Fluorescence in situ hybridization analysis of chromosome 1p36 deletions in human MYCN amplified neuroblastoma

Fluorescence In Situ Hybridization Analysis of Chromosome 1~36 Deletions in Human MYCiV Amplified Neuroblastoma By Hiroaki

Komuro, Memphis,

Marcus

B. Valentine, Susan T. Rowe, Vincent J. Kidd, Shun-ichi Makino, Garrett Susan L. Cohn, and A. Thomas Look Tennessee; Chicago, Illinois; Philadelphia, Pennsylvania; and Tochigi, Japan

Background/fbpose:

Deletion of the short arm of chromosome 1 (Ipi is one of the poor prognostic factors in human neuroblastomas. Recent studies have suggested that one or more of the neuroblastoma tumor suppressor genes reside in this region and have identified the shortest region of overlap (SRO) on 1~36. The purpose of this study was to examine deletions of 1 p in human neuroblastomas by fluorescence in situ hybridization (FISH).

Methods:

Two-color FISH analysis was performed to detect chromosome 1~36 abnormalities in 42 MYCN-amplified neuroblastomas. Four different probes from the 1~36 region, the E2F2, NPPA, DlS160, and CDCSLI loci were used for detection of Ip abnormalities. A repeat sequence probe, which is specific for the heterochromatic region of chromosome 1 (pUC1.77), was used as a control.

D

ELETION of the short arm of chromosome 1 (lp) is the most common cytogenetic abnormality observed in human neuroblastomas. Recent molecular studies have shown loss of heterozygosity (LOH) for lp in about 30% of neuroblastomas and confirmed the shortest region of overlap (SRO) on 1~36.1-2.‘~~ Several lines of evidence indicate that chromosome lp involves two or more tumor suppressor genes that contribute to the pathogenesis of neuroblastomas.4-6 Moreover, the translocation junctions of chromosome lp are clustered at the band 1~36.1-2.~ MYCN gene amplification in neuroblastomas is associated with advanced stages of disease, rapid tumor progression, and a poor prognosis. A strong correlation between allelic loss of chromosome 1~36 and MYCN amplification was found in some studies,1,8 but another study did not support this correlation3 The results of LOH studies by Southern blotting or polymerase chain reaction (PCR) uniformly are not informative because they require the patient to harbor a constitutional microsatellite or restriction fragment length polymorphism (RFLP). Interphase fluorescence in situ hybridization (FISH) analysis is a new technique, providing a useful tool for examining solid tumors that have been difficult to analyze by conventional cytogenetics because of small number of metaphases. Using FISH, rapid analysis is possible in a large number of interphase cells. This provides not only a direct and reliable method to observe hemizygous deletion when patient material is limited, but also recognizes Journaloffediatric

Surgery,

Vol33,

No 11 (November),

199% pp 1695.1698

M. Brodeur,

Results: Large deletions of 1~36 were observed in 31 (73.8%) of 42 tumors, whereas the remaining 11 (26.2%) showed no deletion. In these 11 tumors, a translocation of lp was found in one and a duplication of lp was detected in another. Conclusions: A strong correlation between Ip abnormalities and MYCN amplification was found in this study. MYCNamplified neuroblastomas were found to show large deletions of 1 p encompassing the SRO. FISH provided a rapid and reliable method to detect hemizygous deletions of Ip.

J Pediatr Sorg 33:1695-1696. Saunders Company. INDEX WORDS: Neuroblastoma, fluorescent in situ hybridization.

Copyright

o 1998 by W.B.

chromosome

1 deletion,

tumor subpopulations with different ploidy pattems.9 Here we report an analysis of deletion of chromosome 1~36 in N-myc-amplified neuroblastomas by the use of interphase FISH techniques. MATERIALS

AND

METHODS

Tumor Samples Tumor tissues or bone marrow aspirates from patients with neuroblastomas were obtained from the Pediatric Oncology Group (POG) Neuroblastoma Tumor Bank from December 1992 to March 1994. This study was reviewed and approved by the POG Neuroblastoma Biology Subcommittee and informed consent was obtained on each patient.

From the Departments of Experimental Oncology and Tumor Cell Biology, St Jude Children’s Research Hospital, Memphis, TN: Depanment of Pediatrics, Northwestern University and Children’s Memorial Hospital, Chicago, IL; Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA: Depattment of Pediatrics, The University of Pennsylvania School of Medicine, Philadelphia, PA: Deparhlent of Pediatrics, UniversiQ of Tennessee School of Medicine, Memphis, TN: and Department of Surgery, Jichi Medical School, Tochigi, Japan. Presented at the 31st Annual Meeting of the Pat@ Association of Pediatric Surgeons, Maui, Hawaii, June 9-13, 1998. This work was supported in part by NIH grants CA 31566, CA 71907, CA 39771, and CA 21765 (Cancer Center CORE grant), and by The American Lebanese Syrian Associated Charities (ALSAC), St Jude Children S Research Hospital. Address reprint requests to A. Thomas Look, MD, Department of Experimental Oncology, St Jude Children 5 Research Hospital, 332 N Lauderdale, Memphis, TN 38105-2794. Copyright o 1998 by WB. Saunders Company 0022-3468/98/3311-0027$03.00/O 1695

1696

KOMURO

B

A

0

Staging was done according to the POG classificationto Single cell suspensions were analyzed for the percentage of tumor cells on Wright-stained smears. MYCN gene copy number was detected by fluorescence in situ hybridization (FISH) as described previously.” Forty-two MYCN-amplified neuroblastomas, in which the percentage of tumor was greater than 40%, were analyzed for deletion within chromosome band 1~36 by FISH.

DNA Probes Four genomic clones that are located on the distal short arm of chromosome 1 were used as probes. These loci correspond to E2F2, NPPA, DlS160 and CDC2L1, arranged in centromeric to telomeric order (Fig 2).‘*-i5 A repeat sequence probe that is specific for the heterochromatic region of chromosome 1 @UC 1.77) was used to identify the number of chromosome 1 present in the tumor cellsI

Fluorescence In Situ Hybridization Purified DNAs were labeled by nick translation with either biotin-16dUTP (Boehringer Mannheim, Indianapolis, IN) or digoxigenin-I l-

Fig 2. positions map. The retained, respectively.

ET AL

Summary of FISH results of lp deletion in 42 csses. The of FISH probes used and the SRO are shown on the upper open, shaded, hatched, dotted circles, and bars indicate deleted, duplicated and translocatsd loci, and gene regions,

Fig 1. Two-color FISH analysis of neuroblastoma cells with probes from chromosome band lp26. (A) Interphase nuclei shows E2F2 probe (green) and pUCl.77 control probe fredI. This demonstrates hemizygous deletion of the E2F2 locus because each cell has three control signals fredl but only two E2F2 signals (green). (B) A metaphase spread shows cohybridization of CDC2Ll probe (green) and pUC1.77 probe (red). The CDCPLI probe occupies its usual position near the lp terminus (open arrow). The other allele of CDC2Ll has been moved to an unidentified marker chromosome (closed arrow), indicating a chromosome translocation between CDC2Ll and pUC1.77.

dUTP (Boehringer Mannheim). In two-color FISH experiments two differentially labeled probes were cohybridized overnight at 37°C to interphase or metaphase nuclei fixed on glass slides in a solution containing sheared human DNA, 50% formamide, 10% dextran sulfate, and 2XSSC. Signals were detected by incubating the slides with fluorescein-conjugated sheep antidigoxigenin antibodies (Boebringer Mannheim) and Texas red conjugated avidin (Vector Laboratories, Burlingame, CA), followed by signal amplification with fluoresceinconjugated rabbit antisheep antibodies (Boehringer Mannheim), biotinylated antiavidin, and additional fluorescein-conjugated sheep antidigoxigenin antibodies and Texas red conjugated avidin. Slides were counterstained with 4, 6 diamidino-2-phenylindole (DAPI). Nuclear DNA was visualized by counterstaining with propidium iodide. Fluorescence microscopy was performed with a Zeiss standard microscope equipped with appropriate dual band pass filters.

RESULTS Detection of Deletion of Chromosome 1~36 Loci An analysis was conducted of 42 neuroblastomas with MYCN gene amplification. CDC2Ll and E2F2 were analyzed first, and cases lacking deletions with these two probes were then tested with two internal probes (NPPA and DlS160) to detect microdeletions between these loci. Each probe (labeled green with FITC) was cohybridized with the pUC1.77 control probe from the long arm of chromosome 1 (labeled red with Texas red). The number of signals from each locus were then compared with the number of signals from pUC1.77 in the same interphase nucleus to determine whether the lp locus had been deleted (Fig 1A). Deletions of both E2F2 and CDC2Ll were observed in 31 (73.8%) of 42 tumors, whereas the remaining 11 (26.2%) did not have deletions of either of these loci

CHROMOSOME

1~36

Table

DELETIONS

1697

1. PISH Results and Patient Characteristics in MYCf&Amplffied Neuroblastomas M&phase* +

1 ploss(+)

6

1 ploss(-)

5+

*Presence

IN NEUROBLASTOMA

Sex M

F


>lYr

A

B

C

D

Ds

25

22

9

10

21

1

0

4

3

6

5

6

2

9

23 80

(+) or absence

tThe

POG classification

*Two

of these

showed

staget

Age

-

t--J of metaphase system

102

cells.

was used.‘O

lp abnormalities.

(Table 1). In the 11 neuroblastomas without deletions of either E2F2 or CDC2L1, we tested two additional probes specific for sites between these genes (NPPA and D 1S 160). These loci also were retained, indicating that small interstitial deletions within this region were not detectable with these probes. Diploid tumors were hybridized with an MYCN cosmid and either E2F2 or CDC2Ll in two-color FISH experiments to verify that only tumor cells, and not associated stromal cells, were being analyzed. Accordingly, the neuroblastomas with MYCN amplification could be assigned to two groups: one composed of tumors in which the entire region from E2F2 to CDC2Ll is deleted, and the other composed of tumors devoid of detectable deletions in this region (Table 1). Interphase cells routinely were examined from fresh tumor biopsies; we were also able to identify metaphase cells in 11 of 42 N-myc-amplified samples. The results obtained in the metaphases of these cases confirmed the interphase results for each of 11 cases (indicated + in Table 1). In addition, results obtained in one of the 11 tumors without deletions of the four probes tested showed a translocation of both the CDC2Ll and E2F2 loci from 1~36 to a group C chromosome in metaphase cells prepared a fresh tumor cell specimen (Fig 1B). Thus, the translocation breakpoint in this case was centromeric to E2F2. Metaphase chromosomes from case 41 showed a MYCN-containing homogeneously staining region (HSR) of distal chromosome lp. FISH analysis demonstrated duplicated copies of CDC2L1, DlS160 and NPPA, but not E2F2 within the HSR of both metaphase and intephase cells (Fig 2). DISCUSSION

Deletions of the short arm of chromosome 1 are found in about 30% of neuroblastomas.1-3,8 Both deletion of the short arm of chromosome 1 and amplification of the MYCN gene are poor prognostic markers for this disease.” Previous results of LOH studies on the correlation between lp deletion and MYCN gene amplification were conflicting. Several studies observed a strong correlation between 1~36 LOH and MYCN amplification, but another did not.‘s3T8Recent studies suggest that a second tumor suppressor gene associated with MYCN amp-

lification may be located more centromeric to the shortest region of overlap (SRO) of LOH on chromosome l~.~ A third tumor suppressor locus proximal to band 1~36 might be involved in some neuroblastomas.5 Recent LOH studies defined an SRO on 1~36 that lies telomeric to NPPA and centromeric to CDC2Ll (Fig 2).2 E2F2 is located more than 20 cm centromeric from NPPA.* Our results support the studies of several groups suggesting that deletions in MYCN-amplified neuroblastomas generally are large and emcompass the entire SR0.4-6 Most recent studies on the association between lp deletion and MYCN amplification in neuroblastomas have been performed with a limited number of samples by LOH.1,3,8 Here we examined 42 neroblastomas with MYCN amplification and found large deletions encompassing the SRO on 1~36 in 73.8% of the cases using two-color FISH. The sex, age, and stage for patients with neuroblastomas with and without lp deletion detected by FISH are shown in Table 1. Two of the 11 neuroblastomas that did not show deletion of lp demonstrated lp abnormalities. A duplication of the 1~36 region was detected in one (Fig 2). Thus far, one neuroblastoma cell line (NGP) was also found to have duplication of a segment of 1p36.*J8 The NGP cell line has a t( 1; 15) translocation as well as the duplication. The proximal boundary of the duplication was located within the SRO. In our case the proximal border of duplication was positioned centromeric to the SRO. Further studies are required to determine the significance of duplication in the 1~36 region in neuroblastoma. Another neuroblastoma had a translocation of chromosome lp, the breakpoint of which was located centromerit to E2F2 locus in metaphase cells (Fig 2). One possible explanation for this observation is that a second or a third tumor suppressor gene might be disrupted by this breakpoint. Metaphase cells were not seen in all the samples (Table 1). If one includes the two cases with translocations, at least 33 (78.6%) of 42 MYCN-amplified neuroblastomas were found to show lp abnormalities. Recently, FISH has been used to analyze the structural and numerical abnormalities of chromosomes in various tumors. FISH analysis has several advantages over conventional LOH studies using Southern blotting and PCR techniques to detect allelic loss. One advantage is that only small quantities of tumor cells are required for FISH analysis, which is important because primary tumor biopsies are often limited. It is also a very rapid procedure that takes as little as 2 working days to complete. Normal tissue is not required as a control for FISH analysis, whereas it is essential in LOH studies. A further advantage is that unlike LOH studies, heterozygosity at a particular locus is not needed to identify an allelic loss by

1698

KOMURO

FISH analysis. Furthermore, FISH analysis provides a rapid assessment of ploidy by counting the number of chromosome 1 homologues per cell. Our studies using two-color FISH allowed a rapid, simple, and reliable analysis of lp deletions in neuroblastomas. Further follow-up will be required to determine the prognostic

ET AL

significance of lp deletion in neuroblastomas with MYCN amplification. ACKNOWLEDGMENT The authors thank the Pediatric Oncology Group Neuroblastoma Biology Subcommittee for receiving and approving this research project and for providing neuroblastoma tumor samples.

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some 1 aberrations in neuroblastoma. Cytogenet Cell Genet 63:24-28, 1993 10. Look AT, Hays FA, Shuster JJ, et al: Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: APediatric Oncology Group Study. J Clin Gncol9:581-591,199l 11. Shapiro DN, Valentine MB, Rowe SB, et al: Detection of N-myc gene amplification by fluorescence in situ hybridization: Diagnostic utility for neuroblastoma. Am J Path01 142: 1339-1346, 1993 12. Lee JA, Saito M, Vidal M, et al: The retinoblastoma protein binds to a family of E2F transcription factors. Mol Cell Biol 13:7813-7825, 1993 13. Frossard PM, Coleman RT: Human atrial natriuretic peptides (ANP) gene locus: BglII RFLP Nucleic Acid Res 14:9223, 1986 14. Hudson TJ, Engelstein M, Lee MK, et al: Isolation and chromosomal assignment of 100 highly informative human simple sequence repeat polymorphism. Genomics 13:622-629,1992 15. Lahti JM, Valentine M, Xiang J, et al: Alterations in the PITSLRE protein kinase gene complex on chromosome 1~36 in childhood neuroblastoma. Nature Genet 7:370-375, 1994 16. Christiansen H, Schestag J, Bielke W, et al: Chromosome 1 interphase-cytogenetics in 32 primary neuroblatomas of different clinical stages. Advances in neuroblastoma research 3:99-105, 1991 17. Canon H, van Sluis P, de Kraker J, et al: Alleic loss of chromosome lp as a predictor of unfavorable outcome in patients with neuroblastoma. N Engl J Med 334:225-230, 1996 18. Casciano I, Marchi JVM, Muresu R, et al: Molecular and genetic studies on the region of translocation and duplication in the neuroblastoma cell line NGP at the lp36.13-~36.32 chromosomal site. Oncogene 12:2101-2108, 1996