JP2013138613A - Method for detecting genetic abnormality in neuroblastoma using blood-derived specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a noninvasive method for detecting a neuroblastoma without extracting tumor tissue.SOLUTION: The method is provided for detecting LOH (loss of heterozygosity) on chromosome 11 by performing PCR with a microsatellite sequence as a target in a blood-derived specimen, and methods are provided for detecting the neuroblastoma by detecting the LOH, and predicting a prognosis.

Description

  The present invention relates to a method for detecting Loss of Heterozygosity (11q LOH) or neuroblastoma of chromosome 11 using a blood-derived specimen.

  Neuroblastoma is the most common abdominal solid tumor that occurs in children, and the 5-year survival rate of advanced cases is about 40%, which is still extremely refractory even with advanced medical care. In order to improve the prognosis of neuroblastoma, treatment according to risk is indispensable, but genetic diagnosis using tumor tissue is required for accurate risk classification. In recent years, the risk classification of neuroblastoma includes the age, stage, pathological classification, and presence or absence of MYCN gene amplification (MNA), as well as the loss of Heterozygosity (11q LOH) of chromosome 11. A new classification combining presence or absence was announced. For this reason, MNA and 11q LOH have become indispensable examination items for risk classification, but in order to carry out genetic diagnosis, it is first necessary to collect tumor tissue by surgery, which is often difficult for children who are difficult to perform invasive examinations. It is.

  Recently, research on free nucleic acids derived from tumor tissues present in cancer-bearing patients has been reported as a diagnostic method to replace genetic testing using cancer tissues, and has attracted attention from many clinicians. The present inventors have paid attention to this free nucleic acid in serum and have been researching its application to diagnosis (Non-Patent Documents 1 and 2). The present inventors have already reported the usefulness for diagnosis and risk classification of gene analysis using serum free nucleic acid in neuroblastoma patients. In particular, we have established a detection method using serum free DNA for MNA, which is one of the strong prognostic factors of neuroblastoma, and applied it to diagnosis. This technique can be applied to neuroblastoma cases that are difficult to classify due to poor condition and tumor tissue for genetic diagnosis cannot be collected. Since genetic analysis is possible in time, and it is possible to provide accurate diagnosis and risk classification, diagnosis is requested from facilities nationwide. On the other hand, the diagnostic method for 11q LOH has not been established in Japan despite the diagnostic items essential for risk classification.

Gotoh, et al. J Clin Oncol 23: 5205-5210, 2005 Yagyu, et al. Clin Cancer Res 14: 7011-7019, 2008

  An object of the present invention is to provide a method for detecting 11q LOH by a noninvasive method without collecting tumor tissue. Another object of the present invention is to provide a method for detecting neuroblastoma or predicting its prognosis by detecting 11q LOH.

  11q LOH is found in neuroblastoma cells but not in other cells. Therefore, genetic diagnosis using tumor tissue was considered essential for detection of 11q LOH only in neuroblastoma cells in which this mutation occurred. Surprisingly, however, chromosome 11 of neuroblastoma cells with mutations is present in blood in an amount sufficient to detect 11q LOH, detecting 11q LOH using blood-derived specimens, and neurological species. The present inventors have found that detection and prediction of the prognosis are possible, and have completed the present invention.

The present invention provides a method for detecting 11q LOH or (malignant) neuroblastoma and a method for predicting neuroblastoma prognosis.
Item 1. A method for detecting LOH, comprising detecting a LOH (loss of heterozygosity) on chromosome 11 by performing PCR using a microsatellite sequence as a target in a blood-derived specimen.
Item 2. A method for detecting neuroblastoma, comprising detecting serum LOH (loss of heterozygosity) on chromosome 11 by performing PCR using a microsatellite sequence as a target in serum.
Item 3. A method for detecting malignant neuroblastoma, characterized in that, in serum, LOH (loss of heterozygosity) on chromosome 11 is detected by PCR using a microsatellite sequence as a target.
Item 4. A method for predicting prognosis of neuroblastoma, characterized in that, in serum, LOH (loss of heterozygosity) on chromosome 11 is detected by performing PCR using a microsatellite sequence as a target.
Item 5. Item 5. The method according to any one of Items 1 to 4, wherein LOH on chromosome 11 is detected by a microsatellite analysis method.

  Neuroblastoma can be primary screened by urinalysis.

  On the other hand, because most of the neuroblastomas found in urinalysis regress spontaneously, it is necessary to identify early whether the neuroblastoma is naturally regressing or progressive and has a poor prognosis. It becomes necessary to do.

  According to the present invention, 11q LOH can be easily detected using a blood-derived specimen, so that it is possible to more accurately distinguish spontaneously regressing neuroblastoma from progressive neuroblastoma.

An unbalanced LOH detection is schematically shown. In the case of LOH (+), only one band is detected, and in the case of LOH (−), two bands are detected. 11q LOH analysis by microsatellite analysis and FISH method. a: Results of microsatellite analysis using serum, tumor, and lymphocyte DNA. Two peaks are observed in lymphocyte DNA, whereas one peak is greatly reduced in serum and tumor DNA. b: FISH analysis using primary cultured cells from the same patient. Chromosome 11 has 4 signals (green), whereas the probe made in the MLL gene has 2 signals, and this case is determined to be 11q LOH. c: Schematic diagram of each probe. Results of FISH method for each case. Distribution of LOHindex in serum.

  The blood-derived specimen used in the present invention includes serum and plasma, and serum is more preferable.

  For example, as shown in FIG. 1, when there is a deletion in one chromosome (unbalanced LOH (+)), only one band is detected and either LOH of chromosome 11 of the present invention is detected. When there is no deletion in the chromosome (unbalanced LOH (-)), it can be distinguished by detecting two bands. That is, in the case of normal cells, theoretically, signals indicating the generation of two types of PCR products are detected, but in a system using tumor-derived DNA in which 11q LOH exists, one allele has disappeared. Only one type of signal is detected.

  Chromosome 11 has 8 microsatellite regions. For detection of LOH, PCR primers are designed so that the amplified product contains at least one microsatellite region. As a PCR primer, the primer set shown in Table 1 can be illustrated. The microsatellite region has a large number of CA repeats, and alleles are identified using the polymorphism of the number of CA repeats. Preferred primer sets include the sequences shown in Table 1.

  The length of the PCR reaction product is not particularly limited as long as 11q LOH can be detected, but it is, for example, about 50 to 1000 bp, preferably about 60 to 800 bp, more preferably about 80 to 600 bp, more preferably about 90 to 500 bp. In particular, it is about 100 to 350 bp. When the primer set shown in Table 1 is used, two bands from 174 bp to 196 bp are detected due to the difference in the number of CA repeats for each chromosome, and if there is LOH, one band is missing.

  Since the 11q LOH of the present invention has a normal sequence in cells other than neuroblastoma, for example, lymphocyte cells are separately separated from a blood sample, and the sequence corresponding to the 11q LOH is amplified to obtain an unbalanced LOH ( By using as a standard sample of-), it can be easily detected whether the neuroblastoma is unbalanced LOH (+) or non-unbalanced LOH (-).

  Specifically, the position to be analyzed by LOH is the 23.3 position (11q23.3) of chromosome 11.

In LOH analysis targeting microsatellite arrays, if the LOH index is 0.5 or less or 2 or more, it is judged as LOH, and if it is between 0.5 and 2, it is judged as not LOH (normal) (FIG. 4). .
LOH index = {A / B} / {C / D}
here,
“A”: normal tissue allele 2 peak area “B”: normal tissue allele 1 peak area “C”: tumor tissue allele 2 peak area “D”: tumor tissue allele 1 peak area normal Judgment: 0.5 <LOH index <2
Judged as LOH: 0.5 ≧ LOH index or LOH index ≦ 2

  For detection of PCR products, it is preferable to label one or both of the primers with a fluorescent label or the like. As the label, fluorescein, rhodamine, Texas red, dansyl, lucifer yellow VS, umbelliferyl, rare earth chelate, Cy2, Cy3, Cy5, fluorescein isothiocyanate (FITC), TRITC, ALEXA (registered trademark) (Molecular Probe) And labeling with fluorescent substances such as quantum dots, labeling with biotin, avidin, streptavidin, gold particles, iron particles, magnetic particles, and magnetic beads.

  LOH on chromosome 11 can be detected preferably by a microsatellite analysis method.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not particularly limited by the following examples.

Example 1
1. Methods Cell lines, tumor specimens, and serum Serum at the first visit, cryopreserved tumor specimens, peripheral blood lymphocytes, primary tumor tissue among neuroblastoma patients treated at the Department of Pediatrics, Kyoto Medical University and Kyoto City Hospital We examined 24 cases in which cultured cells were preserved and consent to use specimens for research was obtained.
Extraction of DNA DNA was extracted from 30 μg of tumor specimen and 200 μl of serum at the first visit using the QIAamp DNA Mini kit (Qiagen). For serum, centrifugation was further performed at 15,000 rpm for 10 minutes to remove cellular components, and the supernatant was used.
PCR
A primer set that amplifies three microsatellite regions contained in the 11q LOH region of neuroblastoma that has already been reported was prepared (Table 1). Each forward primer was fluorescently labeled, and the reverse primer was a tailed primer (Applied Biosystems). Using these primer sets, PCR was performed using DNA extracted from serum, tumor, and lymphocytes as a template.

Capillary electrophoresis Each PCR product was subjected to capillary electrophoresis using ABI PRISM 310 genetic analyzer (Applied Biosystems). The fragment size and fluorescence intensity of each PCR product were analyzed with Genescan Software (Applied Biosystems). The ratio of fluorescence intensity (LOH index) of PCR products obtained from serum DNA and lymphocyte DNA was calculated (Fig. 2a).

Fluorescent in situ hybridization
The primary cultured cells of the tumor tissue were Carnoy fixed, developed on a slide glass, and the above fixed cells were subjected to FISH analysis. A probe was created in the MLL gene contained in the 11qLOH region (GSP Laboratory). In addition, two-color FISH was performed using a probe (Abbott Molecular / Visys) set in the chromosome 11 satellite portion as a centromere enumeration probe (FIGS. 2b and 2c). The FISH specimen was observed at 600 times using a BZ-8000 fluorescence microscope (KEYENCE) and subjected to LOH determination.

Statistical analysis
The results of FISH method were divided into two groups, 11q LOH positive group and negative group, and LOH index in each serum was compared using Mann-Whitney U test.

2. result
There were 19 cases that were determined to be 11q LOH negative by the FISH method (LOH negative group), and 5 groups that were determined to be LOH positive (LOH positive group) (FIG. 3). There was a significant difference between the LOH index of serum DNA in the LOH negative group and the LOH index of LOH positive (p = 0.007). The serum LOH index in the LOH negative group is around 1, whereas in the LOH positive group, it is much higher than 2 or much lower than 0.5, and the sensitivity and specificity when the LOH index cutoff values are set to 0.5 and 2.0. Each degree was 100% (Figure 4).

3. Discussion Based on the results of the present inventors, it was considered that the presence or absence of 11q LOH in the tumor can be predicted by microsatellite analysis using tumor-derived DNA extracted from the serum at the first visit of the patient. This indicates that the presence or absence of 11q LOH, which is a strong poor prognosis factor for neuroblastoma, can be diagnosed preoperatively.

  The treatment strategy for neuroblastoma is combined with age, disease stage, and risk classification that considers the presence or absence of genetic prognostic factors. A wide range of multidisciplinary treatments. Recently, the presence or absence of MYCN gene amplification and 11q LOH has been reported as a strong prognostic factor and has become an essential diagnostic item for risk classification. On the other hand, for localized neuroblastoma that developed in infancy, untreated follow-up that expects spontaneous regression without surgery is widely performed, but the search for genetically poor prognostic factors Risk of underestimating the risk from time to time. From these results, if prognostic factors can be determined using serum at the first visit, more detailed risk classification can be performed without surgery. It will be possible to provide.

Claims (5)

A method for detecting LOH, comprising detecting a LOH (loss of heterozygosity) on chromosome 11 by performing PCR using a microsatellite sequence as a target in a blood-derived specimen. A method for detecting neuroblastoma, comprising detecting serum LOH (loss of heterozygosity) on chromosome 11 by performing PCR using a microsatellite sequence as a target in serum. A method for detecting malignant neuroblastoma, characterized in that, in serum, LOH (loss of heterozygosity) on chromosome 11 is detected by PCR using a microsatellite sequence as a target. A method for predicting prognosis of neuroblastoma, characterized in that, in serum, LOH (loss of heterozygosity) on chromosome 11 is detected by performing PCR using a microsatellite sequence as a target. The method according to any one of claims 1 to 4, wherein LOH on chromosome 11 is detected by a microsatellite analysis method.

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