Cytogenetic and array comparative genomic hybridization analysis of a series of hepatoblastomas

Cancer Genetics and Cytogenetics 194 (2009) 82e87

Cytogenetic and array comparative genomic hybridization analysis of a series of hepatoblastomas Eva Stejskalova´a,*, Josef Malis˘a, Jirˇ´ı S˘najdaufb, Karel Py´chab, Helena Urba´nkova´c, Viera Bajciova´d, Jan Stary´a, Roman Kodete, Marie Jarosˇova´c Department of Pediatric Hematology and Oncology, Oncocytogenetics, University HospitaleMotol, V U´valu 84, 15006 Prague 5, Czech Republics b Department of Pediatric Surgery, University HospitaleMotol, Prague, Czech Republic c Department of Hemato-oncology, Palacky University Hospital, Olomouc, Czech Republic d Department of Pediatric Oncology, University HospitaleBrno, Brno, Czech Republic e Institute of Pathology and Molecular Medicine, University HospitaleMotol, Prague, Czech Republic

a

Received 2 February 2009; received in revised form 29 April 2009; accepted 1 June 2009

Abstract

Hepatoblastoma is the most common primary hepatic tumor in children, and only a limited number of detailed karyotypic analyses have been reported to date. In the present study, cytogenetic abnormalities were identified in nine cases of hepatoblastoma from a single institution. Among characteristic chromosomal changes detected were simple numerical aberrations, structural alterations of chromosomes 1, 2, and 8, and the recurrent unbalanced rearrangements der(4)t(1;4)(q25.2;q35.1) and der(6)t(1;6)(q21;q26). Array comparative genomic hybridization was applied in four of the cases. The combined cytogenetic, molecular cytogenetic, and histopathologic analyses are presented here, together with clinical data. The results substantially confirm previous findings of aberrations involving chromosomal loci on 1q, 2 or 2q, 4q, 6q, 8 or 8q, and 20 as significant in the development and clinical course of this disease. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Hepatoblastoma is a rare embryonal malignant disease that occurs almost exclusively in infants and young children in the first 3 years of life. A dramatic improvement in the prognosis for these patients has been achieved with multimodal therapies combining surgery and chemotherapy. Even so, approximately one quarter of the children do not survive the disease [1]. A resectable tumor, a decline in circulating a-fetoprotein levels during chemotherapy, and pure fetal histology are thought to characterize patients with low-risk disease [2]. The genetic basis is not well understood. Unlike solid tumors in general, hepatoblastomas exhibit relatively simple karyotypes, with predominantly numerical changes [3]. Detailed cytogenetic data have so far been reported in only a limited number of hepatoblastomas, partly because of the rarity of these neoplasms [4e13]. The first large series reported is still relatively small (n 5 111), * Corresponding author. Tel.: þ420-224436485; fax: þ420224436417. E-mail address: [email protected] (E. Stejskalova´). 0165-4608/09/$ e see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.06.001

and further studies of abnormalities in histologic subtypes are needed to assess the significance of nonrandom aberrations and to make inferences about their prognostic significance [14]. The gain of partial or complete trisomy of chromosomes 1, 2, 8, and 20 is a characteristic cytogenetic change of possible pathogenetic importance in this tumor [3,4,14]. A recurring structural change der(4)t(1;4) (q12;q34) was first reported by Schneider et al. [15]. One study suggested that gains on 8q and 20 are predictors of poor outcome, but more work is needed to validate these findings [16]. In contrast to many other childhood tumors, relatively little is known about the molecular mechanisms involved in the pathogenesis and progression in hepatoblastomas [1,2,7,17,18]. Similarities with other embryonal tumors, such as Wilms tumor and rhabdomyosarcoma, have been found, suggesting a parallel pathway of genetic steps in initiation, progression, or both [9,10,18]. Cytogenetic analysis is limited by the requirement of suitable cells in metaphase, and alternative methods such as comparative genomic hybridization (CGH) [19] and array-CGH have been introduced [20]. Here we report the cytogenetic and molecular cytogenetic analysis of 9 consecutive hepatoblastomas.

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2. Materials and methods

3. Results

2.1. Histologic and immunologic analysis

The histologic and cytogenetic features of the nine cases, seven primary and two recurrences (lung metastases), are summarized in Table 1. Clonal chromosomal abnormalities were detected in all patients. The most frequent recurrent chromosomal aberration was trisomy of chromosome 20, revealed in five cases, and structural rearrangements of chromosome 1q arm in six cases: a duplication 1q in case 1 (Fig. 1), an isochromosome 1q in cases 5 and 9 (Fig. 2), and unbalanced translocations der(10)t(1;10)(q23;q26) in case 4, der(6)t(1;6)(q21;q26) in case 7 (Fig. 3), and der(4)t(1;4)(q25.2;q35.1) in case 8 (Fig. 4). An overrepresentation of chromosome 2 material was observed in five tumors. M-FISH demonstrated that the additional material on chromosomes 1q and 2q in patient 1 indeed was a duplication (Fig. 5). Total or partial trisomy 8 was seen in four tumors. Two abnormal karyotypically related clones were detected in patients 1 and 4. Numerical aberrations were detected nearly in all patients; they were present as a sole anomaly in patients 3 and 6. Array-CGH was performed in the four cases (patients 6, 7, 8, and 9) for which tumor DNA was available. For patient 6, the cytogenetic findings were confirmed by array-CGH, with no additional unbalanced changes detected. For patient 7, only a normal array-CGH profile was detected. For patient 8, the gain of 1q25.21q44 was revealed, defining the exact breakpoint loci: arr cgh 1q25.2q44(RP5-990P15-GS-167-K11)3. The findings of unbalanced changes of chromosomes 1 and 2 in case 9 are detailed in Figure 6.

Tumor samples from seven primary and two recurrent hepatoblastomas were surgically removed and submitted for analysis. Fresh tissue fragments were divided: one portion was fixed in 10% buffered formolesaline and processed for conventional histology and one portion was submitted for cytogenetic analysis; the remaining portions were snap-frozen in liquid nitrogen for eventual DNA analysis. Histologic and clinical data are presented in Table 1. 2.2. Cytogenetic analysis Fresh tumor samples were submitted routinely to our cytogenetic laboratory, in a sterile transport medium, and were treated as described previously [21]. The karyotypes were described according to the ISCN 2005 [22]. 2.3. Multicolor FISH (M-FISH) Multicolor FISH (M-FISH) was performed using a 24XCyte-MetaSystems kit (MetaSystems, Altlussheim, Germany), according to the manufacturer’s instructions and standard procedures as described previously [21]. 2.4. Array-CGH Array-CGH was performed in four cases, using two different platforms. In three cases, bacterial artificial chromosome (BAC) array slides containing ~3500 BAC clones from the Wellcome Trust Sanger Institute (produced at the Leiden University Medical Center, Leiden, Netherlands) were used; in the fourth case, the CytoChip Focus for Haematology array (BlueGnome, Cambridge, UK) containing 3,242 BAC clones was used. Both platforms cover the human genome at ~1-Mb resolution. Probe preparation was performed, with small modifications, as described by Fiegler et al. [20]. Hybridization and posthybridization washing was performed using an HS 400 Pro hybridization station (Tecan, Mannedorf, Switzerland). Slides were scanned using a GenePix Professional 4200A scanner (Axon Instruments, Foster City, CA), and primary images were processed using GenePix Pro 6.1 software (Axon Instruments). Further analysis was performed using BlueFuse for Microarrays software (BlueGnome), or the signal intensities of replicates of the features were averaged in an in-house routine using spreadsheet software (Microsoft Excel 2000). Spots outside the 20% confidence interval of the average of the replicate were excluded. Only those targets presenting at least two spots within a 20% confidence interval of their average were used. Unbalances of the targets were determined based on log2 ratios of the average of their replicates, and we considered sequences as gained or deleted when outside the range of 60.3.

4. Discussion Cytogenetic and molecular genetic aberrations involving chromosomal loci on 1q, 2 or 2q, 4q, 6q, 8 or 8q, and 20 have frequently been found in hepatoblastoma. Because of the low incidence of this tumor, cytogenetic aberrations have been reported in a limited number of cases, compared with numbers reported for other types of pediatric cancers. Accumulation of detailed karyotypes of more hepatoblastoma cases is needed in order to identify chromosomal loci involved in nonrandom genetic changes implicated in the development of hepatoblastoma [23]. The present results confirm those of previous studies of hepatoblastoma, and thus further support the premise that these abnormalities are nonrandom and are strongly associated with hepatoblastoma. In three patients in our series, centromere aberrations were evident as isochromosomes 1q and 8q. In addition, we confirm that imbalance of the terminal region of 4q may be found as a sole abnormality in hepatoblastoma, as observed by Nagata et al. [23]. This abnormality was originally reported as the first recurrent translocation in hepatoblastoma, an unbalanced translocation between the long arms of chromosomes 1 and 4, probably the most specific abnormality in this tumor, and one

84

Table 1 Histologic, clinical, and cytogenetic data for nine patients with primary or recurrent hepatoblastoma

Histologic subtype

Treatment

Karyotype

2000

F/7 yr

PRETEXT III

embryonal

primary surgery

46,XX,dup(1)(q?44),dup(2)(q?37)[16]/50, idem,þ5,þ7,þ20, þ 22[2]/46,XX[4]

7,260

2R

1999

M/14 mo

PRETEXT II

mixed fetal and embryonal

preoperative CTx (cisplatin)

48,XY,add(6)(q27),þi(8)(q10),þ 20[10]/46,XY[6]

280,000

3P

2001

F/18 mo

PRETEXT II

mixed fetal and embryonal

48,XX,þ8,þ20[4]

280,000

4R

2004

F/17 mo

fetal

48,XX,þ2,þ8,der(10)t(1;10)(q23;q26)[3]/48, idem,der(1)t(1;?17)(p36;q21)[2]/46,XX[2]

27,954

Multiple lung recurrence, alive in 4th CR.

5P

2000

F/9 mo

PRETEXT III, lung metastasis PRETEXT II

preoperative CTx (doxorubicin, cisplatin) preoperative CTx (doxorubicin, cisplatin) primary surgery

48,XX,þi(1)(q10),þ2[6]/46,XX[2]

!7,260

Alive, 1st CR.

6P

2006

F/18 mo

PRETEXT II

51,XX,þ2,þ5,þ8,þ17,þ20[6]/46,XX[1]

280,000

Alive, 1st CR.

7P

2007

M/20 d

PRETEXT II

mixed fetal and embryonal

48,XY,t(1;6)(q21;q26),þder(6)t(1;6)(q21;q26), þ 20[7]/46,XY[5]

85,134

Alive, 1st CR.

8P

2007

F/6 d

PRETEXT III

46,XX,der(4)t(1;4)(q25.2;q35.1)[10]/46,XX[5]

7,260

Alive, 1st CR.

9P

2008

F/3 yr

PRETEXT II

mixed fetal and embryonal mixed fetal and embryonal

preoperative CTx (doxorubicin, cisplatin) preoperative CTx (doxorubicin, cisplatin) primary surgery preoperative CTx (doxorubicin, cisplatin)

47,XX,i(1)(q10),þder(2)[10]

280,000

Alive, on CTx.

Case 1P

mixed fetal and embryonal fetal

a-fetoprotein, mg/L

Outcome Liver relapse 7 yr after diagnosis; liver transplantation; achieved 2nd CR; alive with disease. Lung metastasis 4 mo after completion of CTx; alive, 2nd CR. Alive, 1st CR.

Abbreviations: CR, complete remission; CTx, chemotherapy; F, female; M, male; P, primary operation; R, recurrence of disease. a PRETEXT II, two adjoining sections free and two sections involved; PRETEXT III, two nonadjoining sections free, or just one section free, in the latter case three sections are involved (http://siopel.org/ public/pretext/index.html).

E. Stejskalova´ et al. / Cancer Genetics and Cytogenetics 194 (2009) 82e87

Sex/age

Pretreatment extent of diseasea

Year of Dx

E. Stejskalova´ et al. / Cancer Genetics and Cytogenetics 194 (2009) 82e87

Fig. 1. G-banded karyogram from patient 1 showing the less frequent clone 50,XX,dup(1)(q?44),dup(2)(q?37),þ5,þ7,þ20,þ22. Arrows indicate the duplicated chromosomes 1q and 2q and extra copies of chromosomes 5, 7, 20, and 22.

reported only rarely and in isolated cases of other types of neoplasms [15]. The loss of a putative tumor-suppressor gene on the distal end of 4q is presumed [6,24]. Allelic losses on 4q35 have been associated with a larger tumor size and an aggressive histological tumor type in hepatocellular carcinoma [25]. Little is known about the clinical relevance of these abnormalities. One study suggested that gains on 8q and 20 serve as markers of prognostic significance and could be linked with aggressive disease [16]. We have detected similar changes in patient 2 (Table 1). Another study highlighted the gain of 2q in connection with poor outcome [26]. This association was evident in our patient 1 (Table 1). This patient presented with a stage III histologically highly proliferating and mitotically active tumor at an unusually high age of 7 years. Despite a high-risk treatment protocol (SIOPEL 3), the patient relapsed after 7 years from diagnosis. A liver transplant from a living donor was performed, and the patient relapsed for a second time

Fig. 2. M-FISH karyogram from patient 1, showing the more important clone with a duplication on 1q and 2q.

85

Fig. 3. G-banded karyogram from patient 7 showing 48,XY,t(1;6) (q21;q26),þder(6)t(1;6)(q21;q26),þ20. Arrows indicate the aberrant chromosomes.

after that, with a neoplastic growth on the skin of the calva. Soon afterwards metastases could be seen in the lungs; as of writing, the patient was off treatment with an ongoing slow progression. Cytogenetically, in this case the rearrangement of 1q and 2q was associated with trisomies of chromosomes 5, 7, 20, and 22. Patient 2 presented initially with a tumor without metastases and was subject to treatment according to a low-risk protocol (SIOPEL 2). He relapsed early, 9 months from diagnosis, with a solitary lung metastasis. Unfortunately, cytogenetic analysis could be performed only out of this lung sample (i.e., 4 months after chemotherapy). Typical markers of unfavorable prognosis were detected, including an isochromosome 8q associated with a trisomy 20 (Table l). Patient 4 presented with multiple lung metastases. This presentation is unusual, given that this was histologically a fetal-type tumor, which has a commonly favorable prognosis. Despite preoperative chemotherapy, according to the SIOPEL 3 treatment protocol, lung progression was evident after an initial regression. Cytogenetic analysis could be performed only at the time of the patient’s first lung relapse, with typical hepatoblastoma numerical aberrations marking progression, þ2 and þ8, being detected (Table l). In patients 1, 4, 5, 7, 8, and 9, aberrations of the long arm of chromosome 1 were detected, either resulting in an isochromosome formation or being part of an unbalanced translocation (Table l). A der(6)t(1;6)(q12;q22.2) and a der(6)t(1;6)(q21;q25), with evident variation in breakpoints of the involved chromosomes, was included

Fig. 4. G-banded partial karyogram from patient 8 showing normal chromosomes 1, 2, and 3, one normal chromosome 4, and der(4)t(1;4) (q25.2;q35.1) (arrow).

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E. Stejskalova´ et al. / Cancer Genetics and Cytogenetics 194 (2009) 82e87

Fig. 5. G-banded karyogram from patient 9 showing 47,XX, i(1)(q10),þder(2). Arrows indicate the aberrant chromosomes.

in the largest consecutively karyotyped hepatoblastoma series by Tomlinson et al. [14]. Breakpoints vary also in the recurrent der(4)t(1;4)(q12;q34). Although the breakpoint on 1q seems to be largely restricted to 1q12~q21, the breakpoints on recipient chromosomes vary. The closest structural gene to the 1q breakpoint is NOTCH2, a gene involved in hepatoblastoma development and differentiation. On the distal 4q, the breakpoint range is 4q32~q35 [14]. Our patient 8 presented with a less common version of the der(4)t(1q;4q), with a breakpoint in band 1q25.2,

as the sole abnormality in a diploid karyotype (Table l). Array-CGH has revealed a gain in the region 1q25.2~q44 representing ~25 Mb. The same breakpoint was reported earlier by Tomlinson et al. [14] and by Schneider et al. [15]. A plausible hypothesis for these variations is that a specific hybrid product is less important than the disruption of a critical sequence in the pathogenesis of hepatoblastoma [14]. There is considerable variation in breakpoints on chromosome 2q as well. Earlier studies have reported breaks frequently occurring between 2q21 and 2qter [12,13]. According to Yeh et al. [3], the majority of breaks occurs at 2q35~q37, a region that has been shown to nonrandomly rearrange in rhabdomyosarcoma. Our most notable finding is array-CGH analysis of patient 9, which specified the breakpoints on the additional derivative chromosome 2. The short arm of this chromosome consists of region 2p25.3pter and the long arm is composed of two regions, 2q11.2q24.3 and 2q33.1qter, with breaks at q24.3 and at q33.1. Thus, an interstitial deletion was unveiled by array-CGH on the long arm of chromosome 2, in addition to the deletion on 2 p observed with cytogenetics. Trisomy 1q has also been associated with another embryonal tumor of childhood, Wilms’ tumor, and with astrocytoma. It is associated with a negative prognosis in those diseases. Because hepatoblastoma was detected prenatally in patient 8, our findings further support the possibility that structural chromosomal abnormalities involving 4q may be one of the early events in the development of this tumor, as proposed by Nagata et al. [23]. In summary, the present results bring additional evidence on the importance of abnormalities of chromosomal regions 1q, 2 and 2q, 4q, 6q, and 8 and 8q in the development of hepatoblastoma and in the clinical course of the disease.

Acknowledgments The authors thank Mrs. H. Mendlova´, D. Honsova´, J. S˘ulcova´, and J. Balca´rkova´ for their invaluable technical assistance. This work was supported by a grant from the internal grant agency of the Ministry of Health (IGA MZ NR/9050-3).

References

Fig. 6. Array comparative genomic hybridization result for patient 9, with partial profile of chromosomes 1 and 2 (BlueGnome array, Cambridge, UK): arr cgh 1p36.33p12(RP5-890O3 / RP11-418J17)1,1q21.3q25.3(RP11 3 P 2 0 / R P 1 1 - 2 9 3 B 7 ) 3 , 1 q 3 1 . 3 q 4 4 ( R P 1 1 - 7 5 C 2 3 / R P 1 1 978I15)3,2p25.3p24(RP11-1N7 / RP11-17L5)3,2q11.2q24.3(RP11-34 G16 / RP11-5J4)3,2q33.1q37.3(RP11-530J6 / RP11-367H1)3.

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