JP3901684B2 - Examination method of body fluid of neuroblastoma - Google Patents

Description

  The present invention relates to a method for diagnosing neuroblastoma. More specifically, the present invention relates to a diagnostic method that serves as an indicator of early detection, treatment, and recurrence of neuroblastoma based on gene amplification.

  Neuroblastoma (NB) is the most common extracranial malignancy in children and is characterized by a wide range of clinical behaviors ranging from spontaneous healing to rapidly progressing fatal conditions despite multidisciplinary treatment. Clinical heterogeneity has been reported to be closely associated with various biological, pathological and genetic characteristics in neuroblastoma. Among several genetic abnormalities, MYCN amplification is strongly associated with rapid tumor progression and unfortunate consequences. Amplification of MYCN is observed in 4 to 8% of early cases of neuroblastoma and about 30% of advanced cases, and the amplification cases are highly malignant. At present, the presence or absence of amplification of MYCN is an indispensable evaluation item for selection of a treatment protocol based on risk classification (see Non-Patent Documents 1 to 6).

  The examination of neuroblastoma in Japan has been conducted for the purpose of early detection and early treatment by measuring catecholamine metabolites contained in urine in all infants 6 to 7 months old. The cases discovered by the examination include a considerable number of cases that do not require active treatment, and such mass screening has been temporarily stopped. However, advanced neuroblastoma is a disease that is difficult to treat, and an examination method that can identify neuroblastoma with high malignancy is desired.

  Interestingly, it has been reported that the peripheral blood of patients with malignant gastrointestinal cancer contains about 30 times as much DNA as healthy donors (see Non-Patent Document 7). Furthermore, it has been reported that tumor-related abnormalities such as loss of heterozygosity and mutation of p53 gene were detected using serum DNA of patients with malignant tumors (see Non-Patent Documents 8 to 10). thing). These reports suggest that DNA in serum circulating throughout the body of cancer-bearing patients can be released primarily from tumors rather than normal tissues. Thus, tumor associated DNA present in peripheral blood may be applicable to predict tumor MYCN status.

Recently, it has been reported that a high level of MYCN DNA is present in the peripheral blood of a neuroblastoma patient in which MYCN has been amplified (see Non-Patent Document 11).
Science, 224: 1121-1124, 1984 N. Engl. J. Med., 313: 111-1116, 1985 J. Pediatr. Hematol. Oncol., 19: 93-101, 1997 J. Clin. Oncol., 11: 1466-1477, 1993 Eur. J. Cancer, 33: 2113-2116, 1997 Cancer, 86: 364-372, 1999 Cancer, 51: 2116-2120, 1983 Clin. Cancer Res., 5: 2689-2692, 1999 Clin. Cancer Res., 5: 2297-2303, 1999 Cancer Res., 59: 3251-3256, 1999 Cancer Res., 62: 3646-3648, 2002

  However, in Non-Patent Document 11, the amount of MYCN in serum is evaluated by PCR without placing a control gene, and this assay may be influenced by the quality of template DNA or the change in the number of second chromosomes. . Therefore, an object of the present invention is to provide a simple and accurate method for diagnosing neuroblastoma.

  As a result of intensive investigations taking into consideration the relationship between the clinical state of neuroblastoma and genetic abnormalities, the present inventors are able to diagnose neuroblastoma not only using tumor tissue but also using DNA in body fluids. As a result, the present invention has been completed.

That is, the method for examining a neuroblastoma body fluid of the present invention is
Contained in a body fluid from a subject, detecting the MYCN gene and N- acetylglucosamine kinase (nagk) gene,
A step of calculating a ratio of the MYCN gene for the NAGK gene, and
Determining the condition of the subject based on the amount ratio,
It is characterized by including.

  The body fluid is preferably serum.

  The detection step is preferably performed by polymerase chain reaction (PCR).

  According to the method for diagnosing neuroblastoma of the present invention, by setting an appropriate control gene and examining the copy number of MYCN per haploid, the degree of malignancy and treatment of neuroblastoma at the time of diagnosis and treatment course of neuroblastoma Stratification, determination of therapeutic effect and markers of recurrence can be provided, which is a very advantageous method.

  In the diagnostic method of the present invention, when the measurement object is a body fluid derived from a subject, particularly serum, it can be collected and stored before treatment, during treatment, and after treatment, and the burden on the patient is small. Diagnosis of neuroblastoma can be performed easily and accurately.

  In the diagnostic method of the present invention, when a gene located on the short arm of human chromosome 2 and located outside the amplicon of the MYCN gene, particularly the NAGK gene (2p12), is selected as the single copy control gene, the MYCN / NAGK ratio is calculated. A neuroblastoma marker can be provided as a more specific value.

  In the diagnostic method of the present invention, when the detection step is performed by PCR widely used in this technical field, it is possible to provide a diagnostic method with better quantification.

  In the present specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the double-stranded DNA. There is no particular limitation. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing cDNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. The “gene” or “DNA” includes not only “gene” or “DNA” represented by a specific base sequence (SEQ ID NO: 1) but also a biological function equivalent to the protein encoded by these. “Gene” or “DNA” encoding a protein (eg, homologs (such as homologs and splice variants), variants and derivatives) is encompassed. Specifically, the “gene” or “DNA” encoding such a homologue, variant or derivative is, for example, a hybrid with a complementary sequence of the specific base sequence shown in SEQ ID NO: 1 under stringent conditions. Examples include “gene” or “DNA” having a base sequence for soybean.

  For example, as a gene encoding a homologue of a human-derived protein, genes of other species such as mouse and rat corresponding to the human gene encoding the protein can be exemplified, and these genes (homologues) are homologous genes (http: / /www.ncbi.nlm.nih.gov/HomoloGene/).

  The gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.

  The MYCN gene in the present invention is also called an N-MYC or N-myc gene, and a human-derived gene (GenBank accession No. NM — 005378) is preferable.

  Accordingly, when the term “MYCN gene (DNA)” is used in the present specification, the human MYCN gene (DNA) represented by the specific base sequence (SEQ ID NO: 1) and its homologues, unless otherwise specified by the SEQ ID NO. It is used to include genes (DNA) encoding mutants and derivatives. Specifically, the human MYCN gene described in SEQ ID NO: 1, its mouse homolog, rat homolog, and the like are included.

The neural blastoma, observed cases chromosome number of tumor cells is changing, tumors are the number of a particular chromosome and has a triploid increases is relatively prognosis is good, normal (46 Those close to chromosomes are said to have a poor prognosis. Therefore, in the present invention, a control gene that can be quantified by removing such factors is provided when a sample in which the MYCN gene is apparently amplified with an increase in the number of chromosomes is also examined. That is, a preferred control gene is located at the same locus as the MYCN gene (second chromosome short arm in humans) and outside the amplicon of the MYCN gene (2p24). In the present invention, N-acetyl is a gene that satisfies the above conditions, is not amplified in various cancers, is present in a region that is neither amplified nor deleted in neuroblastoma, and is not affected by fluctuations in the number of second chromosomes. The glucosamine kinase (NAGK) gene (2p12) is particularly preferred.

The method for examining a neuroblastoma using a body fluid according to the present invention includes the following steps.

(1) contained in a body fluid from a subject, step subject detecting the MYCN gene and N- acetylglucosamine kinase (nagk) gene, especially but not limited to, Lord children from birth to about 7 years of age The target is also from puberty to adults. Prior to performing the determination method of the present invention, or at the same time as the determination method, a preliminary examination may be performed by another diagnostic method.

  The body fluid is not particularly limited as long as it contains tumor-derived DNA, and includes blood, serum, plasma, cerebrospinal fluid, pleural effusion, ascites or urine. preferable.

  As a method for detecting the MYCN gene and the control gene, a known gene amplification method is used without limitation. Polymerase chain reaction (PCR), Loop-Mediated Isothermal Amplification (LAMP method, http://loopamp.eiken.co.jp) /), Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN (registered trademark) method, http://www.takara-bio.co.jp/), etc. Any of the gene amplification methods can be performed using a commercially available kit according to the manufacturer's instructions, and a gene amplification product can be detected and quantified in real time by using a probe labeled with a fluorescent dye. Among them, it is preferable to use the most popular real-time PCR, but it is not limited to this.

  As the primer set used for the gene amplification method, a suitable sequence can be appropriately selected from the MYCN gene sequence and the control gene sequence according to each amplification method. At this time, it is preferable to check a registered SNP in a database or the like and select a primer sequence from a region having no SNP. As examples of primer sets that can be used for real-time PCR, MYCN gene amplification primer sets (SEQ ID NOs: 2 and 3) and NAGK gene amplification primer sets (SEQ ID NOs: 5 and 6) are described in the Sequence Listing and Examples. .

(2) Step of calculating the ratio of the amount of the MYCN gene to the control gene The M / N is calculated from the amounts of the MYCN gene (M) and the control gene (N) detected in the step (1). For example, when the NAGK gene is selected as a control gene and there is no amplification of the MYCN gene, the serum M / N is about 1.0, and in the case of highly advanced neuroblastoma, the serum M / N is about 100. Up to about 800. A preferable cut-off value is 2.0 to 2.2.

(3) Step of determining the state of the subject based on the quantitative ratio In this step, the state of the subject can be determined based on the quantitative ratio of the MYCN gene to the control gene calculated in the step (2).

a) When used for primary screening of patients with neuroblastoma Examples show that with this diagnostic method, significant amounts of genomic DNA can be detected in body fluids even in early stage patients . Therefore, the present diagnostic method is useful as a primary screening because a patient having MYCN gene amplification can be screened from the initial stage to the advanced stage.

b) When used for stratification of neuroblastoma treatment By using this diagnostic method, tumors are evaluated by evaluating the M / N ratio in DNA in body fluids without being affected by fluctuations in the number of chromosomes 2. Amplification of the MYCN gene in DNA can be detected. By using this diagnostic method, it is possible to predict the copy number of the MYCN gene at the time of diagnosis prior to biopsy or tumor resection.

c) When used for the determination of neuroblastoma treatment results Using this diagnostic method, the body fluid before treatment and the body fluid after treatment of the same patient having MYCN gene amplification are compared, and the M / N before treatment is compared. If the ratio and the M / N ratio after treatment are significantly reduced, it indicates that the treatment was successful. Therefore, this diagnostic method can be used to determine the therapeutic outcome of neuroblastoma.

d) When used to determine the prognosis of neuroblastoma This diagnostic method can be used to follow a patient being treated with amplification of the MYCN gene. Examination is performed in comparison with the M / N ratio before treatment, and it is suggested that there is a therapeutic effect from the time when the value significantly decreased. Therefore, this diagnostic method can be used to determine the prognosis of neuroblastoma.

e) When used as an index of recurrence of neuroblastoma Since this diagnosis method can detect patients at an early stage, it is also useful for early detection of recurrence in follow-up after remission of MYCN-amplified patients. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

Diagnostic method based on amplification of MYCN gene in serum of patients with neuroblastoma Sample Preparation 53 patients diagnosed with neuroblastoma at Kyoto Medical University Hospital were enrolled in this study with informed consent from each patient. Tumor specimens were surgically excised and stored immediately at -80 ° C. Peripheral blood was collected from each patient prior to surgery. In addition, informed consent was obtained from 15 non-cancer-bearing children and blood samples were collected as normal controls. Serum was prepared by centrifuging and centrifuging at 1000 g for 10 minutes and stored at −20 ° C. until use.

  The groupings of neuroblastoma patients were determined by Southern blotting, 11 patient groups with MYCN amplification and 42 patient groups with no amplification. The age of the patients at the time of diagnosis was 27 patients less than 1 year old and 26 patients 1 year old. Table 1 shows the stage distribution of these patients.

２．ＤＮＡの単離
ＤＮＡを、ＱＩＡｍｐ tissue and blood kits（ＱＩＡＧＥＮ製）を用いて製造業者のプロトコールに従って、組織および血清試料からそれぞれ抽出した。

2. DNA Isolation DNA was extracted from tissue and serum samples, respectively, using QIAmp tissue and blood kits (QIAGEN) according to the manufacturer's protocol.

3. Real-time quantitative PCR
Real-time quantitative PCR was performed by the TaqMan (registered trademark) method using an ABI Prism (registered trademark) 5700 sequence detection system (manufactured by Applied Biosystems). Clin. Chem., 43: 752-758, 1997, using a PCR mixture containing TaqMan® universal PCR master mix (Applied Biosystems), 200 nmol / l of each probe, and 100 nmol / l of fluorogen probe. It carried out according to the method of description. In addition to the MYCN sequence, an N-acetylglucosamine kinase gene (NAGK: GenBank accession No. NM — 017567) located on chromosome 2p12 was simultaneously measured as a single copy control gene.

The primer and TaqMan probe sequences used for detection of MYCN are as follows:
MYCN-forward, 5 '-GTGCTCTCCAATTCTCGCCT-3' (SEQ ID NO: 2)
MYCN-reverse, 5 '-GATGGCCTAGAGGAGGGCT-3' (SEQ ID NO: 3)
MYCN-probe, 5 ′ FAM-CACTAAAGTTCCTTCCACCCTCTCCT-TAMRA 3 ′ (SEQ ID NO: 4).

Primer and TaqMan probe sequences used for detection of NAGK are as follows:
NAGK-forward, 5 '-TGGGCAGACACATCGTAGCA-3' (SEQ ID NO: 5)
NAGK-reverse, 5 '-CACCTTCACTCCCACCTCAAC-3' (SEQ ID NO: 6)
NAGK-probe, 5 ′ VIC-TGTTGCCCGAGATTGACCCGGT-TAMRA-3 ′ (SEQ ID NO: 7).

  All PCR reactions were performed in duplicate, and after 1/5 cycle at 95 ° C., PCR amplification was performed in 50 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. A standard curve is obtained for each PCR performed on samples containing 20 ng / l salmon sperm DNA and healthy donor DNA in 4-fold dilutions (20, 5, 1.25, 0.3125 and 0.078125 ng / l). Respectively. The dose of target gene in each sample was interpolated using the standard curve. Next, the MYCN copy number of the sample DNA was determined by the amount ratio (M / N ratio) of MYCN to NAGK. The M / N ratio in serum DNA and tumor DNA was measured for each patient.

[result]
1. Prediction of MYCN status in tumors by measuring M / N ratio in serum DNA (Figure 1)
FIG. 1 is a graph showing the distribution of M / N ratio in various patients. Neuroblastoma patients with MYCN amplification (mean ± SE: 300.05 ± 68.25) are neuroblastoma patients with no MYCN amplification (1.01 ± 0.12, p <0.005) and non- Serum M / N ratio is significantly higher than cancer-bearing patients (0.99 ± 0.10, p <0.005). Differences were analyzed with Welch's t-test. There was no significant difference in serum M / N ratio between neuroblastoma patients with no MYCN amplification and non-cancer-bearing children (p> 0.5, according to Student's t-test). The cut-off value 2.11 was determined as the average of non-cancer-bearing + 3SD. Of the 42 neuroblastoma patients in which MYCN was not amplified, 5 (11.9%) were found to have a high serum M / N ratio slightly above the cut-off value.

2. Correlation of M / N ratio between serum DNA and tumor DNA in MYCN amplified patients (FIG. 2)
FIG. 2 shows the relationship of M / N ratio between serum DNA and tumor DNA in MYCN-amplified patients, and was evaluated by linear regression analysis. There was a significant correlation between the M / N ratio of serum DNA and the M / N ratio of tumor DNA (r ² = 0.548, p <0.001). The serum M / N ratio of stage 1 patients with MYCN amplification was relatively low compared to the tumor M / N ratio (188.9 vs. 521.6). However, the serum M / N ratio was high enough to distinguish it from patients who had not amplified MYCN at the time of diagnosis.

3. Serum M / N ratio potential marker for therapeutic effect and recurrence (Figure 3)
Serum M / N ratio was subsequently analyzed in 5 patients (with MYCN amplification) where serum samples were available at multiple time points in time after the start of treatment. In 3 patients with complete remission (cases 1-3), the serum M / N ratio decreased to the normal range, and in 1 patient with complete remission for 11 years (case 3), a significant increase indicating recurrence And consistently low (FIGS. 3A-C). In contrast, one patient (case 4) who failed to achieve complete remission and the tumor became resistant to treatment afterwards had a serum M / N ratio not reduced to the normal range, A significant high level was maintained until death (FIG. 3D).

  Another patient (case 5) who had once reached remission but developed brain metastasis after a 2-year incubation period had a serum M / N ratio of 3 weeks before diagnosis and 6 weeks before diagnosis of brain metastasis by MRI, respectively. 6 and 8.9. Although these values were significantly lower compared to the initial level, they had already increased beyond the normal range. However, when the patient developed disseminated bone metastases after 2 years, the serum M / N ratio had risen to a corresponding level at the first visit (FIG. 3E).

It is a distribution map of M / N ratio in serum DNA for every patient group. It is the figure which plotted M / N ratio in the serum DNA and tumor DNA of the patient who has MYCN amplification. FIG. 5 is a bar graph showing the serum M / N ratio of 5 patients with MYCN amplification for each course of treatment. In each case, the leftmost column shows the serum M / N ratio at the time of diagnosis, and the serum M / N value is examined over time over the rightmost column.

Claims (3)

Contained in a body fluid from a subject, detecting the MYCN gene and N- acetylglucosamine kinase (nagk) gene,
A step of calculating a ratio of the MYCN gene for the NAGK gene, and
Determining the condition of the subject based on the amount ratio,
A method for examining neuroblastoma with a body fluid , comprising:   The method according to claim 1, wherein the body fluid is serum. The detection step The method according to claim 1 or 2 carried out by polymerase chain reaction (PCR).

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