Highly sensitive analysis for N-myc amplification in neuroblastoma based on fluorescence in situ hybridization

Highly Sensitive Analysis For N-myc Amplification in Neuroblastoma Based on Fluorescence In Situ Hybridization By Tatsurou

Tajiri, Kumiko Shono, Masazumi

Yoshimitsu Tsuneyoshi, Fukuoka,

Background/Purpose:The N-mycamplification status in neuroblastoma has been evaluated previously for the whole tumor by the Southern blot method. The aim of this study is to evaluate the effectiveness of the fluorescence in situ hybridization (FISH) method to analyze N-myc amplification in neuroblastoma and compare the findings with those using the Southern blot method. Methods: In 26 neuroblastoma primary tumors and metastatic lesions, the N-myc amplification status was evaluated by both the Southern blot method and FISH method. Resu/ts:Of the 22 samples with no N-mycamplification using Southern blot, no cells with N-myc amplification using FISH were present in 21 of the samples. However, one metastatic liver lesion showed 16% of the nuclei to display more than 10

N

EUROBLASTOMA is the most common extracranial solid tumor in children; however, its development is still unclear.’ The prognosis in neuroblastoma is variable, and many findings have shown both clinical and biological factors that can correlate with outcome. To select the optimal chemotherapy treatment, it is essential to accurately identify the presence of neuroblastoma cells with unfavorable prognostic factors to determine the degree of malignancy in neuroblastoma. The amplification of the N-myc gene is the most unfavorable prognostic factor in neuroblastoma.2 The N-myc amplification status has been evaluated previously in the whole tumor by the Southern blot method.3s4 However, this method is not able to detect intratumor heterogeneity. The fluorescence in situ hybridization (FISH) method has been found able to evaluate the status of N-myc amplification in individual neuroblastoma cells5 In the current study, to evaluate the effectiveness of the FISH method for analyzing the prognostic factors in neuroblastoma, we investigated the status of N-myc amplification in 26 neuroblastoma primary tumors and metastatic lesions using both FISH and the Southern blot method. MATERIALS

AND

METHODS

Patients and Samples Twenty-six neuroblastoma samples from 22 patients were obtained at the Department of Pediatric Surgery, Kyushu University and associated hospitals. Of the 26 samples, 22 samples were primary tumors, and 4 JournalofPediatricSurgery,Vol34,

No 11 (November),

1999: pp 1615-1619

Fujii, Shinichi and Sachiyo

Noguchi, Suita

Yoshiaki

Kinoshita,

Japan

copies of N-myc based on FISH analysis. In the 4 remaining samples with N-myc amplification using the Southern blot method (17 copies, 15 copies, 6 copies, and 3 copies), the rates of cells with more than 10 copies of N-myc based on a FISH analysis were 79%, 68%, 94%, and 9%, respectively. Conclusions: The FISH method can detect more accurately N-myc amplification than the Southern blot method either when the rate of cells with N-myc amplification is low or intratumor heterogeneity is present. J Peciiatr Surg 34:1615-1619. Copyright o 7999 by W.B. Saunders Company.

INDEX WORDS: toma, N-myc.

Fluorescence

in situ hybridization,

neuroblas-

were metastatic lesions. Twenty-four of 26 samples were obtained at the time of initial diagnosis. One primary tumor sample and another sample from metastatic tumor to the liver were obtained after chemotherapy had been administered. The clinical features of the 22 neuroblastoma patients included the following. The age range varied from 1 month to 2 years of age. Of the 22 patients, 17 cases were detected by mass screening tests at 6 months of age. Staging was based on the international neuroblastoma staging system (INSS). Of the 22 patients, 10 cases were stage 1, 3 cases were stage 2A, 4 cases were stage 3, 2 cases were stage 4, and 3 cases were stage 4s. Of the 22 patients, all are alive, and 7 patients have residual disease. The follow-up period ranged from 4 months to 3 years.

Southern Blot Method of N-myc

Gene

DNA was extracted from frozen samples using the standard methods. The number of copies of the N-myc gene was determined as described previously.6 Human placental DNA was used as the single copy amount.

FISH Method of N-myc Gene Fresh tumor single cells were suspended in 75 mmol/L potassium chloride for 10 minutes. Fixation was in 3:l methanol-glacial acetic acid, and the nuclei were dropped onto cleaned glass slides. The nuclei on slides were denatured immediately before hybridization in 70%

From the Departments of Pediatric Surgery and Pathology, Kyushu University, Fukuoka, Japan. Presented at the 32nd Annual Meeting of the Pact& Association of Pediatric Surgeons, Beijing, China, May 9-14, 1999. Address reprint requests to Tatsurou Tajiri, MD, Department of Pediatric Surgery, Kyushu University, 3-1-I Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Copyright 0 1999 by WB. Saunders Company 0022-3468/99/3411-0007$03.00/O 1615

1616

TAJIRI

formamide, 2 times standard saline citrate (SSC) at 75°C for 2 minutes, and then were dehydrated through an ethanol series of 70%, 100% for 5 minutes. The hybridization buffer (10 pL, Vysis) containing 10 ng aliquot of IV-myc probe (Vysis, LSI n-myc spectrum Orange) was denatured for 5 minutes at 75”C, chilled on ice, and then applied on the slide. Hybridization was performed overnight at 37°C under sealed coverslips. The slide was washed first in 50% formamide 2 times SSC at 33°C for 10 minutes, followed by 4 times SSC, 0.1% Triton X100-4 times SSC and 4 times SSC for 2 minutes each using a shaker. Finally, the slide was washed in 2 times SSC for 2 minutes. The nuclei were counterstained with 30 uL of DAPI (0.25 pg/mL) containing 5 pL of antifade solution. The signals representing the N-myc gene were countered for 100 cells on one slide. ‘Ihe images were photographed using a fluorescence microscope (Olympus, BX60) and Provia 400 (Fuji, IS0 400).

ET AL

Kb

RESULTS N-myc Copy Number by Southern Blot N-myc amplification analyzed by Southern blot (22 samples, 20 patients) showed no amplification of the N-myc gene (Table 1). Four samples (3 patients) showed the findings of N-myc amplification. In case 19, stage 4, Table 1. The Comparison of IV-myc Amplification Status the FISH Method and Southern Blot Method

Case No.

stage (INS3

1 2

1 1

3 4 5

1 1 1

6

1

7 8

1 1

9

1

10 11 12

1 2A 2A

13 14

2A 3

15 16

3 3

17 18 19

3 4 4

Siteof Samples

Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary Primary*

MS Cases

N-myc Copy No. by Southern Blot

Between

Analysis of N-myc by FISH Rate of Cells With More Than 10 Copies of N-myc (%)

Outcome

Yes Yes

1 1

0 0

Alive Alive

(DF) (DF)

Yes Yes Yes

1 1 1

0 0 0

Alive Alive Alive

(DF) (DF) (DF)

Yes

1

(DF)

1 1

0 0

Alive

Yes Yes

0

Alive Alive

(DF) (DF)

Yes

1 1

0 0

Alive Alive

(DF) (DF)

1 1

0 0

Alive Alive

(RD) (DF)

No Yes

1 1

Alive Alive

(DF) (RD)

Yes Yes Yes

1 1 1

0 0 0 0 0

Alive Alive Alive

(RD) (RD) (RD)

Alive Alive

(RD) (DF)

No Yes Yes

No No

17 3

79 9

Lvwh node 4s

Primary Liver*

Yes

15 6 1

68

20

94 16

Alive

(RD)

21

4s

Primary Liver

Yes

1

0 0

Alive

(DF)

Primary Liver

No

1 1 1

0 0

Alive

(DF)

22

4s

Abbreviations: MS, mass ease free. *After chemotherapy.

screening;

RD. residual

disease;

DF, dis-

Fig 1. Southern blot of Kmyc. (Lane 1) Human placental DNA as single copy control. (Lane 2) Primary tumor after chemotherapy in case 19; 3-fold. (Lane 3) Left axillary metastatic lymph node in case 19; 15-fold.

the sample from the left axillary metastatic lymph node obtained at the initial diagnosis showed 15 copies of the N-myc gene, whereas a sample of primary tumor after the aggressive chemotherapy showed 3 copies of the N-myc gene (Fig 1). In case 20, stage 4s the sample from the right adrenal tumor at the initial operation showed 6 copies of N-myc, whereas a sample of the liver metastasis after chemotherapy showed 1 copy of N-myc. The Analysis by FISH of the N-myc Gene Of the 22 samples with no amplification of N-myc by Southern blot, no cells with more than 10 copies of N-myc amplification using FISH were present in 21 samples (Table 1, Fig 2A). However, a metastatic liver lesion in case 20 showed 16% of the nuclei with more than 10 copies of N-myc based on the FISH analysis (Fig 2B). Two samples (case 18 and lymph node of case 19) with over 10 copies of N-myc amplification using the Southern blot had cells with more than 10 copies of N-myc based on a FISH analysis at 79% and 68%, respectively. The primary tumors in case 20 with 6 copies of N-myc by Southern blot method showed 94% of the nuclei with more than 10 copies of N-myc using FISH method. In case 19, the FISH analysis of the primary tumor after chemotherapy showed 9% of the nuclei with more than 10 copies compared with 3 copies of N-myc based on the Southern blot (Fig 2C). These results suggest that a discrepancy thus exists between the findings of the Southern blot method and those of the FISH method.

FISH ANALYSIS

OF IV-myc

1617

IN NEUROB~STOMA

Fig 2. Fl6H of fU-myc. (A) Case 13: No cell with more than 10 copies is present. (B) The metestatic sample contain more than ‘IO copies of I\l-mycas displayed in the figure. ICI The primary tumor after with more than 10 copies as shown in the figure is 9%.

The Clinical Course of a Case of a Discrepancy Between FISH Method and Southern Blot Method for N-myc AmpliJcation Status

Figure 3 shows the clinical course of case 20 with a discrep~cy between the FISH and Southern blot methods. This case was diagnosed as right adrenal neuroblastoma with multiple liver metastatic lesions by mass screening when the patient was 6 months of age. The histological diagnosis of the primary tumor was ganglioneuroblastoma with favorable histology by Shimada’s classification (Southern blot: the copy number of N-myc

Primary tumor extirpation and liver biopsy

D regimen : CPA, VCR, THP-ADR, CDDP new Al : CPA, VP-l 6, THP-ADR, CDDP new AZ: CPA, THP-ADR, Carboplatin new A2

new Al

~~~~~~~~~~~

4

+

. . 1 Time in years from initiation of therapy Fig 3.

The clinical

of case 20: 16% of cells in the of case 19: The rate of cells

was 6. FISH: the rate of cells with N-myc amplification was 94%). Other biological prognostic factors of the primary tumor were diploid, deletion of 1~36, and the low expression of TrkA gene. The histological findings of the liver metastatic lesion was almost the same as the primary tumor. Mild chemo~erapy (CPA 300 mg/mgz, VCR 1.5 mg/mg2, l-week interval) was administered after complete resection of primary tumor, because the patient was stage 4s under 1 year of age, and the N-myc amplification of the primary tumor was less than 10 copies based on the Southern blot findings. However, the

Liver biopsy

(CPA, VCR) D regimen

liver lesion chemotherapy

course

of case 20.

+

1 2

1618

TAJIRI

size and number of multiple liver metastases did not change, so intensive multiple drug chemotherapy (CPA 800 mg/mg*, VCR 1.5 m/mg*, THP-ADR 30 mg/mg*, CDDP 60 mg/mg*, 4-week interval) was started. After 3 courses of intensive chemotherapy, a liver biopsy was performed because of no change in the size of the liver metastases. A number of viable ganglioneuroblast cells were present in the liver biopsy sample, which was similar to the findings in the liver metastasis at the primary operation (Southern blot: the copy number of N-myc was 1, FISH: the rate of cells with N-myc amplification was 16%. As a result, more intensive chemotherapy (CPA 1200 mg/mg*, VP-16 100 mg/mg* X 5, THP-ADR 40 mg/mg*, CDDP 90 mg/mg*, 4-week interval) was administered after the liver biopsy. The intensive chemotherapy has been continued for 15 months, however, without change in the size and number of liver metastases. DISCUSSION

The amplification of N-myc is the most unfavorable prognostic factor in patients with neuroblastoma.6,7 In many established neuroblastoma treatment protocols, it is mandatory to evaluate N-myc gene amplification.8 In the protocol of the study group in Japan regarding the treatment of advanced neuroblastoma, when N-myc is amplified more than lo-fold, patients receive high-dose, multiple-drug chemotherapy.g It is therefore important to identify accurately the level of N-myc amplification to avoid either under or over treating the patients. In this study, most of the results obtained using the Southern blot method were consistent with those obtained by the FISH method. However, in several samples, a discrepancy was observed between the two methods. In the primary tumor of case 19 and the liver metastatic lesion of case 20, according to the results of FISH, these samples were found to contain cell components with N-myc amplification of more than 10 copies. In these samples, the Southern blot method was unable to detect an N-myc amplification of more than 10 copies, because the number of cells with N-myc amplification was low. Analysis by the FISH method showed no cells with more than 10 copies of N-myc to be present in the liver

ET AL

metastases of both cases 21 and 22. The metastases disappeared after a resection of the primary tumor and mild chemotherapy. By contrast, in case 20, the presence of a small number of neuroblast cells with more than 10 copies of N-myc in the tumor might be the reason why the liver metastases were refractory to chemotherapy. In case 19, a metastatic lymph node was composed of immature neuroblast cells, whereas the majority of the primary tumor after chemotherapy was composed of ganglioneuroblast cells and a small part of the tumor contained immature neuroblast cells. There was a possibility that the immature neuroblast cells had an N-myc amplification of more than 10 copies, whereas the ganglioneuroblast cells had no amplification of N-myc. If this hypothesis is true, then the primary tumor of case 19 might show heterogeneity in the tumor. An analysis by FISH method of N-myc may be able to detect many casesof heterogeneity in the tumor.lOFurthermore, regarding the casesof more than 1 copy and less than 10 copies of N-myc based on Southern blot, it may be important to investigate cells with more than 10 copies of N-myc in the tumor to evaluate the degree of tumor biological aggressiveness. The accuracy of N-myc amplification of tumor cells cannot be determined by the standard FISH method, because tumor cells with 1 copy of N-myc cannot be distinguished from the peripheral blood leukocytes and normal stromal elements. To analyze the percentage of tumor cells in the clinical samples, it may be necessary to perform the Giemsa staining or tumor cell-specific staining using antibody-neuroblastoma such as the antiGD~ on aliquots of each tumor suspension. The disialoganglioside (3~2 is a glycolipid expressed on the surface of neuroblastoma. This immunochemistry can be performed within 1 day. l l The FISH method can more accurately detect N-myc amplification than the Southern blot method either when the rate of cells with N-myc amplification is low, or intratumor heterogeneity is present. Diagnostic analyses based on a combination of the FISH and Southern blot methods are thus considered to be effective for evaluating the degree of aggressiveness in neuroblastoma.

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5. Shapiro DN, Valentine MB, Rowe ST, et al: Detection of N-myc gene amplification by flurescence in situ hybridization. Am J Path01 142:1339-1346, 1993 6. Tsuda T, Obara M, Hirano H: Analysis of N-myc amplification in relation to disease stage and histologic types in human neuroblastomas. Cancer 60:820-826, 1987 7. Brodeur GM, Seeger RC, Schwab M, et al: Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224:1121-1124,1984 8. Cohn SL, Look AT, Johsi VV, et al: Lack of correlation of N-myc gene amplification with prognosis in localized neuro-

FISH ANALYSIS

OF A’-myc

IN NEUROBLASTOMA

blastoma: A Pediatric Oncology Group Study. Cancer Res 55:721-726, 1995 9. Kaneko M, Ntshihira H, Mugishima H, et al: Stratification of treatment of stage 4 neuroblastoma patients based on N-myc amplification status. Med Pediatr Oncol31: l-7, 1998 10. Lorenzana AN, Zielenska M, Thomer P, et al: Heterogeneity of

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MYCN amplification on a child with stroma-rich neuroblastoma (ganglioneuroblastoma). Pediatr Path01 Lab Med 17:875-883, 1997 11. Strehl S, Ambros PF: Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma. Cytogenet Cell Genet 63:24-28,1993