In situ detection of DNA fragmentation and expression of bcl-2 in human neuroblastoma: Relation to apoptosis and spontaneous regression

In Situ Detection of DNA Fragmentation and Expression of bd-2 in Human Neuroblastoma: Relation to Apoptosis and Spontaneous Regression By Takaharu

Oue, Masahiro

Fukuzawa, Takeshi Kusafuka, Yosuke Susumu Nagahara, and Akira Okada Osaka, Japan

0 Spontaneous regression occurs in some cases of neuroblastoma, especially stage IVS. The incidence of neuroblastoma has been reported to be increasing since the mass screening program was introduced in Japan, This would indicate that the screening is detecting regressing tumors, However, the mechanism of regression is still unknown, To evaluate the hypothesis that the regression might be related to apoptosis, the authors examined apoptosis by in situ end-labeling of fragmented DNA and expression of the apoptosis-suppressing protein bcl-2. Materials and Methods; One hundred eighteen neuroblastoma cases were available for examination. Eighty (67.8%) were detected by the mass screening program. Serial sections were cut from paraffin-embedded tumors. A modified TdT-mediated dUTP nick end-labeling (TUNEL) method was performed to detect apoptosis. lmmunohistochemical analysis was performed to detect bcl-2 expression. Results: In cases under 1 year of age or with a favorable clinical stage, the incidence of apoptosis was significantly high. Expression of b&-2 was associated with N-nryc amplification and unfavorable histology (Shimada classification). Tumors in patients under 1 year of age often had areas where cellularity was markedly decreased, and apoptosis was often observed while bcl-2 expression was reduced. In such cases, there was a negative correlation between occurrence of apoptosis and bcl-2 expression. This suggests that apoptosis may be related to spontaneous regression in neuroblastoma. Copyright o 1996 by W.B. Saunders Company INDEX WORDS: labeling.

Neuroblastoma,

apoptosis,

b&2,

nick

end-

EUROBLASTOMA is one of the most common malignant neoplasms in children. The prognosis of patients over 1 year of age with advanced neuroblastoma remains poor in spite of aggressive antitumor therapy, whereas cases under 1 year of age have a good prognosis even in advanced stages. In Japan, the incidence of neuroblastoma has increased since the introduction of a mass screening program,l which indicates that the screening is detecting regressing tumors at least in some cases. It is well recognized that some neuroblastomas regress spontaneously, especially stage IVS.2-4 However, the mechanism of regression is still unknown. Recently, a concept of programmed cell death, or apoptosis, has been recognized as a potentially important process during development or maintenance of tissue organization.5-12 It is a mode of cell death in which cells are deIeted through an endogenous, physiological process. Because apoptosis has

N

Journa/ofPediafricSurge~,

Vol31,

No 2 (February),

1996: pp 251-257

Kohmoto,

Kenji lmura,

been shown to play an important role in the development of the nervous system,13 we hypothesized that the spontaneous regression of neuroblastoma might be the result of apoptosis. To assess this hypothesis, we examined apoptosis by in situ end-labeling of fragmented DNA14,15 and expression of the apoptosissuppressing protein bcl-2 in neuroblastoma. MATERIALS

AND METHODS

Patients Samples of primary neuroblastoma were available from 118 patients who had undergone extirpation or biopsy at Osaka University Hospital and its affiliated hospitals. There were 66 boys and 52 girls; the mean age was 13 months (range, 2 days to 6 years). Eighty cases were detected through the mass screening program. Histologically, 104 tumors were neuroblastoma and 14 were ganglioneuroblastoma. According to Evans’ staging system,i6 44 patients had stage I, 31 had stage II, 18 had stage III, 18 had stage IV, and 7 had stage IVS.

Tissue Preparation The neuroblastoma specimens were fixed in 10% buffered formalin immediately after surgical resection and embedded in paraffin blocks. Portions of the samples were frozen in liquid nitrogen, stored at -8O’C and examined for N-rrzyc amplification. Serial sections of 3 km in thickness were cut from the same blocks, mounted on poly-L-lysine-coated slides and used for H&E staining, in situ end-labeling of DNA, and bcl-2 immunostaining.

In Situ End-Labeling of Fragmented DNA The chromatin of apoptotic cells is cleaved at internucleosomal sites. generating a “ladder” of DNA fragments when analyzed by agarose gel electrophoresis. To detect this fragmented DNA, we performed the modified terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method,i4 using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). Paraffin-embedded sections (3 pm) were deparaffinized and then incubated with proteinase K (20 pg/mL) in phosphate-

From the Depaflmem of Pediairic Surgery, Osaka University Medical School, the Osaka City General Hospital, and the Osaka Medical Cemter and Research Institute for Maternal and Child Health, Osaka, Japan. Presented at the 28th AnrzuaJ Meeting of the Pacific Association of Pediatric Surgeons, Huatulco, Oaxaca, Mexico, May 14-18, 1995. Address reprint requests to Takaham Oue, MD, Department of Pediatric Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Copyright 0 1996 by WB. Saunders Company 0022-3468J96f3102-0010$03.00l0 251

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OUE ET AL

buffered saline (PBS) for 15 minutes at 25C. PBS containing 0.01% Hz02 was applied to neutralize endogenous peroxidase. The sections were incubated with TdT enzyme in the presence of digoxigenin-d-UTP. After washing in PBS, the sections were incubated with antidigoxigenin antibody conjugated with peroxidase. Diamino-benzidine-hydrogen peroxidase was used to detect the presence of the peroxidase, and a light Mayer’s hematovlin counterstain was applied.

Irnrnunohistochemistry

of bcl-2

Immunostaining was performed with the standard streptavidinbiotin immunoperoxidase method (LSAEI kit; Dako, Kyoto, Japan). Good immunostaining was achieved using a microwave oven to pretreat the dewaxed sections. The sections were placed in 10 mmol/L citrate buffer (pH 6.0) and processed twice, for 5 minutes, in a microwave oven and then rinsed in PBS. The sections were stained with monoclonal antibody against bcl-2 (1:50 dilution; Dako) overnight, at 4C, then counterstained with light Mayer’s hematoxylin.

Evaluation of Staining Apoptosis was quantitated by determining the percentage of positively stained cells within a field of view at a magnification of 400x. At least 1,000 tumor cells were counted from 10 to 20 randomly chosen fields per slides assayed, and the counts were averaged to obtain the apoptotic index. To evaluate the bcl-2 expression, at least 1,000 tumor cells were counted from 10 to 20 representative fields (areas in which the reaction was clearly positive). In these fields, all the reactive nuclei were considered to be positive, regardless of the intensity of staining, and the fraction of positive cells was determined. The bcl-2 lab&g index was expressed as the number of positive cells per 100 tumor cells.

Statistical Analysis Data were analyzed by computer, using StatView statistical sofhvare (Abacus Concepts, Berkeley, CA). The statistical significance of each correlation was determined by the unpaired t test. Linear regression analysis was used to test significance of correlations. P values of < .05 were considered significant. RESULTS

In Situ End-Labeling of Fragmented DNA The modified TUNEL method readily detected apoptotic cells in almost all neuroblastoma tumors, at various rates. Most positive cells showed morphological characteristics of apoptosis such as condensation of chromatin, reduction in nuclear size, nuclear fragmentation, or formation of apoptotic bodies (Fig 1). However, some appeared to have almost normal nuclei. Most positive cells appeared isolated and scattered, but among the 93 cases under 1 year of age, accumulation of positive cells was observed in 46 (Fig 2). Table 1 shows the correlation between apoptotic index and clinical variables. The apoptotic index was significantly higher in cases with a favorable clinical stage (I, II, and IVS). It also was significantly higher for children under 1 year of age than for those over 1

year of age. Most tumors in patients aged 6 months to 1 year had a high apoptosis rate. According to Shimada’s classification, the apoptotic index for the favorable-histology group compared was significantly higher than that of the unfavorable-histology group. i7 There were no statistically significant differences in histological type, N-myc amplification, or DNA ploidy, but the apoptotic index of the N-myc-amplified cases was generally lower than that of nonamplified cases. Immunostaining

of bcl-2

Immunostaining using a monoclonal anti-bcZ-2 antibody in the present study showed that almost all neuroblastoma cases were bcZ-2-positive. The percentage of bcZ-2-positive cells (bcl-2 labeling index) varied from specimen to specimen and ranged from 0.2% to 97%. The staining was localized in the cytoplasm of neuroblastoma cells. Nuclear staining was not seen in any tumor specimen. Large cells that displayed differentiation to ganglionic cells showed intense bci-2 expression, some advanced-stage cases over 1 year of age showed intense staining even in small blastemal cells (Fig 3). The correlation between bcZ-2 labeling index and prognostic features of neuroblastoma was assessed (Table 2). The rate of bcZ-2 expression correlated with tumor histology. The bcl-2 labeling index was significantly lower among cases of ganglioneuroblastoma or favorable histology (Shimada classification) than among those of neuroblastoma or unfavorable histology. There were no statistically significant differences in age, clinical stage, or DNA ploidy. The bcZ-2 labeling index of N-myc-amplified tumors was higher than that of nonamplified tumors, but the difference was not statistically significant. Relationshk Between In Situ End-Labeling and bcl-2 Expression Cases under 1 year of age often showed “loose areas” in which the cell population was markedly decreased and cellularity was low (Fig 4). In such area, expression of bcl-2 was often reduced and apoptotic cells often were observed by in situ endlabeling of fragmented DNA. In these cases, there was a negative correlation between apoptosis and bcl-2 expression. DISCUSSION

Neuroblastoma is one of the most common malignant neoplasms in children. The prognosis of patients with advanced neuroblastoma who are over 1 year of age remains poor in spite of aggressive antitumor therapy.18 In Japan, the nationwide urinary VMA

APOPTOSIS

AND

bd-2

EXPRESSION

253

IN NEUROBLASTOMA

Fig 1. Typical staining of modified TUNEL method in neuroblastoma morphological characteristics of apoptosis, such as nuclear fragmentation or condensation of chromatin (B, C, D).

(original magnifications and formation of apoptotic

x500). Most of the positive cells showed bodies (A), reduction in mklear size (A, B)

(vamllylmandehc acid) and HVA (homovanilhc acid) mass screening program for 6 month old infants began in 1985. l9 Since then, the biological features of cases detected through mass screening were found to be different from those not detected in this manner. The prognosis for cases detected by mass screening is good, even in advanced-stage cases, and N-myc amplification is seldom detected.*9-21 The incidence of neuroblastoma has increased since the introduction of the mass screening program; it is suspected that the screening is detecting some of the regressing tumors.2z Apoptosis, or programmed cell death where cells are delated physiologically, is important in the development or maintenance of tissue organization.s-12 Cells that undergo apoptosis have a series of characteristic morphological features, including nuclear condensation, reduction of nuclear size, membrane blebbing, and nuclear and cellular fragmentation, and the cell finally fragments into apoptic bodies.7 A biochemical hallmark of apoptosis is a characteristic form of

DNA fragmentation in which the genome is cleaved at internucleosomal sites, showing a ladder of DNA fragments when analyzed by agarose gel electrophoresis.u Apoptosis has been shown to play an important role in the development and differentiation of the nervous system.r3 A large number of cells in the embryonic nervous system are eliminated by apoptosis during development. Apoptosis also is recognized in neuroblastoma cell lines in some conditions.23-25 We hypothesized that the spontaneous regression of neuroblastoma might be induced by apoptosis. Recently, Gavrieli and Wijsman described a new method to visualize DNA fragmentation in conventional histological slides. 14,15These methods could label the numerous nick-ends of fragmented DNA generated in apoptosis, ie, in situ end-labeling. To examine apoptosis in neuroblastoma, we performed the modified TUNEL method described by Gavrieli et al.14 The presence of DNA strand breaks is not always unique to apoptosis, thus this evidence should

254

OUE ET Al

Fig 2. Staining of modified TUNEL method in neuroblastoma [original magnification and scattered, but in some cases there was accumulation of positive cells (B).

Table

1. Relationship

Between

and Clinical Clinical Value

Apoptotic

Index

Variables

NO.

ApopMc

93 25

0.767 k 0.535 0.339 k 0.372

.0006

Histology Neuroblastoma Ganglioneuroblastoma

104 14

0.700 2 0.535 0.509 k 0.515

.2826

Shimada Classification Favorable Unfavorable

103 15

0.754 2 0.532 0.198 k 0.185

.0002

82 36

0.817 x! 0.529 0.372 2 0.406

.OOOl

3* 66

0.265 iz 0.182 0.792 k 0.067

.0995

31* 67

0.660

zk 0.726

0.707

IL 0.480


yr

> 1 yr

Clinical staging I, II, IVS Ill, lVA,B N-myc Amplified Not amplified DNA ploidy Diploid Aneuploid

Index

NOTE. Apoptotic index is defined as the percentage stained cells according to the modified TUNEL method Statistical *N-myc evaluated

significance was determined amplification was evaluated in 98 patients.

by the unpaired in 69 patients;

f Value

.7107 of positively (mean k SD).

r test. DNA ploidy

was

x100).

in most

cases,

positive

cells appeared

isolated

(A)

be confirmed by other findings.15 In this study, most positive cells showed morphological characteristics of apoptosis such as condensation of chromatin, reduction in nuclear size, nuclear fragmentation, or formation of apoptotic bodies. There were no signs showing necrosis, such as lymphocyte infiltration or granulation. These findings may indicate that in situ endlabeling of fragmented DNA appeared to be a suitable technique for identi@ing cells at the stages of apoptosis in neuroblastoma. Apoptosis was detected more frequently in cases with a favorable clinical stage and cases between 6 months and 1 year of age. Most tumors were detected through mass screening. These cases often have loose areas in which the cell population is markedly decreased and cellularity is low, and a concentration of apoptotic cells often was observed. It is suggested that apoptosis may have reduced the cellularity in these loose areas and may be involved in the spontaneous regression of some tumors. The bcf-2 gene was first discovered because of its involvement in the t(l4;18) chromosomal translocations found in the majority of non-Hodgkin’s lymphomas.26 bcl-2 might enhance cellular surviva1 by bltxk-

APOPTOSIS

AND k/-2

EXPRESSION

IN NEUROBLASTOMA

255

Fig 3. Typical staining of k/-2 in neuroblastoma (original magnification cells. Large cells that displayed differentiation to ganglionic cells showed age and in the advanced stage, even small blastemai cells showed intense

Table

2. Relationship Between be/-2 and Clinical Variables

Clinical Value

Expression

NO.

Labeling Index

P Value

be
yr

93

67.0 YL 19.8

>I

yr

25

69.0 SC 27.6

104

69.7 iz 20.6

14

50.8 z!z 21.8

103

65.6 2 21.2

15

80.1 2 20.4

82

70.9 2 21.9

36

70.1 2 21.9

Histology Neuroblastoma Ganglioneuroblastoma Shimada Classification Favorable Unfavorable Clinical staging I, II, IVS Ill, lVA,B N-myc amplification Amplified Not amplified DNA ploidy Diploid Aneuploid

3*

amplification in 98 patients.

68.3

31*

70.2 2 22.0

67

67.0 2 19.2

was evaluated

.0018

.0142

.2436

92.8 2 11.1

66

.0574

zt 21.8

NOTE. Labeling index is defined as the percentage stained cells according to bd-2 immunostaining (mean cal significance was determined by the unpaired r test. *N-myc evaluated

,672

in 69 patients;

.4686

of positively k SD). StatistiDNA

ploidy

was

x 100). The staining was localized to the cytoplasm intense expression of bd-2 (A). in some cases that staining (B).

of neuroblastoma were over 1 year of

ing programmed cell death.27,2sbcl-2 may be associated with neural cell survival and differentiation and functions as a regulator of programmed cell death.29 Castle et al investigated bd-2 expression in neuroblastoma30 and reported that 40% of the specimens stained positively for bd-2. They showed that bcl-2 expression correlates with features of poor prognosis and is strongly associated with unfavorable histology (Shimada classification) and N-WZ~Camplification. They suggested that bcl-2 may play an important role in the genesis or progression of malignant neuroblastoma. In our study, the bcl-2 labeling index also was associated with unfavorable histology according to the Shimada classification. There were only three N-myc-amplified cases, and the bd-2 labeling index for these was higher than that of nonamplified cases, but the difference was not significant. In addition, the apoptotic index was low for unfavorable-histology or N-myc-amplified cases. These findings suggests that bcl-2 may block apoptosis in the malignant cases of neuroblastoma. In cases under 1 year of age, bcl-2 expression often was reduced, especially in the loose areas. In these

OUE ET AL

256

Fig 4. H&E staining (A), TUNEL staining (g), and bd-2 immunostaining (C) of the same area of the tumor, detected through mass screening [original magnification x 100). Cases under 1 year of age often showed “loose areas” in which the cell population was markedly decreased and cellularity was low. In such areas, bd-2 expression often was reduced and apoptotic cells often ware observed. There was a negative correlation between apoptosis and bd-2 expression.

lesions, accumulation of apoptotic cells often was detected, which suggests an inverse relationship between bcl-2 expression and apoptosis. Perhaps in these cases, reduction of bci-2 expression causes apoptosis, which reduces cellularity in loose areas and may cause spontaneous regression. It has been reported that neurotrophic factors such as nerve growth factor (NGF) regulate the survival of postmitotic neurons, and their deficiency may cause programmed cell death of dependent neurons.24T31 Recently, NGF receptor expression has been associated with good prognosis in cases of neuroblastoma.32 These findings may indicate that NGF regulates the survival and programmed cell death in cases of

favorable neuroblastoma. bcl-2 reportedly prevents neural cell death induced by withdrawal of NGF from cultured sympathetic neurons.33 What regulates the apoptosis and bcl-2 expression in neuroblastoma is not known, but NGF may be one of candidate that is a factor in this regulation. Our findings may suggest that bcl-2 expression and apoptosis are related to the spontaneous regression of neuroblastoma. ACKNOWLEDGMENT We thank Dr Kobayashi (Department of Pathology, Osaka City General Hospital) and Dr Nakayama (Department of Pathology, Osaka Medical Center and Research Institute for Maternal and Child Health) for providing the neuroblastoma specimens.

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AND bd-2

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