CZ301540B6 - Method of determining prognosis of B-cell chronic lymphocytic leukemia - Google Patents

Abstract

The present invention relates to a method of determining prognosis of B-cell chronic lymphocytic leukemia from a biological sample taken out of a patient body wherein the method is characterized in that there is determined expression of a lymphocyte-activation gene 3 (LAG3), LPL and ZAP70. Expression determination of these genes can result in determination of the disease prognosis with up to 95 percent certainty. High expression of these genes correlates with the presence of a non-mutated gene for IgVH and thus with a worse disease development and shorter survival of patients.

Description

The present invention relates to a method for determining the prognosis in patients diagnosed with CELL chronic lymphocytic leukemia.

BACKGROUND OF THE INVENTION

B-cell chronic lymphocytic leukemia (B-CLL) is characterized as a monoclonal expansion of mature B cells that accumulate in peripheral blood and lymphatic organs (Chiorazzi N et al., N. Engl J. Med. 2005; 352: 804- 815). Until recently, only Raag (Rai KR et al., Blood 1975; 46: 219-234) or Binet (Binet JL et al., Cancer 1981; 48: 198-206) has been used in clinical practice to determine prognosis. However, this empirical system is currently not sufficient. Patients are often diagnosed at a very early stage of B-CLL, and on the basis of simple staging it is not possible to distinguish patients who will progress rapidly from patients with endometrial disease. B-CLL is a clinically highly heterogeneous disease and the survival time varies greatly in individual patients. Moreover, chemotherapy of the early stages of the disease does not bring the expected benefit to the patient; on the contrary, the survival time or development of secondary malignancies is often shortened. Therefore, only B-CLL patients without clinical signs of disease are often recommended. Especially problematic is the early detection of aggressive disease often recommends only monitoring. In particular, early detection of an aggressive form of the disease, which is more common in younger patients, is problematic. Over the past decade, B-CLL has received great attention and results in a set of several parameter groups that characterize B-CLL in detail and allow prediction of disease progression.

The mutation status of the immunoglobulin heavy chain (IgVH) gene appears to be the most important prognostic marker so far in B-cell chronic lymphocytic leukemia 30 mie. The presence of an unmutated IgVH gene (nucleotide homology with germline greater than 98%) is associated with poorer course and shorter patient survival (8 years vs. 25 years in IgVH mutated patients) (Damle RN et al., Blood 1999; 94: 1840-1847; HamblinTJ et al. Blood 1999; 94: 1848-1854). However, the determination of this parameter necessary for determining the prognosis and treatment of the disease is highly technically, time consuming and costly, and it is therefore an attempt to replace its detection with another easily determinable marker or group of markers. The mutation status of the IgVH gene is determined as follows: RNA is isolated from the tumor cells, which is then transcribed into cDNA. The cDNA is amplified with primers specific for individual VH families. In the monoclonal type of disease, the PCR product is directly sequenced. In polyclonal patients, individual clones must be distinguished by cloning and sequencing. In both cases, the sequences obtained are then compared to the germline sequence in the database and the degree of homology determined. This assay is performed once in monoclonal patients and is considered a factor that does not change during disease. Polyclonal patients are continuously monitored for the likely increase in the most aggressive clone.

It is an object of the present invention to replace the current IgVH mutation status determination with a set of genes whose expression would differentiate both prognostics of a different group. Currently, some genes are known whose expression level correlates with the presence or absence of a mutated IgVH gene. One such marker discovered in B-CLL by DNA chip analysis is ZAP70. This tyrosine kinase is physiologically expressed in T cells and its high expression correlates with the presence of an unmutated IgVH gene (Rosenwald A et al., J Exp. Med. 2001; 194: 1639-1647; Wiesner A et al., Blood 2003; 101: 4944-951). Another gene with different expression distinguishing both groups is LPL (lipoprotein lipase). This gene was also detected by chip analysis (Oppezzo P et al., Blood 2005; 106: 650-657; Bilban M et al., Leukemia

2006; 20: 1080-1088) and its high expression correlates with the presence of an unmutated IgVH gene.

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LAG3 (CD223) (lymphocyte-activation gene 3) is a membrane protein structurally similar

CD4, both at the gene and protein levels. LAG3 is expressed on T cells and NK cells upon activation (Triebel F et al., Journal of Experimental Medicine 1996; 171: 13935 1405). LAG3 was also found to be expressed on B cells after activation by T lymphocytes (Kisielow M et al., European Journal of Immunology 2Q05; 35 (7); 2081-2088). The protein consists of two subunits - the 70 kDa transmembrane strand oligomerizes with the second truncated 54 kDa extracellular fragment, the remaining 16 kDa strand with the transmembrane domain is released as soluble LAG3 (LiN et al., Journal of Immunology 2004; 173 (11): 6806-6812 ). The function of this protein is not yet fully understood. It probably participates in a number of regulation of immune processes. For example, signaling on T cells via LAG3 inhibits the activation of CD4 + and CD8 + T cells (Macon-Lemaitre L and Triebel F, Immunology 2005; 115 (2): 170-178) and regulates their expansion (Workman CJ and Vignali DA, European Journal of Immunology 2003; 33 (4): 970-979). Furthermore, this protein affects e.g. maturation of dendritic cells (Andreae Set al., Journal of Immunology 2002; 168 (8): 3874-3880) and regulates dendritic cell macrophage differentiation from monocytic precursors (Buisson S and Triebel F, Immunology 2005; 114 ( 3): 369-374), etc. The literature reports the association of high LAG3 gene expression with unmutated IgVH status and poor disease prognosis associated with the need for treatment (Chuang et al., Blood (ASH Annual Meeting Abstracts) 2005; 106 (11), abstract

2952; Kotaskova J. et al., Haematologica 2007; 92, suppl. 1,390, abstract 1056).

SUMMARY OF THE INVENTION

The present invention relates to a new set of prognostic markers that make it possible to determine the prognosis of a patient with B cell chronic lymphocytic leukemia (B-CLL) with up to 95% confidence.

SUMMARY OF THE INVENTION The present invention provides a method for determining the prognosis of B-cell chronic lymphocytic leukemia from a biological sample taken from a patient, which comprises determining the expression of lymphocyte-activation gene 3 (LAG3), ZAP70 and LPL genes in B-CLL cells of the sample. . The biological sample may be a peripheral blood sample, a bone marrow sample or a tissue sample, in particular the tissue sample may be a lymph node sample.

It is a feature of the invention that detection of the gene mRNA level can be used to determine gene expression.

Based on DNA chip analysis of gene expression of B-CLL cells in order to define new prognostic markers, approximately 50 genes with different expression levels were detected distinguishing patients with mutated and non-mutated IgVH and different clinical course of the disease.

A set of 3 markers, ZAP70, LPL and LAG3, was found to allow 95% confidence to determine the prognosis of a B-CLL patient and to determine appropriate treatment based on this prognosis. Simple detection of the expression of this set of genes could reliably complement or replace the demanding determination of the mutation status of the IgVH gene, which would save time and money. In addition to the primary diagnosis of leukemia patients, this methodology could also be applied to post-treatment patients whose therapy may suppress the dominant B cell clone (Fig. 2).

Detection of the gene mRNA level of a given gene is preferably used to determine gene expression, for example by transcribing it into cDNA and determining by real-time quantitative RT-PCR (RT-qPCR). The determination of the expression of said genes, eg by RT-qPCR, can be used to detect the progression of a new B cell clone with different IgVH mutation status. For patients with low expression, the assay should be repeated periodically for the same reasons.

The set of LPL, ZAP70 and LAG3 genes is a suitable set of prognostic markers to determine the prognosis of B-CLL and the mutation status of IgVH.

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Overview of pictures

Giant. 1 depicts Kaplan-Meier curves expressing time (in days) from disease diagnosis to initiation of therapy in groups of patients with high and low LAG3 gene expression in B-CLL cells. On the x-axis is Time in days, on the y-axis the proportion of patients.

Giant. 2 depicts fragmentation analysis of the IgVH gene. Left patient sample prior to therapy dominant clone with mutated IgVH from VH 3-30 family, right same patient six months after the end of therapy - dominant clone changed. The new clone carries unmutated IgVH from the io VH1-69 family. Capture based on monitoring of LAG3, LPL, and ZAP 70 expression during disease.

Giant. 3 shows differentially expressed genes between groups of B-CLL cells with different mutational status of the IgVH gene detected by DNA chips. Columns represent individual samples, rows represent individual genes. "M" - mutated IgVH gene (n = 10), "N" ^- unmutated IgVH gene (n = 9). Up-regulated genes are highlighted in red, down-regulated genes are highlighted in green.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

In our study, we performed DNA microarray analysis of gene expression of B-CLL cells to define new prognostic markers in B-CLL. Total RNA was isolated from B-CLL cells using RNeasy Mini Kit (Qiagen). The quality and concentration of the total RNA obtained was determined spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). Only samples with good parameters (RIN> 8, concentration> 100 ng / µl) were used for further analyzes. For analysis of gene expression by DNA microarrays, RNA was linearly amplified and fluores30 was priced. The Low RNA Input Linear Amplification Kit (Agilent) was used for linear amplification. The manufacturer's protocol has been modified to allow the production of amplified RNA incorporating a modified aminoallyl-UTP nucleotide. The protocol includes several steps: 1) transcription of RNA into double stranded cDNA incorporating the T7 promoter, 2) linear amplification of RNA by in vitro transcription by T7 RNA polymerase with incorporation of aminoallyl35 UTP, 3) purification of amplified RNA. The achieved RNA amplification level was monitored spectrophotometrically. For fluorescence labeling of amplified RNA, a proprietary protocol using aminoreactive fluorescent dyes (DY-547-NHS-ester and DY-647-NHS-ester; Dyomics) was used, which covalently binds to the primary amino groups, in this case the amino group, in an alkaline environment. aminoallyl-UTP. To run the labeling reaction, it was amplified

RNA was purified again. Two-color hybridization technique was used for DNA microarray analysis. Prior to hybridization, a sample of the amplified fluorescently labeled patient RNA was mixed with an equal amount of similarly prepared reference human RNA (Stratagene) labeled with another fluorescent dye. For hybridization, the RNA mixture was chemically fragmented (average fragment length 200bp). Hybridization was carried out for 17 hours at 65 ° C. Following hybridization, DNA microarrays were washed to remove unbound RNA, dried by centrifugation, and scanned with a ScanArray Express fluorescence scanner (Perkin Elmer). The image data was then converted to a numerical form for further analysis. The conversion of image data to numerical (image analysis) was performed by QuantArray (Perkin Elmer). To reduce non-biological sources of variability, raw data was transformed and normalized by R and R Bioconductor (Gentleman RC et al., Genome Biol. 2004; 5 (10): R80). Two types of algorithms were used to find the genes based on which it is possible to divide individual samples into classes (groups). The first belongs to the “unsupervised (machine) leaming” group and is able to divide the samples into clusters without their prior knowledge (Hierarchical Clustering, K-Means / Medians Clustering, Shelf Organizing Maps - for 55 grams of TIGR MultiExperimentViewer). The second type is “supervised”

-3GB 301540 B6 (machine) leaming ', searching for genes capable of distinguishing between two pre-known groups (TIGR MultiExperimentViewer -Support Vector Machines) (Saeed AI et al., Biotechniques

2003; 34 (2): 374-8). Normalized data were statistically processed using SAM (Significance

Analysis of Microarrays) (Tusher VG et al., Proc. Natl Acad Sci USA 2001; 98 (9); 5116-21).

Expression analysis of B-CLL cells was performed on Human IA Arrays (Agilent) chips. The selected DNA chips are all genomic and carry 60 base oligonucleotide probes. We detected - 50 genes (Fig. 3) with different expression levels differentiating patients with mutated and unmutated IgVH and different clinical course of the disease. In addition to the already known prognostic markers, we detected LAG3 (lymphocyte-activation gene 3), which has not yet been published in connection with ΒΙΟCLL and mutation status. Its high expression correlates with unmutated IgVH status and poor prognosis associated with the need for treatment. Thus, we have found that the LAG3 gene correlates significantly with the severity of the disease and its determination provides significant information about the disease prognosis and the patient's expected survival.

Example 2

Peripheral blood of a patient diagnosed with B-CLL was collected in anti-coagulant collection tubes (heparin). Mononuclear cells were separated by gradient centri20 fugation (Ficoll-Paque PLUS, GE Healthcare), preceded by incubation with a cocktail of bispecific antibodies (anti CD2, CD3, CD16, CD36, CD56, CD66b at one end and anti-glycophorin A at the other end) (RosetteSep® B Cell Enrichment Cocktail, StemCell). The unwanted cells form formations called rosettes with bispecific antibodies along with red blood cells. The density of the rosettes formed is higher than that of the unwanted cell itself and sedimentes during gradual centrifugation. Total RNA was isolated from the separated B-CLL cells by RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). The quality and concentration of the total RNA obtained was determined spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). Only samples with good parameters (RIN> 8, concentration> 50ng / µl) were used for further analyzes. SuperScriptTM Ι Reverse System was used for cDNA synthesis

Transcriptase with oligo (dT) 12-18 primer (Invitrogen). Each sample was analyzed in triplicate using a TaqMan® Gene Expression Assay system (Applied Biosystems) according to the manufacturer's instructions. DNA amplification was then detected with a 7300 Real Time PCR System (Applied Biosystems) and the data obtained was analyzed using the Sequence Detection System (SDS) software version 1.3.1. The result was a cycle number at which the fluorescence reached the set threshold (CT). Relative gene expression was normalized to the expression of the housekeeping GAPDH gene, which was selected based on the stability of expression across samples.

Example 3

The patient's B-CLL peripheral blood was transferred to a test tube and blood volume was measured. Next, RosetteSep® Human B Cell Enrichment Cocktail (50 µl / ml blood) was added, the mixture was swirled and incubated for 20 minutes at room temperature. At least an equal volume of 2% FBS (fetal bovine serum in phosphate buffered saline) was then added, mixed and layered with 6 ml of diluted blood to 3 ml of Ficoll-Paque Plus® brought to room temperature. Centrifuge was performed for 30 minutes / 400 g. The lymphocyte layer was transferred to a clean tube (15 cm) and supplemented with 2% FBS per 10 ml, the number of tubes was selected so that the separated lymphocytes were diluted at least 2x, the tube contents mixed well. Then centrifugation was carried out for 10-15 minutes / 200 g, the supernatant was aspirated and the sediment was suspended in 1 ml of cooled lysis buffer (lymphocyte lysis - 154.9 mM NH 4 Cl, 9.9 mM NH 4 HCO 3, 1 mM EDTA). 10 ml and inverted. Multiple tube sediments were pooled at this step. Centrifugation was performed for 10-15 minutes / 200 g, aspirated supernatant, and the sediment suspended in 2% FBS and transferred to a microtube. After another

The supernatant was aspirated by centrifugation for 10 minutes / 200 g and the sediment was lysed in 350 μί RLT buffer (lysis buffer, part of RNeasy Mini Kit from Qiagen for total RNA isolation).

Total RNA was isolated from the separated B-CLL cells by RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). The quality and concentration of the total RNA obtained was determined spectrophotometrically (NanoDrop) and electrophoretically (BioAnalyzer 2100). Only samples with good parameters (RTN> 8, concentration> 50 ng / μΙ) were used for further analyzes. SuperScript ™ ΙΠ Reverse Transcriptase with oligo (dT) 12-18 primer (Invitrogen) was used to synthesize cDNA according to the manufacturer's instructions. 500 ng of total RNA was transcribed. The obtained 20 μΐ cDNA was also diluted with RNAse-free water to a final volume of 50 μί. Each sample was analyzed in triplicate using a TaqMan® Gene Expression Assay (Applied Biosystems) according to the manufacturer's instructions. DNA amplification was then detected with a 7300 Real Time PCR System (Applied Biosystems) and the data obtained was analyzed using the Sequence Detection System (SDS) software version 1.3.1. The result was a cycle number at which i5 fluorescence reached the set threshold (Ct - fluorescence threshold cycle). Relative gene expression was normalized to the expression of the housekeeping GAPDH (Δ Ct) gene. Obtained data were analyzed by nonparametric Wilcoxon test.

LAG3, LPL, and ZAP70 gene expression was determined by RT-qPCR in the 94 B-CLL set

2 patients (see Example 2). Based on the results documented in Fig. 1,2 and Tab. 1, a set of LAG3, LPL and ZAP70 genes whose expression distinguishes non-mutated IgVH patients from mutated IgVH patients, i.e. patients with different disease prognosis, has been found to be a suitable set for determining the disease prognosis in a patient with high confidence.

Table 1 shows the summary results of the RT-qPCR LAG3, LPL, and ZAP70 gene expression assays in a set of 94 B-CLL patients. According to the expression of individual genes (LPL, LAG3, ZAP70), the samples were divided into positive (gene expression higher than specified) and negative (gene expression lower than specified). The positivity limit for LAG3 was 8.8 Δ Ct, for LPL 8.4 Δ Ct and for ZAP70 4.7 Δ Ct. The summary for all three genes of interest was generated by placing samples classified as positive for all three genes in the group of positive, the others were evaluated as negative. In the table, samples are grouped according to the mutation status of the IgVH gene and further positivity / negativity for the determined genes. The numbers in bold represent the actual number of cases, in brackets they are expressed as a percentage (100% - number of samples in a given group with the same IgVH status). The last row of the table represents the percentage of correctly classified 35 samples (100% - all samples examined).

Tab. 1

gene expression LPL ZAP70 LAG3 3 genes IgVH UNMUT (n = 40) POSITIVE 39 (97.5%) 39 (97.5%) 4Q (ioo,%) 38 (95.0%) NEGATIVE 1 (2.5%) 1 (2.5%) 1 (0%) 2 (5.0%) IgVHMUT (n = 54) POSITIVE 9 (16.7%) 22 (40.7%) 39 (66.7%) 3 (5.6%) NEGATIVE 45 (83.3%) 32 (59.4%) IB (33.3%) 51 (94.4%) Percentage of correctly determined mutation status 89% 80% 62 95%

Claims (3)

A method for determining the prognosis of B-cell chronic lymphocytic leukemia (B-CLL) from a biological sample taken from a patient, characterized in that the expression of LAG3, LPL and ZAP70 genes in the B-CLL cells of the sample is determined. 10 Method according to claim 1, characterized in that detection of the mRNA level of the gene is used to determine gene expression. Method according to claim 1 or 2, characterized in that the biological sample is a peripheral blood sample, a bone marrow or a tissue sample, preferably the tissue sample is a lymphatic sample. 15 nodules.