CZ2015106A3

CZ2015106A3 - Method of absolute quantification of miRNA expression, especially miR-34a and/or miR-150 and use thereof in diagnostics and prognostic of B-cell malignancies

Info

Publication number: CZ2015106A3
Application number: CZ2015-106A
Authority: CZ
Inventors: Marek Mráz; Kateřina Černá; Šárka Pospíšilová; Jiří Mayer
Original assignee: Masarykova Univerzita; FakultnĂ nemocnice Brno
Priority date: 2015-02-17
Filing date: 2015-02-17
Publication date: 2016-07-27
Also published as: CZ306080B6

Abstract

Předkládané řešení poskytuje způsob absolutní kvantifikace exprese mmiRNA, s výhodou vybrané z miR-34a a/nebo miR-150, kdy se v biologickém vzorku odebraném z těla pacienta stanoví exprese miRNA, s výhodou vybrané z miR-34a a/nebo miR-150, spolu s expresí endogenní kontroly, kterou je malá jadérková RNA či stabilně exprimovaná miRNA, a zároveň se stanoví exprese syntetických standardů miRNA, s výhodou vybrané z miR-34a a/nebo miR-150, a endogenní kontroly o známých počtech molekul. Řešení dále poskytuje použití tohoto způsobu kvantifikace v in vitro diagnostice B-buněčných malignit, prognostice B-buněčných malignit a predikci léčebné odpovědi pacientů s B-buněčnými malignitami, zejména CLL, identifikaci abnormalit TP53.The present invention provides a method for the absolute quantification of mmiRNA expression, preferably selected from miR-34a and / or miR-150, wherein miRNA expression is determined in a biological sample taken from the body of the patient, preferably selected from miR-34a and / or miR-150, together with the expression of endogenous control, which is a small nuclear RNA or stably expressed miRNA, and the expression of the miRNA synthetic standards, preferably selected from miR-34a and / or miR-150, and endogenous control of known number of molecules is determined. The solution further provides the use of this method of quantification in in vitro diagnosis of B-cell malignancies, prognostics of B-cell malignancies, and prediction of the therapeutic response of patients with B-cell malignancies, particularly CLL, to identify TP53 abnormalities.

Description

A method of absolute quantification of miRNA expression, particularly miR-34a and / or miR-150, and its use in the diagnosis and prognosis of B-cell malignancies

FIELD OF THE INVENTION The present invention relates to a novel methodology for the absolute quantification of miRNAs, preferably selected from miR-34a and / or miR-150, allowing diagnostic use of miRNAs, preferably selected from miR-34a and / or miR-150. It further describes the use of this methodology for prognosis determination in patients diagnosed with CLL and other B-cell malignancies follicular lymphoma, FL; dietary large B-cell lymphoma, DLBCL; Burkitt's lymphoma, BL; mantle cell lymphoma, MClJ determining their therapeutic response to therapy and determining TP53 mutation status in these patients. Background Art

Chronic lymphocytic leukemia (CLL) is characterized as a monoclonal expansion of mature B-lymphocytes that accumulate in peripheral blood and lymphatics {Chiorazzi N et al. N Engl J Med. 2005; 352: 804-815). Until recently, only Raie staging (Rai KR et al. Blood 1975; 46: 219-234) or by Binet (Binet JL et al. Cancer 1981; 48: 198-206) was used to determine prognosis. However, this empirical system is currently insufficient. Patients are often diagnosed at a very early stage of CLL and cannot be distinguished from patients who will rapidly progress from indolent patients due to simple staging. CLL and other B-cell lymphomas are clinically highly heterogeneous and survival time varies greatly between patients. For this reason, only monitoring is often recommended in CLL and some FL patients without clinical signs of disease. Particularly problematic is the early recognition of an aggressive form of the disease, which is more common in younger patients. Over the past decade, CLL has received great attention, resulting in a set of several parameter groups that characterize CLL in detail and predict disease progression. There is only a very limited number of prognostic markers available in other B-cell lymphomas.

The most significant prognostic marker to date is the mutant status of the immunoglobulin heavy chain gene (IgVH) in CLL. The presence of an unmutated IgVH gene (nucleotide homology with a germline greater than 98%) is associated with a worse course and shorter survival of patients (8 years vs. 25 years in patients with mutated IgVH; Damle RN et al. Blood 1999; 94: 1840- 1847, Hamblin TJ et al., Blood 1999; 94: 1848-1854). However, the determination of this parameter necessary to determine the prognosis and treatment of the disease is highly technical, time-consuming and costly. At the same time, clinically most desirable is to determine the prediction of the therapeutic response to a given therapeutic regimen, which virtually none of the markers used so far reliably. For FL, the Follicular Lymphoma International Prognostic Index (FLIPI) is practically exclusively in clinical practice, based on a combination of clinical examination, patient age and biochemical parameter (lactate dehydrogenase level; Solal-Celigny P et al. Blood 2004) ; 104: 1258-1265). In the case of DLBCL, a technically very demanding and hardly reproducible methodology is used based on the determination of gene expression of several genes for the division of patients into groups called ABC vs.. B cell GC phenotype (RosenwaldA et al. NEnglJMed. 2002; 346: 1937-1947).

MiRNAs are 19 to 25 nucleotides long evolutionarily conserved gene expression regulators that control a number of physiological and pathological processes at the post-transcriptional level in the cell. The disease in which miRNA association was first observed in 2002 was chronic lymphocytic leukemia. Due to the association of miRNA expression and cancer, it is possible to distinguish not only individual oncological diseases but also to differentiate subtypes within one disease based on the expression of different miRNA expression.

MiR-34a has been identified in earlier studies as a miRNA significant for the biology of CLL and B-cell malignancies (Zenz T et al. Blood 2009; 113: 3801-3808; Mraz M et al Leukemia 2009; 23: 1159-1163; Djikstra MK et al., Leukemia 2009; 23: 625-627) and its involvement in the p53 pathway, whose deregulation has a significant effect on tumors, has also been described. miR-150 was previously described as the most expressed miRNA in CLL (Mraz M et al. Blood. 2014; 124: 84-95), but the possibility of using it as a prognostic marker in other B cell malignancies and in combination with miR-34a has not been investigated .

The association of miR-34a and TP53 has already been observed and association with prognosis of disease has been suggested (Asslaber et al. Blood; 115 (21): 4191-7; Dufour et al. Blood 2013; 121 (18): 3650-7). However, these studies were not prospective; cohorts of patients were not in a single treatment regimen and included small amounts of poorly characterized samples in TP53 abnormalities. These studies used only relative determination of miRNA expression, which in principle cannot be reproduced by a constant in another laboratory. Thus, these studies cannot be considered as the basis for the diagnostic and predictive use of miR-34a in CLL. However, our proposed absolute quantification of miR-34a and the data obtained from prospectively collected samples from patients treated with the most frequent therapeutic protocol (fludarabine + cyclophosphamide + rituximab = FCR) makes it possible to detect patients with poor treatment response, worse prognosis and abnormalities in TP53 with high specific and sensitivity (deletions and mutations in more than 20% of cells; specificity 82%, sensitivity 89%). For the use of miR-34a as a diagnostic marker, a reproducible method for quantifying it is needed.

SUMMARY OF THE INVENTION The present invention provides a method for the absolute quantification of miR-34a and / or miR-150, wherein the expression of a miRNA is determined in a biological sample taken from the patient, preferably selected from miR-34a and / or miR-150, together with the expression of endogenous control, while expression of the miRNA synthetic standards, preferably selected from miR-34a and / or miR-150, and endogenous control of known molecular numbers are determined.

As endogenous control, due to the nature of the miRNA, a small nuclear RNA (snoRNA; e.g., RNU38B, RNU6B, RNU44, RNU48) and / or stably expressed miRNγ (e.g., miR-16) can serve. We designed synthetic standards, according to the evolutionary conserved sequences of given miRNAs and snoRNAs for miR-34a, miR-150, and endogenous control. We analyzed these known molecular number standards together with the samples and then recalculated the exact copies of miR-34a and miR-150 per million endogenous control molecules.

For tent expression, we have advantageously used miRNA transcription to copyDNA (cDNA) and real-time quantitative polymerase chain reaction (qRT-PCR) detection. The advantage of miRNA as a marker is its easy accessibility (eg from the collection of peripheral blood), the possibility to isolate miRNAs due to their high stability even from long-term archived x FFPE blocks and the possibility of using miRNA circulating in body fluids (plasma, serum, urine, sputum).

In particular, the application of the invention is possible in the in vitro diagnosis of CLL and other B-cell malignancies, CLL prognostics and prediction of therapeutic response (eg, FCR therapy) of CLL patients and B-cell malignancies, and the method of the invention is useful for rapid and easy detection of TP53 abnormalities in a biological sample taken from the patient. For example, a sample of peripheral blood, placenta, serum, urine, sputum, but also a sample obtained from an FFPE block can serve as this biological sample. It is a further object of the present invention to use the method of the invention to determine the prognosis of a B-cell malignant patient by first prognostic group distribution and miR-34a and / or miR-150 expression levels (sample numbers) and statistically assessing the patient population. survival thresholds for individual prognostic groups and prognosis are then quantitated for expression of miR-34a and / or miR-150 in a sample taken from the patient and determined to belong to the appropriate prognostic group. Expression of miR-34 / miR-150 below the threshold (given number of molecules in the sample) indicates a more aggressive course of the disease and thus a shorter survival time. The threshold (number of molecules in the sample) is obtained by statistically evaluating samples of patients with aggressive and indolent disease progression (see, eg, Example 2, threshold = 2047 copies miR-34a / million copies RNU38B; Fig. 2). Further, the use of miR-34a expression assays for predicting response to FCR treatment is contemplated. The prediction is performed based on the expression level of miR-34a in a sample taken from the patient's body (eg, peripheral blood). Low basal expression in the sample, as well as minimal induction of miR-34a expression in the follow-up of the patient, indicate a worse response to therapy, more aggressive disease progression, and reduced time to disease progression. Basal expression level and induction during therapy is determined by statistical analysis of miR-34a expression in patients with aggressive and indolent disease (see, eg, Example 4 normalized expression of [NE] miR-34a in patients with complete remission prior to therapy, Day 1 = 30% RNU38B expression, induction during therapy [day 3] = min 50% RNU38B expression; Fig. 4a). Currently, some markers are known whose expression correlates with the prognosis of B-cell lymphomas. The most significant factor is the TP53 mutation, with patients with TP53 abnormalities exhibiting the most aggressive course of disease (Zenz T et al. Leuk Lymphoma. 2009; 50: 510-513; Xie Y et al. Am J Clin Pathol. 2014: 141: 593-604; Sanchez-Gonzales B et al Leuk Lvmvhoma, 2009; 50: 455-456). To determine TP53 abnormalities, fluorescence in situ hybridization (FISH) has traditionally been used to determine the deleted regions of chromosome 17 where the p53 gene lies and sequencing is used to determine mutations. Based on the level of miR-34a, which is the direct target of p53, patients with TP53 abnormalities can be easily and quickly identified based on qRT-PCR. These patients generally show the lowest levels of miR-34a. By monitoring the change in miR-34a level, the selection of these abnormalities during progression of the disease can also be followed to detect patients with potentially aggressive disease prior to full manifestation of abnormalities. The identification is performed by determining the absolute number of miR-34a molecules in the biological sample taken from the patient. If the threshold is not reached, the presence / development of the TP53 abnormality can be assumed over time. The threshold is determined by analyzing a group of patients with / without TP53 abnormalities, the sensitivity and specificity of the method being determined on the same set of patients.

Similarly, expression levels of miR-34a and / or miR-150 can be determined from lymphoma tissue FFPE blocks. By assessing the expression and belonging to the high / low expression group, prognosis of patients with other B-cell malignancies (e.g., FL, DLBCL) and predicting transformation into more aggressive forms of the disease can be determined. For example, the quantification method of the invention can be used by determining the number of copies of miR-34a and / or miR-150 in a sample taken from the patient's FL, while reducing the copy number during the disease indicates FL transformation to DLBCL. By the aforementioned process, it is possible to detect and quantify circulating miRNA and determine the prognosis of cancer patients by dividing patients into high and low expression groups.

In addition, the present invention provides a method for determining the success of replacement miRNA therapy by substituting miRNA expression (BaderAG Front Genet. 2012; 3: 120; Di Martino MT et al. Clin Cancer Res. 2012; 18: 6260-6270). It consists in determining the expression of miR-34a (or other miRNA replacement) in a sample taken from the patient's body, with an increase over baseline levels found at the beginning of replacement therapy or earlier therapy therapies to be successful. Accordingly, the invention provides a method for screening absolute levels of miR-34a, the expression of which allows differentiation of aggressive patients regardless of other known prognostic markers (including both CLL groups with different IgVH status) and at the same time predicts response to therapy (specifically, FCR).

The most abundant miRNA in CLL is miR-150 (Mraz Met al. Blood. 2014; 124: 84-95), the combination of which with miR-34a yields original and otherwise unattainable stratification of patients. Furthermore, we can also utilize the levels of miR-34a or the combination of miR-34a and miR-150 to predict treatment response in FL, DLBCL, BL and MCL. The combination of stratification by miR-150 and miR-34a levels is an independent predictor in multivariate patient survival analysis.

Thus, a methodology for absolute quantification has been developed that can serve not only to determine the absolute copy number of miR-34a and / or miR-150, but also to track patients over time and detect abnormalities in TP53 during selection. Quantitative determination of miR-34a and / or miR-150 expression may also allow monitoring of miRNA success of replacement therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. la. Ct Cycle Cycle (or miRNA copy number) covered by synthetic standards (Example 1).

FIG. lb. The range of molecules covered by synthetic standards (Example 1).

FIG. 2. Patient survival depending on miR-34a expression level (Example 2).

FIG. 3. Patient survival versus miR-150 expression level in combination with miR-34a (Example 3).

FIG. 4a. MiR-34a expression in patients with different final response to therapy (KR = complete remission, n = 31; PR = partial remission, n = 9; SO = stable disease and worse response, n = 10) (Example 4).

FIG. 4b. PFS (progression-survival survival) in the fludarabine group of patients (n = 50) (Example 4).

FIG. 5a. The miR-34a level observed in different CLL cytogenetic subgroups (Example 5).

FIG. 5b. The decrease in miR-34a copy number in 12 patients during disease progression accompanied by abnormalities in TP53 (Example 5).

FIG. 6a. Survival of FL patients depending on basal miR-150 level (Example 6).

FIG. 6b. MiR-150 level in patients after transformation from FL to DLBCL (Example 6). EXAMPLES Example 1 In our study, we analyzed miR-34a expression in CLL cells to define the role of miR-34a in CLL biology and to determine the appropriate methodology for its absolute quantification. In contrast to relative quantification, absolute quantification allows accurate determination of miRNA copy numbers in a given sample and the possibility to do so reproducibly in different laboratories. To calculate the number of copies, we prepared standard dilution series of known number of molecules that would cover the required number of qRT-PCR cycles. For the application of qRT-PCR in the miRNA region, modification is required due to the specific properties of miRNA. The biggest obstacle is the length of the miRNA itself, which corresponds to the length of commonly used primers, with shorter primers not applicable. To overcome the problem of too low a melting point of primers in the duplex with miRNA, we use, for example, commercially available stem-loop or hairpin primers. The TaqMan® MicroRNA Assay (Life Technologies) detection system is based on the use of specially designed reverse transcription primers to extend the miRNA and subsequently the specific probes for qRT-PCR. Reverse transcription is used to synthesize ss cDNA (= single strand) isolated total RNA via TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies). The RNA input amount is approximately 4 ng of total RNA in a total reaction volume of 7.5 μΐ. As endogenous control for miRNA expression normalization, we use the small nucleotide RNA RNU38B and the miR-34a and / or miR-150 and RNU38B (according to miRBase v. 19; Sigma Custom Synthesis) to determine the number of miR-34a and / or miR-150 molecules. Aldrich). Analogously, RNU6B, RNU44, RNU48 and other snoRNA can be used as control RNA. miR-34a: 5 '- UGG CAG UGU CUU AGC UGG UUG U - 3' miR-150: 5 '- UCU CCC AAC CCU UGU ACC AGU G - 3' RNU38B: 5 '- CCA GUU CUG CUA CUG ACA GUA AGU GAA From these synthetically prepared RNAs, we prepare a cDNA that is diluted 1: 7 with nuclease-free water and which covers almost 20 cycles in qRT-PCR amplification and the amount of molecules in the reaction from about 70 to 18.106. molecules (Fig. 1a, b). For cDNA synthesis we use the TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies). Analyze each sample in triplicate using TaqMan® Gene Expression Assay (Life Technologies) according to the manufacturer's instructions. The cDNA amplification is then detected by a 7900 Real Time PCR System (Life Technologies). Data is evaluated in SDS v2.4 (Life Technologies) and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, the outliers are discarded. Subsequently, we recalculate the number of miRNA molecules per million endogenous RNU38B control molecules. The use of this endogenous control was validated together with the possible use of other small snoRNAs, eg RNU6B, RNU44, RNU48, and stably expressed miRNAs (miR-16). Example 2

Peripheral blood of a patient diagnosed with CLL is collected into anticoagulant collection tubes (heparin / EDTA). Mononuclear cells are separated by gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare) prior to incubation with a cocktail of bispecific antibodies (RosetteSep® B Cell Enrichment Cocktail, StemCell).

From the separated CLL lymphocytes (CD19 +, CD5 +), lysate is prepared in TRI Reagent (Sigma Aldrich). Using BCP (Molecular Research Center, Inc.), We isolate total RNA of CLL cell lysis. Determine the quality and concentration of total RNA obtained spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). For further analysis, we use only samples with good parameters (RIN> 8, concentration> 100 ng / µl). For the determination of the amount of miRNA in the sample, we use the procedure of Example 1. On one plate, we collect together samples for the miRNA of interest and endogenous control from the sample. cDNA is applied in triplicate. To avoid contamination, we include duplicates in which the cDNA is replaced by nuclease-free water. For board comparison, we use an internal standard, which is a sample of patient RNA (ie, the same RNA, detection of the same endogenous control and miRNA). Data is evaluated in SDS v2.4 and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, the outliers are discarded. Subsequently, we recalculate the number of miRNA molecules per million endogenous RNU38B control molecules.

Based on miR-34a levels in the CLL group (n = 158), we were able to divide the patients into two different prognostically different groups with different survival (median 16.7 months vs. not reached; p = 0.0002; HR: 3.30 [Cl: 1.69- Fig. 2: independent of other prognostic factors commonly used in CLL (dell7pl3, delllq, gender, age, IgVH; Table 1).

Tab. 1. Multivariate analysis of prognostic factors.

Example 3

From the separated CLL lymphocytes (CD19 +, CD5 +), lysate is prepared in TRI Reagent (Sigma Aldrich). BCP (Molecular Research Center, Inc.) isolates total RNA from CLL cell lysate. Determine the quality and concentration of total RNA obtained spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). For further analysis, we use only samples with good parameters (RIN> 8, concentration> 100 ng / µl). We use qRT-PCR to determine absolute gene expression. On a single plate, we collect together the samples for the miRNA of interest and the endogenous control of the sample together with a number of standards for determining the absolute number of miR-34a, miR-150 and RNU38B molecules. cDNA is applied in triplicate. To avoid contamination, we include duplicates in which the cDNA is replaced by nuclease-free water. For board comparison, we use an internal standard, which is a sample of patient RNA (ie, the same RNA, detection of the same endogenous control and miRNA). Data is evaluated in SDS v2.4 and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, we exclude this outliers.

Based on the concomitant level of miR-34a and miR-150 in the CLL group (n = 100), we were able to divide the patients into prognostically different groups with different survival (median 138 months vs. 257 months) independent of the other prognostic factors commonly used in CLL (p <0.05 HR: 2.2 [Cl 1.1-4.9]; FIG. 3). Example 4

From the separated CLL lymphocytes (CD19 +, CD5 +), lysate is prepared in TRI Reagent (Sigma Aldrich). BCP (Molecular Research Center, Inc.) isolates total RNA from CLL cell lysate. Determine the quality and concentration of total RNA obtained spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). We use qRT-PCR to determine absolute gene expression. On a single plate, we collect together samples for the miRNA of interest and endogenous control from the sample together with a number of standards for determining the absolute number of miR-34a and RNU38B molecules. cDNA is applied in triplicate. To avoid contamination, we include duplicates in which the cDNA is replaced by nuclease-free water. For board comparison, we use an internal standard, which is a sample of patient RNA (ie, the same RNA, detection of the same endogenous control and miRNA). Data is evaluated in SDS v2.4 and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, we exclude this outliers. In this way, we analyzed a group of 50 CLL patients in therapy for whom we observed the resulting response to therapy over time. Patients with low miR-34a prior to initiation of therapy (Day 1; NE miR-34a = 15% RNU38B expression) and lowest induction during therapy (Day 3; NO = 30% RNU38B expression) showed the worst response to therapy and shorter time to its failure (Fig. 4 a, b). Example 5

From the separated CLL lymphocytes (CD19 +, CD5 +), lysate is prepared in TRI Reagent (Sigma Aldrich). Using BCP (Molecular Research Center, Inc.), We isolate total RNA of CLL cell lysis. Determine the quality and concentration of total RNA obtained spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). We use qRT-PCR to determine absolute gene expression. On the same plate, we always place samples for the miRNA of interest and endogenous control from the sample together with a number of standards for the absolute number of miR-34a and RNU38B molecules. cDNA is applied in triplicate. To avoid contamination, we include duplicates in which the cDNA is replaced by nuclease-free water. For board comparison, we use an internal standard, which is a sample of patient RNA (ie, the same RNA, detection of the same endogenous control and miRNA). Data is evaluated in SDS v2.4 and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, the outliers are discarded. Subsequently, we recalculate the number of miRNA molecules per million endogenous RNU38B control molecules. In this way we have characterized a group of 161 CLL patients. In patients with abnormalities in TP53 (dell7pl3 / mut p53), we detected the lowest level of miR-34a compared to groups of CLL patients with other cytogenetic abnormalities determined in CLL (delllq, trisomy 12, normal karyotype, separate dell3ql4; Fig. 5a). Based on this analysis, the ability of low levels of miR-34a to identify patients with abnormalities in TP53 can be predicted. Using ROC analysis, we determined the number of miR-34a molecules (threshold value) identifying patients with TP53 abnormality with a specificity of 82% and a sensitivity of 89%. The absolute number of miR-34a molecules also allowed us to track patients over time and to identify patients with disease progression accompanied by abnormalities in TP53 (Figure 5b). As described above, we analyzed samples of 12 patients (sample 1-12) with progressive disease progression accompanied by the emergence of abnormality vTP53. During this selection, we observed a decrease in the copy number of miR-34a in the sample when switching from wt-TP53 (wild-type; sample without abnormalities) to the TP53 abnormality phase (Fig. 5b). Example 6

We isolate the miRNA from FFPE tissue blocks according to the recommendations and using the chemicals of the High Pure miRNA Isolation Kit (Roche). To isolate paraffin RNA, the tissue is dewaxed first and then digested by proteases. We bind RNA in the presence of the guotidine salt of guanidine thiocyanate to glass fibers in a filter of the column. The bound RNA is cleaned of impurities by a series of washing steps and then eluted with a low salt concentration solution (water without nucleases). MiRNs are obtained by simply changing the binding enhancer of the buffer to support the binding to the column. By mixing a small amount of this buffer, long RNA molecules are captured while the miRNA passes through. Subsequently, we increase the binding enhancer concentration and capture the miRNA in the new column.

Determine the quality and concentration of total RNA obtained spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). We use qRT-PCR to determine absolute gene expression. On a single plate, we collect together the samples for the miRNA of interest and the endogenous control from the sample together with a number of standards for the absolute number of miR-34a, miR-150 and RNU38B molecules. cDNA is applied in triplicate. To avoid contamination, we include duplicates in which the cDNA is replaced by nuclease-free water. For board comparison, we use an internal standard, which is a sample of patient RNA (ie, the same RNA, detection of the same endogenous control and miRNA). Data is evaluated in SDS v2.4 and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, the outliers are discarded. Subsequently, we recalculate the number of miRNA molecules per million endogenous RNU38B control molecules.

By this approach, we were able to divide FL patients into groups of different survival (100 months follow-up: low miR-150: 52% survivors, high miR-150: 79% survivors; p = 0.007; HR = 3.0 [0 = Fig. 1.3-6.8]; Fig. 6a). Analogously, based on the number of miR-34a and / or miR-150 copies from FFPE blocks, prognosis of other B-cell malignancies and other oncological diseases can be determined and the transformation of FL into more aggressive form of DLBCL (Fig. 6b). Example 7 The serum (or plasma) of cancer patients is lysed in QIAzol Lysis Reagent (Quiagen). After the addition of chloroform, the interface of the aqueous phase containing RNA and the lower and boundary organic phases containing DNA and proteins are established in the lysate. Remove the upper aqueous phase and add ethanol. Next, transfer the sample to the RNeasy MinElute column where the RNA binds to the membrane and the phenol and other contaminants are washed away. The isolated RNA is then eluted in a small amount of nuclease-free water. Determine the quality and concentration of total RNA obtained spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). We use qRT-PCR to determine absolute gene expression. On a single plate, we always put together samples for the miRNA of interest and endogenous control from a given sample along with a number of standards to determine the absolute number of miRNA molecules and endogenous control. cDNA is applied in triplicate. To avoid contamination, we include duplicates in which the cDNA is replaced by nuclease-free water. For board comparison, we use an internal standard, which is a sample of patient RNA (ie, the same RNA, detection of the same endogenous control and miRNA). Data is evaluated in SDS v2.4 and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, the outliers are discarded. Subsequently, we recalculate the number of miRNA molecules per million of endogenous control molecules.

Based on these analyzes, we can determine the number of copies of circulating miRNAs in cancer patients and determine the prognosis accordingly. Example 8 Tumor tissue (cells) before / during / after miRNA replacement therapy is taken into RNAlater (Life Technologies) to maintain RNA quality in the sample. We isolate the miRNA from these samples according to the recommendations and using the chemicals of the High Pure miRNA Isolation Kit (Roche). We bind RNA in the presence of the guotidine salt of guanidine thiocyanate to glass fibers in a filter of the column. The bound RNA is cleaned of impurities by a series of washing steps and then eluted with a low salt concentration solution (water without nucleases). MiRNs are obtained by simply changing the binding enhancer of the buffer to support the binding to the column. By mixing a small amount of this buffer, long RNA molecules are captured while the miRNA passes through. Subsequently, we increase the binding enhancer concentration and capture the miRNA in the new column.

Determine the quality and concentration of total RNA obtained spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). We use qRT-PCR to determine absolute gene expression. On a single plate, we always put together samples for the miRNA of interest and endogenous control from a given sample along with a number of standards to determine the absolute number of miRNA molecules and endogenous control. cDNA is applied in triplicate. To avoid contamination, we include duplicates in which the cDNA is replaced by nuclease-free water. For board comparison, we use an internal standard, which is a sample of patient RNA (ie, the same RNA, detection of the same endogenous control and miRNA). Data is evaluated in SDS v2.4 and then exported in Excel spreadsheet format (Microsoft Office XP). If the triplicate deviation is greater than 0.3 cycle, the outliers are discarded. Subsequently, we recalculate the number of miRNA molecules per million of endogenous control molecules.

By the mentioned procedure we can determine the success of replacement therapy based on the number of miRNA copies from the tumor tissue. successful recovery of miRNA expression based on increasing number of molecules over time.

Claims

PATENT CLAIMS

A method for determining a patient's prognosis with a B-cell malignancy characterized in that prognostic grouping and miR-34a and miR-150 expression thresholds and prognostic survival threshold values are first obtained by statistical evaluation of a patient sample; the prognosis determination is then quantitated by the expression of miR-34a and miR-150 in a biological sample taken from the patient's body and determined to belong to the appropriate prognostic group; wherein expression of miR-34a and miR-150 is determined in a biological sample taken from the body of the patient, along with expression of an endogenous control that is a small nuclear RNA or stably expressed miRNA, and expression of the miR-34a and miR-150 synthetic standards is determined, and endogenous control of known molecular numbers.

A method of determining B-cell malignancy response prediction for FCR treatment, wherein basal expression of miR-34a is determined in a biological sample taken from the body of the patient, along with expression of endogenous control that is a small nuclear RNA or stably expressed miRNA, while expressing the expression of miR-34a synthetic and endogenous control of known molecular numbers, wherein miR-34a basal expression below the threshold obtained by statistically evaluating a sample of patients predicts poor treatment response, whereas expression higher than threshold predicts good response for treatment.

3. A method for monitoring the transformation of FL into DLBCL in a patient, comprising determining the copy number of miR-34a and / or miR-150 in a sample taken from the patient's body suffering from FL by expressing expression in a biological sample taken from the patient's body miR-34a and / or miR-150, together with the expression of endogenous control, which is small nuclear RNA or stably expressed miRNA, and expression of the miR-34a and / or miR-150 synthetic standards and endogenous control of known molecular numbers is determined, and converting the number of copies per million of endogenous control molecules; wherein the reduction in copy number during the disease indicates the transformation of FL into DLBCL.

A method according to any one of claims 1 to 3, characterized in that detection of the level of miRNA by its transcription into cDNA and detection by qRT-PCR is used to determine expression.

The method of any one of claims 1 to 3, wherein the small core RNA is selected from the group consisting of RNU38B, RNU6B, RNU44, RNU48, and / or stably expressed miRNA is miR-16.

A method according to any one of claims 1 to 3, wherein the biological sample taken from the patient is selected from the group consisting of a peripheral blood sample, plasma, serum, urine, sputum, FFPE block samples.