is given for demonstration purposes only. *on - no amplification.

Table 10. Examples of “gray zone” results, i.e. suspicious for the presence of MRD. There is partial compliance with positive criteria

Patient No. Insert type Ct NPM1 W (average) Ct NPM1 M (average) ΔCt AN (%) Comments 148-170 A 22.67 39.7 (39.8; 39.6) 17.03 0.004 Two replicates with a spread of less than 1 cycle have an increase after cycle 39, but ΔCt is less than 19 cycles. 143-680 A 20.66 36.2 (one rep) - - Only one repetition satisfies the positive criteria 145-912 B 18.7 38.42 (38.57; 38.27) 19.72 0,0006 Two repetitions with a spread of less than 1 cycle have an increase to 39 cycles, but ΔCt is more than 19 cycles. 149-447 A 24.6 39.53 (39.7; 39.4, 39.5) 14.93 0,016 Three replicates with a spread of less than 1.5 cycles have an increase after cycle 39, but ΔCt is less than 19 cycles.

The amplification curves illustrating the results of MRD determination are shown in Figure 5.

The claimed method showed the following results

The analytical sensitivity of the method was 10 ^-5 . The sensitivity of the NPM1 mutation (type A) determination by the AS-PCR method in the form of a calibration (standard) curve is shown in Figure 6.

The diagnostic specificity was 98.7%. Clinical sensitivity and accuracy were not assessed due to the lack of a reference method with similar analytical sensitivity for MRD assessment. All primary patients (n - 55) with proven mutation of the NPM1 gene type A, B, D had a positive result in the primary study.

The selection of temperature conditions for maximum PCR specificity is shown in Figure 7.

Graphs of RT-PCR curves and the value of threshold cycles depending on the annealing temperature of primers. The maximum specificity of determination was achieved at a temperature of 65°C. (Figure 7).

To confirm the capabilities of this technique, we present our 2-year experience of using it in clinical practice. To date, 152 primary patients have been analyzed who had a mutation (+4 bp insertion) in exon 12 of the NPM1 gene proven by fragment analysis. The possibility of MRD monitoring was shown in 87.5% (133 out of 152). That is, 19 patients in our cohort had a non-standard type of NPM1 insertion, which was confirmed by the NGS method. Forty patients were monitored for MRD using this method. The summary results of tumor cell clearance are shown in Figure 8. It is shown that only by the third course of PCT the number of MRD-positive patients decreases to 12.5%. The summary results of tumor cell clearance (% of MRD positive patients) after the 1st, 2nd and 3rd courses of PCT are presented (Figure 8).

The essence of the group of inventions is explained by examples of specific implementation, which do not limit the scope of the invention.

Clinical case 1. Patient Sh., 37 years old. Diagnosed with acute myeloid leukemia. During the initial examination, a mutation of the NPM1 gene was detected by fragment analysis (insertion + 4 nucleotides in exon 12). An initial study was conducted by RT-PCR for the presence of insertions of type A, B, D and the possibility of further determination of MRD. Response: NPM1 type A mutation was detected, the allele load was 47%, MRD determination is possible. During the study after the second course of chemotherapy, MRD was detected, the allele load was 1.9%, which indicated primary resistance to the therapy. Doctors revised the treatment tactics and switched to second-line therapy.

Figure 9. RT-PCR results demonstrate a positive result for MRD determination after the 2nd course of chemotherapy (clinical case No. 1).

Case 2. Patient S., 28 years old. Was observed in long-term remission of acute myeloid leukemia. During the next examination, a questionable result (positive without determining the quantity) of the NPM1 gene mutation was found using a patented method. The patient was taken under observation and a positive response was received within a month, with a further increase in the allelic load. According to international recommendations, with a 10-fold increase in the allelic load of the NPM1 mutation in the blood or bone marrow, it is recommended to switch to salvage therapy, since such an increase is inevitably accompanied by a relapse within an average of 3 months. The patient began to select an unrelated donor. AML relapse was diagnosed in March 2022, six months after the initial detection of the NPM1 mutation. Thus, doctors had time to begin donor selection and think over the therapy. This clinical case very well illustrates the high analytical sensitivity of the technique, since MRD immunophenotyping (MRD-IF) became positive only before the development of a relapse (Figure 10).

Results of real-time PCR for the determination of NPM1, DNMT3A, FLT3-TKD mutations and flow cytometry data as an illustration of clinical case No. 2 (Figure 10).

Below we provide examples of the implementation of the claimed invention in the form of a comparative analysis using DNA and RNA as an example.

Patient 3. The amplification cycles of the wild-type primer and primers for mutations of type A, B, and D are given for DNA and RNA isolated from one bone marrow sample. The type B insertion is determined. The calculated values of the allele load (%) for DNA and the relative expression of the mutant allele (%) for RNA are given in accordance with the calculation formulas for DNA and RNA given in the section on accounting for PCR results in real-time analysis of this description.

Table 11 illustrates the amplification cycles (Ct) for DNA and RNA, the allelic load of the mutant allele (%) for DNA, and the relative expression of the mutant allele (%) for RNA in Patient 3.

Primer DNA Ct RNA (cDNA) Ct Allelic load and relative expression of the mutant allele (%)
for DNA and RNA W 22 26.3 A 29.5 30.5 B 27.03 29.45 13.3% and 10.13% D 38 38.46

The amplification curves illustrating this example are shown in Figure 11.

Patient 4. The amplification cycles of the wild-type primer and primers for mutations of type A, B, and D are given for DNA and RNA isolated from one bone marrow sample. The type A insertion is determined. The calculated values of the allele load (%) for DNA and the relative expression of the mutant allele (%) for RNA are given in accordance with the calculation formulas for DNA and RNA given in the section on accounting for PCR results in real-time analysis of this description.

Table 12 illustrates the amplification cycles (Ct) for DNA and RNA, the allelic load of the mutant allele (%) for DNA, and the relative expression of the mutant allele (%) for RNA in Patient 4.

Primer DNA Ct RNA (cDNA) Ct Allelic load and relative expression of the mutant allele (%)
for DNA and RNA W 20.44 24.12 A 24.55 25.95 22.45% and 28.12% B 29.8 31.78 D 28 28.76

The amplification curves illustrating this example are shown in Figure 12.

Patient 5. The amplification cycles of the wild-type primer and primers for mutations of type A, B, and D are given for DNA and RNA isolated from a single bone marrow sample. The type D insertion is determined. The calculated values of the allele load (%) for DNA and the relative expression of the mutant allele (%) for RNA are given in accordance with the calculation formulas for DNA and RNA given in the section on accounting for PCR results in real-time analysis of this description.

Table 13 illustrates the amplification cycles (Ct) for DNA and RNA, the allelic load of the mutant allele (%) for DNA, and the relative expression of the mutant allele (%) for RNA in patient 5.

Primer DNA Ct RNA (cDNA) Ct Allelic load and relative expression of the mutant allele (%)
for DNA and RNA W 22.94 24.65 A 30.72 28.74 B 31.67 31.78 D 25.7 26.17 42.5% and 34.87%

The amplification curves illustrating this example are shown in Figure 13.

Patient 6. The amplification cycles of the wild-type primer and the primers for the A-type mutation are presented for DNA and RNA isolated from a single bone marrow sample. The calculated values of MRD (%), i.e., the allelic load (%) for DNA and the relative expression of the mutant allele (%) for RNA are given in accordance with the calculation formulas for DNA and RNA given in the section on accounting for PCR results in real-time analysis of this description.

Table 14 illustrates the amplification cycles (Ct) for DNA and RNA, MRD value (%) for DNA and RNA in patient 6.

Primer DNA Ct RNA (cDNA) Ct MOB (%)
DNA and RNA W 19.4 23.46 A 35.07 (35.9; 36.7; 34.6) 35.83 (34.9; 36.1; 36.5) 0.01% and 0.02%

The amplification curves illustrating this example are shown in Figure 14.

Patient 7. The amplification cycles of the wild-type primer and the primers for the B-type mutation are presented for DNA and RNA isolated from a single bone marrow sample. The calculated values of MRD (%), i.e., the allele load (%) for DNA and the relative expression of the mutant allele (%) for RNA are given in accordance with the calculation formulas for DNA and RNA given in the section on accounting for PCR results in real-time analysis of this description.

Table 15 illustrates the amplification cycles (Ct) for DNA and RNA, MRD value (%) for DNA and RNA in patient 7.

Primer DNA Ct RNA (cDNA) Ct % MOB
DNA and RNA w 19.04 23,24 B 36.08 (36.35; 36.72; 35.17) 38.43 (39.02;37.84) 0.004% and 0.003%

The amplification curves illustrating this example are shown in Figure 15.

The essence of the invention is explained in graphic materials:

Figure 1. Presentation of methods for primary confirmation of the presence of a mutation in the NPM1 gene (Sanger sequencing, fragment analysis, NGS - next generation sequencing). The sensitivity of determination by these methods does not exceed 10 ^-2 ;

Figure 2. Applicant's data on the incidence of type A, B, D insertions and non-standard insertions in Russian patients with AML;

Figure 3. Schematic representation of the arrangement of primers/probes;

Figure 4. Illustration of the primary RT-PCR study of NPM1 gene mutations. Amplification curves and variants of interpretation of the analysis results for standard insertions of types A, B, D and a non-standard insertion;

Figure 5. Amplification curves illustrating the result of MRD determination;

Figure 6. Sensitivity of NPM1 mutation detection by AS-PCR (type A). Calibration (standard) curve;

Figure 7. Graphs of RT-PCR curves and the value of threshold cycles depending on the annealing temperature of primers. The maximum sensitivity and specificity of the determination was achieved at a temperature of 65°C;

Figure 8. The summary results of tumor cell clearance (% of MRD positive patients) after the 1st, 2nd and 3rd courses of chemotherapy are presented;

Figure 9. RT-PCR results demonstrate a positive result for MRD determination after the 2nd course of chemotherapy (clinical case No. 1);

Figure 10. Results of real-time PCR for the determination of NPM1, DNMT3A, FLT3-TKD mutations and flow cytometry data as an illustration of clinical case No. 2.

Figure 11. Amplification curves illustrating the primary RT-PCR study of NPM1 gene mutations (insertions of type A, B, D) on DNA and RNA material in patient 3.

Figure 12 Amplification curves illustrating the primary RT-PCR study of NPM1 gene mutations (insertions of type A, B, D) on DNA and RNA material in patient 4.

Figure 13 Amplification curves illustrating the primary RT-PCR study of NPM1 gene mutations (insertions of type A, B, D) on DNA and RNA material from patient 5.

Figure 14 Amplification curves illustrating the determination of MRD by RT-PCR ( NPM1 type A insert) on DNA and RNA material from patient 6.

Figure 15 Amplification curves illustrating the determination of MRD by RT-PCR ( NPM1 B type insert) on DNA and RNA material from patient 7.

Sources of information taken into account when preparing the description of the invention:

1) Kaprin A.D. Clinical guidelines. Acute myeloid leukemia. Adults. 2020. https://oncology-association.ru/wp-content/uploads/2020/09/ostrye_mieloidnye_lejkozy.pdf.

2) Malignant neoplasms in Russia in 2021 (incidence and mortality). Ed. by A.D. Kaprin, V.V. Starinsky, A.O. Shakhzadova. Moscow: P.A. Herzen Moscow Oncology Research Institute - branch of the National Medical Research Center of Radiology of the Ministry of Health of the Russian Federation, 2018.

3) Falini B., Nicoletti I., Martelli M.F., Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): Biologic and clinical features. Blood. 2007; 109:874-885. doi: 10.1182/blood-2006-07-012252.

4) Heath E.M., Chan S.M., Minden M.D., Murphy T., Shlush L.I., Schimmer A.D. Biological and clinical consequences of NPM1 mutations in AML. Leukemia. 2017; 31: 798-807. doi: 10.1038/leu.2017.30.

5) Ossenkoppele G., Schuurhuis G.J. MRD in AML: Does it already guide therapy decision-making? Hematol. Am. Soc. Hematol. Educ. Program. 2016; 2016: 356-365. doi: 10.1182/asheducation-2016.1.356.

6) Tomlinson B., Lazarus H.M. Enhancing acute myeloid leukemia therapy-Monitoring response using residual disease testing as a guide to therapeutic decision-making. Expert Rev. Hematol. 2017; 10: 563-574. doi: 10.1080/17474086.2017.1326811.

7) Hourigan C.S., Gale R.P., Gormley N.J., Ossenkoppele G.J., Walter R.B. Measurable residual disease testing in acute myeloid leukemia. Leukemia. 2017; 31: 1482-1490. doi: 10.1038/leu.2017.113.

8) Mosna F., Capelli D., Gottardi M. Minimal residual disease in acute myeloid leukemia: Still a work in progress? J. Clin. Med. 2017; 6: 57. doi: 10.3390/jcm6060057.

9) Buccisano F., Maurillo L., Del Principe M.I., Di Veroli A., De Bellis E., Biagi A., Zizzari A., Rossi V., Rapisarda V., Amadori S., et al. Minimal residual disease as a biomarker for outcome prediCtion and therapeutic optimization in acute myeloid leukemia. Expert Rev. Hematol. 2018; 11: 307-313. doi: 10.1080/17474086.2018.1447378.

10) Coltoff A., Houldsworth J., Keyzner A., Renteria A.S., Mascarenhas J. Role of minimal residual disease in the management of acute myeloid leukemia-A case-based discussion. Ann. Hematol. 2018;97:1155-1167. doi:10.1007/s00277-018-3330-9.

11) Shayegi N, Kramer M, Bornha¨user M, et al; Study Alliance Leukemia (SAL). The level of residual disease based on mutant NPM1 is an independent prognostic factor for relapse and survival in AML. Blood. 2013; 122(1):83-92.

12) Hubmann M, Köhnke T, Hoster E, Schneider S, Dufour A, Zellmeier E, Fiegl M, Braess J, Bohlander SK, Subklewe M, Sauerland MC, Berdel WE, Büchner T, Wörmann B, Hiddemann W, Spiekermann K. Molecular response assessment by quantitative real-time polymerase chain reaCtion after induCtion therapy in NPM1-mutated patients identifies those at high risk of relapse. Haematologica. 2014; 99 (8):1317-1325. doi: 10.3324/haematol.2014.104133.

13) Balsat M, Renneville A, Thomas X, et al. PostinduCtion minimal residual disease prediCts outcome and benefit from allogeneic stem cell transplantation in acute myeloid leukemia with NPM1 mutation: a study by the Acute Leukemia French Association Group. J Clin Oncol. 2017; 35(2):185-193.

14) Krönke J, Schlenk RF, Jensen K-O, et al. Monitoring of minimal residual disease in NPM1-mutated acute myeloid leukemia: a study from the German-Austrian acute myeloid leukemia study group. J Clin Oncol. 2011; 29: 2709-2716.

15) Ivey A., Hills R.K., Simpson M.A., Jovanovic J.V., Gilkes A., Grech A., Patel Y., Bhudia N., Farah H., Mason J., et al. Assessment of minimal residual disease in standard-risk AML. N.Engl. J. Med. 2016; 374: 422-433. doi: 10.1056/NEJMoa1507471

16) Lambert J, Lambert J, Nibourel O, Pautas C, Hayette S, Cayuela JM, Terré C, Rousselot P, Dombret H, Chevret S, Preudhomme C, Castaigne S, Renneville A. MRD assessed by WT1 and NPM1 transcript levels identified distinCt outcomes in AML patients and is influenced by gemtuzumab ozogamicin. Oncotarget. 2014 Aug 15;5(15):6280-8. doi: 10.18632/oncotarget.2196.

17) Dvorakova D., Racil Z., Jeziskova I., Palasek I., Protivankova M., Lengerova M., Razga F., Mayer J. Monitoring of minimal residual disease in acute myeloid leukemia with frequent and rare patient-specific NPM1 mutations. Am. J. Hematol. 2010;85:926-929. doi: 10.1002/ajh.21879.

18) Schuurhuis G.J., Heuser M., Freeman S., Béné M.C., Buccisano F., Cloos J., Grimwade D., Haferlach T., Hills R.K., Hourigan C.S., et al. Minimal/measurable residual disease in AML: A consensus document from the European LeukemiaNet MRD Working Party. Blood. 2018; 131: 1275-1291. doi: 10.1182/blood-2017-09-801498.

19) Heuser M, et al. 2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party. Blood. 2021;138(26):2753-2767. doi: 10.1182/blood.2021013626.

20) Gorello P, Cazzaniga G, Alberti F, Dell'oro MG, Gottardi E, Specchia G et al. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations. Leukemia 2006; 20: 1103-1108.

21) Chou WC, Tang JL, Wu SJ, et al. Clinical implications of minimal residual disease monitoring by quantitative polymerase chain reaction in acute myeloid leukemia patients bearing nucleophosmin (NPM1) mutations. Leukemia. 2007 May;21(5):998-1004. doi: 10.1038/sj.leu.2404637. Epub 2007 Mar 15.

22) Vester B, Wengel J. LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 2004; 43: 13233-13241.

23) Latorra D, Campbell K, Wolter A, Hurley JM. Enhanced allele-specific PCR discrimination in SNP genotyping using 3' locked nucleic acid (LNA) primers. Hum Mutat 2003; 22(1):79-85. DOI: 10.1002/humu.10228. PMID: 12815597.

24) Morandi L, de Biase D, Visani M, et al. Allele specific locked nucleic acid quantitative PCR (ASLNAqPCR): an accurate and cost-effective assay to diagnose and quantify KRAS and BRAF mutation. PLoS One 2012; 7(4):e36084. DOI: 10.1371/journal.pone.0036084. PMID: 22558339.

25) Abdelhamid E, Besbes S, Renneville A, et al. Minimal Residual Disease assessment of IDH1/2 mutations in Acute Myeloid Leukemia by LNA-RQ-PCR. Tunis Med 2016; 94(3):190-7. PMID: 27575502.

26) Albitar A. Ma W, DeDios I, et al. Positive selection and high sensitivity test for MYD88 mutations using locked nucleic acid. Int J Lab Hematol 2016; 38(2):1 33-40. DOI: 10.1111/ijlh.12456. PMID: 26797804.

27) Thiede S, Creutzig E, Illmer T, Schaich M, V Heise, G Ehninger, O Landt. Rapid and sensitive typing of NPM1 mutations using LNA-mediated PCR clamping. Leukemia. 2006 OCt;20(10):1897-9. doi: 10.1038/sj.leu.2404367.

28) Thiede C, Koch S, Creutzig E, Steudel C, Illmer T, Schaich M et al. Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood 2006; 107: 4011-4020.

29) Sidorova Yu.V., Sorokina T.V., Biderman B.V., Nikulina E.E., Kisilichina D.G., Naumova E.V., Pochtar M.E., Lugovskaya S.A., Ivanova V.L., Kovaleva L.G., Ptushkin V.V., Nikitin E.A., Sudarikov A.B. Determination of minimal residual disease in patients with B-cell chronic lymphocytic leukemia by patient-specific PCR. Clinical laboratory diagnostics. 2011; No. 12: 22-35.

30) Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraCtion: twenty-something years on. Nat Protoc 2006;1(2):581-5. doi: 10.1038/nprot.2006.83.

31) van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ. Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspeCts. Leukemia. 2003;17(6): 1013-34. doi: 10.1038/sj.leu.2402922.

--->

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<---

Claims (48)

1. A method for detecting mutations of the NPM1 gene type A, B, D in a test sample and determining MRD, including: - conducting RT-PCR for primary testing to determine mutations of the NPM1 gene types A, B, D; - PCR-RT for MRD testing; - determination of MRD as a percentage, in the case of a sample containing DNA, using the formula for calculating the allelic load (AL) of the mutant allele 100*AO/(1+AO), where AO is the result of calculating the allelic ratio, while the allelic ratio (AR) is calculated using the formula 5/2ΔCt, where ΔCt is the difference in amplification cycles for the mutant and wild allele, in the case of a sample containing RNA, using the formula for calculating the relative expression of the mutant allele 100/2ΔCt, where ΔCt is the difference in amplification cycles for the mutant and wild allele, wherein a set of reaction mixtures is used for RT-PCR, comprising four mixtures: a mixture of NPM1 W oligonucleotides with the sequences SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 5; a mixture of NPM1 A oligonucleotides with the sequences SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 5; a mixture of NPM1 B oligonucleotides with the sequences SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 5; a mixture of NPM1 D oligonucleotides with the sequences SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 5, intended for amplification of the studied DNA or RNA, which are specific for the wild-type NPM1 gene and for each type of mutation of the NPM1 gene A, B, D, after detection of the NPM1 mutation type A, B, D. 2. The method according to item 1, wherein DNA or RNA isolated from the patient’s bone marrow cells or blood is used as the test sample. 3. The method according to item 2, where the use of DNA as a sample allows the examination of archival material from frozen bone marrow cells or blood of patients. 4. The method according to item 1, wherein to identify the type of NPM1 mutation A, B, D for the primary study during the preparation and implementation of RT-PCR, 8 tubes compatible with the real-time PCR device are prepared for each test sample, for the negative control sample and for the positive control sample. 5. The method according to item 4, wherein a DNA sample from a healthy donor is used as a negative control sample. 6. The method according to claim 4, wherein a DNA sample with an NPM1 mutation of type A, type B or type D is used as a positive control sample. 7. The method according to item 4, where the PCR mixture is prepared in 0.5 or 1.5 ml test tubes based on the calculation: 2.5 x (8N+1) µl PCR buffer 2.0 x (8N+1) µl MgCL ₂ 2.0 x (8N+1) µl dNTP 0.5 x (8N+1) µl TaqPol 13 x (8N+1) µl deionized water, where N is the number of samples tested, taking into account negative and positive control samples. 8. The method according to item 7, wherein the tubes with the PCR mixture are shaken for 3-5 s and centrifuged for 1-3 s in a microcentrifuge/vortex with a centrifugal acceleration of 8000-14000g. 9. The method according to claim 4, characterized in that 2 μl of the corresponding oligonucleotide mixture is added to each of the 8 tubes for real-time PCR: separately 2 tubes for NPM1 W with a mixture of oligonucleotides with the sequences of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, separately 2 tubes for NPM1 A with a mixture of oligonucleotides with the sequences of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, separately 2 tubes for NPM1 B with a mixture of oligonucleotides with the sequences of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, separately 2 tubes for NPM1 D with a mixture of oligonucleotides with the sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. 10. The method according to item 4, where 20 μl of the PCR mixture and 3 μl of DNA or 3 μl of cDNA of the test control sample at a concentration of 20-80 ng/μl are added to each of the 8 test tubes. 11. The method according to claim 10, wherein DNA is pre-diluted at a concentration of 100-400 ng/μl to a volume of 5 μl DNA and 20 μl deionized _H2O , and cDNA is pre-diluted to a volume of 5 μl cDNA and 20 μl deionized _H2O . 12. The method according to item 10, wherein the test tubes are centrifuged for 1-3 seconds with a centrifugal acceleration of 8000-14000g in a microcentrifuge. 13. The method according to item 1, wherein to identify the type of mutation NPM1 A, B, D for the primary study when conducting RT-PCR, the type of mutation in the biological material is determined based on the amplification reaction with primers NPM1 A, NPM1 B and NPM1 D according to the numerical values of the threshold cycle Ct on the device and is determined by which rise in the PCR fluorescence curve began earlier. 14. The method according to claim 1, wherein when detecting the type of NPM1 mutation A, B, D, the allelic load of the mutant allele for DNA or the relative expression of the mutant allele for RNA is calculated. 15. The method according to claim 13, wherein the allele load of the mutant allele for DNA or the relative expression of the mutant allele for RNA is determined based on the difference in the Ct amplification cycles between the NPM1 W primer and the NPM1 A, NPM1 B or NPM1 D primer. 16. The method according to item 1, wherein the real-time PCR is carried out observing the following modes: denaturation at 95° for 5 min, then 50 amplification cycles, including: denaturation at 95° for 30 sec; annealing at 65° for 30 sec; synthesis at 72° for 20 sec. 17. The method according to item 1, where the MRD assessment is carried out after the 2nd course of induction, before maintenance therapy, at the end of therapy or before allo-HSCT, every 3 months after the end of therapy and also at any other points of therapy and times as indicated. 18. The method according to claim 1, wherein 6 tubes compatible with the real-time PCR device are prepared for determining the MRD for each test sample, for the negative control sample and for the positive control sample. 19. The method according to claim 18, wherein DNA or cDNA of healthy donors is used as negative controls, and a positive sample with a mutation of NPM1 type A, or type B, or type D is used as a positive control. 20. The method according to claim 1, wherein the mixture for RT-PCR is prepared in 0.5 or 1.5 ml tubes based on: 2.5 x (6N+1) μl PCR buffer; 2.0 x (6N+1) μl _MgCL2 ; 2.0 x (6N+1) μl dNTP; 2.0 x (6N+1) μl oligonucleotide mixture 0.5 x (6N+1), μl TaqPol, 13 x (6N+1) μl deionized water, where N is the number of samples to be tested, taking into account negative and positive control samples. 21. The method according to item 20, wherein the test tubes with the mixture are shaken for 3-5 s on a vortex and centrifuged for 1-3 s on a microcentrifuge with a centrifugal acceleration of 8000-14000g. 22. The method according to claims 18 and 20, characterized in that 2 μl of the corresponding oligonucleotide mixture is added to each of the 6 tubes for real-time PCR: separately 3 tubes for NPM1 W with a mixture of oligonucleotides with the sequences SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, separately 3 tubes with a mixture of oligonucleotides to NPM1 A with the sequences SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, or with a mixture of oligonucleotides to NPM1 B with the sequences SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, or with a mixture of oligonucleotides to NPM1 D with the sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. 23. The method according to item 18, where 20 µl of the mixture is added to the test tubes and 3 µl of DNA at a concentration of 200-300 ng per µl or 3 µl of cDNA of the test or control sample is added to each of the 6 test tubes. 24. The method according to claim 1, wherein during amplification the amount of DNA in one reaction should be more than 500 ng, and the amount of cDNA should exceed 10,000 copies of ABL1. 25. The method according to item 23, wherein centrifugation of the test tube is carried out for 1-3 s in a microcentrifuge with a centrifugal acceleration of 8000-14000g. 26. The method according to item 23, wherein the PCR reaction is carried out observing the following temperature conditions: denaturation at 95° for 5 min; then 50 amplification cycles, including: denaturation at 95° for 30 sec; annealing at 65° for 30 sec; synthesis at 72° for 20 sec. 27. A set of reaction mixtures intended for the amplification of the studied DNA or RNA, which are specific for each type of mutation of the NPM1 A, B, D gene, comprising: a mixture of NPM1 W oligonucleotides intended for the determination of cells without mutation, with the sequences of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 5; a mixture of NPM1 A oligonucleotides intended for the determination of cells with type A mutation, with the sequences of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 5; a mixture of NPM1 B oligonucleotides intended to determine cells with a type B mutation, with the sequences of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 5 and a mixture of NPM1 D oligonucleotides intended to determine cells with a type D mutation, with the sequences of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 5. 28. Kit no. 27, wherein the NPM1 W mixture, intended for the detection of cells without mutation, consists of the forward primer SEQ ID NO: 1 with a final concentration of 5 pmol per μl, the reverse primer SEQ ID NO: 6 with a final concentration of 5 pmol per μl, the TaqMan probe, SEQ ID NO: 5, labeled with FAM and BHQ1, with a final concentration of 3.5 pmol per μl. 29. The kit according to claim 27, wherein the NPM1 A mixture intended for detecting cells with mutation A consists of the forward primer SEQ ID NO: 2 with a final concentration of 5 pmol per μl, the reverse primer SEQ ID NO: 6 with a final concentration of 5 pmol per μl, the TaqMan probe, SEQ ID NO: 5, labeled with FAM and BHQ1, with a final concentration of 3.5 pmol per μl. 30. The kit according to claim 27, wherein the NPM1 B mixture intended for detecting cells with mutation B consists of the forward primer SEQ ID NO: 3 with a final concentration of 5 pmol per μl, the reverse primer SEQ ID NO: 6 with a final concentration of 5 pmol per μl, the TaqMan probe, SEQ ID NO: 5, labeled with FAM and BHQ1, with a final concentration of 3.5 pmol per μl. 31. The kit according to claim 27, wherein the NPM1 D mixture intended for detecting cells with the D mutation consists of the forward primer SEQ ID NO: 4 with a final concentration of 5 pmol per μl, the reverse primer SEQ ID NO: 6 with a final concentration of 5 pmol per μl, the TaqMan probe, SEQ ID NO: 5, labeled with FAM and BHQ1, with a final concentration of 3.5 pmol per μl. 32. A set of oligonucleotides for amplification of the NPM1 gene of the wild type and with mutations of type A, B, D, including the following oligonucleotides: - a direct primer to the DNA sequence of wild-type Homo sapiens, SEQ ID NO: 1, consisting of 22 nucleotides and containing an LNA-modified nucleotide at positions 19 and 21; - a direct primer to the DNA sequence of Homo sapiens mutant type A, SEQ ID NO: 2, consisting of 23 nucleotides and containing an LNA-modified nucleotide in positions 16 and 20; - a direct primer to the DNA sequence of Homo sapiens mutant type B, SEQ ID NO: 3, consisting of 23 nucleotides and containing an LNA-modified nucleotide in positions 17 and 20; - a direct primer to the DNA sequence of Homo sapiens mutant type D, SEQ ID NO: 4, consisting of 23 nucleotides and containing an LNA-modified nucleotide at position 20; - TaqMan probe to the Homo sapiens DNA sequence, SEQ ID NO: 5, consisting of 28 nucleotides and containing BHQ1 at the 3' end and FAM at the 5' end; - reverse primer to the DNA sequence of Homo sapiens, SEQ ID NO: 6, consisting of 22 nucleotides.