Chromosomal 3 and 8 status within hepatic metastasis of uveal melanoma.
Abstract: * Context.--Several studies have evaluated clinical, histopathologic, cytogenetic, and molecular prognostic variables in uveal melanoma. However, it is not known whether the primary tumor cells maintain these aggressive attributes at the metastatic sites.

Objective.--To determine the status of chromosomes 3 and 8q and c-myc amplification using fluorescence in situ hybridization on hepatic metastatic lesions of primary uveal melanoma.

Design.--Ten patients with uveal melanoma with needle core biopsy-confirmed hepatic metastasis. Representative paraffin blocks were selected based on review of hematoxylin-eosin-stained sections. Fluorescence in situ hybridization was performed for detection of monosomy 3 and amplification at the 8q24 MYC locus using standard methods. The tricolor chromosome enumeration probe 8 (CEP8)/ IGH/MYC and the Urovysion probe consisting of CEP3, CEP7, CEP17, and 9P21 probes were used. A total of 200 interphase cells were scored.

Results.--Hepatic metastasis was confirmed in each case by needle core biopsy. Fluorescence in situ hybridization analysis revealed chromosome 3 monosomy in 5 of the 8 cases that could be satisfactorily evaluated. Aneusomy of chromosome 8 was observed in 2 cases. MYC amplification was observed in 5 samples. In a single case where the primary tumor was treated by enucleation, the chromosomal monosomy 3 and aneusomy of chromosome 8 were present both in the primary tumor and its hepatic metastatic lesion.

Conclusions.--The presence of cytogenetic changes within the metastatic lesions confirms that chromosome 3 monosomy and aneusomy of chromosome 8 are not just markers of metastatic potential of the primary tumor but are also present within the hepatic metastatic lesions.

(Arch Pathol Lab Med. 2009;133:1223-1227)
Article Type: Report
Subject: Metastasis (Development and progression)
Melanoma (Development and progression)
Biopsy, Needle (Methods)
Liver cells (Properties)
Cytogenetics (Research)
Histology, Pathological (Research)
Authors: Singh, Arun D.
Tubbs, Raymond
Biscotti, Charles
Schoenfield, Lynn
Trizzoi, Pierre
Pub Date: 08/01/2009
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 College of American Pathologists ISSN: 1543-2165
Issue: Date: August, 2009 Source Volume: 133 Source Issue: 8
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230247106
Full Text: Several studies have evaluated clinical, histopathologic, cytogenetic, and molecular prognostic variables in uveal melanoma. (1) Uveal melanoma consistently demonstrates specific chromosomal alterations, such as the loss of 1 copy of a chromosome (monosomy) or deletions and gains affecting chromosomes 3, 6, and 8. (2-12) Among the cytogenetic changes, monosomy 3 and chromosome 8q duplication are most significantly associated with the poor prognosis. (13,14) MYC gene located on chromosome 8q24 is overexpressed in 70% of the primary tumors, (15,16) and its expression correlates with improved survival. (17) However, it is not known whether the primary tumor cell maintains these cytogenetic changes at the metastatic sites. We have therefore conducted this study to determine the status of chromosomes 3 and 8q and c-myc amplification using fluorescence in situ hybridization on hepatic metastatic lesions of primary uveal melanoma.

MATERIALS AND METHODS

Ten patients with uveal melanoma who underwent needle core biopsy for hepatic metastasis were studied. Institutional review board approval was obtained. Representative paraffin blocks were selected based on review of hematoxylin-eosin-stained sections with confirmed uveal melanoma metastases. Fluorescence in situ hybridization was performed for detection of monosomy 3 and amplification at the 8q24 MYC locus using methods that were described previously. (1) Briefly, unstained 4-[micro] thick paraffin sections of formalin-fixed tissue were prepared on electrostatically charged slides and compared to consecutive presections and postsections stained by hematoxylin-eosin to confirm the presence of tumor in the unstained sections and to specifically circle areas of interest on the reverse side of the unstained slides using etching with a diamond pen. Slides were deparaffinized in xylene and graded alcohols and immersed in molecular-grade Milli Q water (Millipore Corp, Billerica, Massachusetts) for 5 minutes. The slides were immersed in target retrieval solution (Dako, Carpinteria, California) and allowed to incubate at 95[degrees]C for 40 minutes, then removed from the water bath and allowed to cool in the retrieval solution at room temperature for 20 minutes. Slides were then rinsed in distilled water for 5 minutes, the excess water was wiped from the slide surface, and the slide was placed on a flat surface for protease digestion. Proteinase K solution (Dako) was added at a 1:5000 dilution using 150 [micro]L of the enzyme and allowed to incubate for 8 minutes at room temperature. The slides were then placed on a plastic rack and rinsed several times during 5 minutes with water, and then they were dehydrated in graded alcohol. The slides were then allowed to completely air dry, and probe was applied immediately to the sections. The tricolor chromosome enumeration probe 8 (CEP8)/ IGH/MYC probe (Abbott Molecular, Des Plaines, Illinois) and the Urovysion probe consisting of CEP3, CEP7, CEP17, and 9p21 probes (Abbott Molecular) were used for detection of monosomy 3 (using CEP7 as the reference locus) and amplification at the 8q24 MYC locus. A total of 10 [micro]L of probe mixture (7 [micro]L of hybridization buffer, 2 [micro]L of purified molecular-grade water, and 1 [micro]L of probe) was then applied to each slide and covered with a 22 X 22 mm coverslip. Doubles were removed from the target area, and the coverslip edges were sealed over with cement. The target and probe were codenatured by placing the slides on the surface of the Abbott Molecular HYBright using a melting temperature of 73[degrees]C and a melting time of 5 minutes. The slides were then placed in a humidified chamber and hybridized at 37[degrees]C overnight. The slides were then removed from the hybridization chamber, the cement was removed from the coverslips by soaking the slides in 2X sodium chloride-sodium citrate buffer for 5 minutes at room temperature, and the coverslip was removed gently from the surface. The slides were then immersed in 0.4X sodium chloride-sodium citrate buffer/0.3% nonylphenylpolyethylene glycol for 2 minutes, then rinsed in 2X sodium chloride-sodium citrate buffer/0.1% nonylphenylpolyethylene glycol for 3 to 5 seconds, and then rinsed in 2X sodium chloride-sodium citrate buffer, followed by distilled water rinsing. The slides were allowed to air dry completely in darkness, and 10 [micro]L of Vectashield with 4'-6-diamidino-2-phenylindole counterstain (Vector Laboratories, Burlingame, California) was applied to the target area of the slide and coverslipped with a 22 X 22 mm coverslip. The bubbles underneath the coverslip were removed and then sealed with rubber cement. The slides were scored using a 4'-6-diamidino-2-denylindole/tetramethylrhodaminoisothiocyanate/fluorescein isothiocyanate triple-bandpass filter using 4'-6-diamidino-2-phenylindole as a surrogate hematoxylin stain for confirmation of appropriate areas for counting. A total of 200 interphase cells were then scored to determine the percentage of signals. For the determination of 8q24/MYC amplification, only cells having at least two 8q24 signals were counted. For the assessment of monosomic states for chromosome 3, to control for truncation artifact, only tumor cell nuclei with at least 2 CEP7 signals were counted. The CEP3:CEP7 ratio was then calculated, and the loss of CEP3 was determined to be present if the CEP3: CEP7 ratio was 0.7 or lower. CEP8 was reported as aneusomic when the tumor averaged 3 or more of chromosome 8 per cell. MYC amplification was reported as present when the ratio of MYC probe to CEP 8 probe was 2.0 or higher.

RESULTS

There were a total of 10 patients (8 men and 2 women) in this study, with a mean age of 50.3 years (range, 25-68 years; Table 1). Most of the primary tumors were medium sized (90%) and one was large according to the Collaborative Ocular Melanoma Study criteria. (18) Most primary tumors were treated with plaque radiotherapy (80%), and one each was treated with proton beam radiation and enucleation. The time to metastasis varied from 0 months (present at the time of ophthalmic diagnosis) to more than 61 months. As of June 30, 2008, 7 patients were dead because of metastasis.

Hepatic metastasis was confirmed in each case by needle core biopsy. Fluorescence in situ hybridization analysis revealed chromosome 3 monosomy in 5 of the 8 cases that could be satisfactorily evaluated using CEP3 and CEP7 (Table 2). Of the 3 cases with normal complement of chromosome 3, aneusomy of chromosome 8 was observed in 2 cases. In the remaining single case, there was not enough tissue left to perform the diagnostic study. Additional evaluation of chromosomal region 8q24 with MYC amplification in 9 samples revealed amplification in 5 samples.

There was no correlation between MYC amplification and aneusomy of chromosome 8.

[FIGURE 1 OMITTED]

In a single case where the primary tumor was treated by enucleation, the chromosomal monosomy 3, 8q aneusomy, and MYC amplification were present both in the primary tumor (Figure 1) and its hepatic metastatic lesion (Figure 2).

COMMENT

Metastasis is a complex, multistep process that includes invasion, survival in the circulatory system, and colonization of distant organs. (19) There are several genetic determinants of cancer metastasis that can be considered as tumorigenic genes, metastasis initiation genes, metastasis progression genes, and metastasis virulence genes. (20)

Although recent studies have focused on the cytogenetic determinants within the primary tumors,12 there is a paucity of data from the metastatic lesions. Because uveal melanoma preferentially metastasizes to liver, (21) we identified 10 cases with needle core biopsy-confirmed hepatic metastases of uveal melanoma, which formed the basis of this study. Fluorescence in situ hybridization analysis of the hepatic metastasis revealed chromosome 3 monosomy in 5 of the 8 cases that could be satisfactorily evaluated, and in 3 cases with normal complement of chromosome 3, aneusomy of chromosome 8 was observed in 2 cases. In the remaining single case, there was not enough tissue left to perform the diagnostic study. Monosomy 3 was associated with and chromosome 8q aneusomy in 3 cases, and the remaining 2 cases had normal complement of chromosome 8q. There was no correlation between MYC amplification and aneusomy of chromosome 8. (16) Our results are comparable with previously published limited data on a small number of hepatic metastatic lesions that were evaluated by Giemsa banding (22,23) or comparative genomic hybridization (Table 3). (24)

On the basis of studies of the primary tumor, it is currently believed that in the pathogenesis of uveal melanoma, changes in chromosomes 3 occur early, and 8q amplification is a secondary change associated with tumor progression (8,10,25,26) and development of a metastatic phenotype. (9,24,27,28) Our observations of the cytogenetic changes within the metastatic lesions confirm that chromosome 3 monsomy and aneusomy of chromosome 8 are not just markers of a primary tumor's metastatic potential, but are also observed within the hepatic metastatic lesions. These conclusions are further supported by observations in a single case from our series that was treated with enucleation (and, hence, the primary tumor tissue as available for fluorescence in situ hybridization analysis) and that showed similar cytogenetic changes of monosomy 3, 8q aneusomy, and MYC amplification within the primary tumor and in the metastatic lesion. Our observations are further supported by detection of monosomy 3 in the circulating tumor cells in patients with uveal melanoma. (29)

[FIGURE 2 OMITTED]

This work was supported by a Research to Prevent Blindness Challenge Grant to the Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine.

References

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(2.) Prescher G, Bornfeld N, Becher R. Nonrandom chromosomal abnormalities in primary uveal melanoma. J Natl Cancer Inst. 1990;82(22):1 765-1769.

(3.) Sisley K, Cottam DW, Rennie IG, etal. Non-random abnormalities of chromosomes 3, 6, and 8 associated with posterior uveal melanoma. Genes Chromosomes Cancer. 1992;5(3):197-200.

(4.) Wiltshire RN, Elner VM, Dennis T, Vine AK, Trent JM. Cytogenetic analysis of posterior uveal melanoma. Cancer Genet Cytogenet. 1993;66(1):47-53.

(5.) Horsman DE, White VA. Cytogenetic analysis ofuveal melanoma: consistent occurrence of monosomy 3 and trisomy 8q. Cancer. 1 993;71(3):811-819.

(6.) Singh AD, Boghosian-Sell L, Wary KK, et al. Cytogenetic findings in primary uveal melanoma. Cancer Genet Cytogenet. 1994;72(2):109-1 15.

(7.) Prescher G, Bornfeld N, Friedrichs W, Seeber S, Becher R. Cytogenetics of twelve cases of uveal melanoma and patterns of nonrandom anomalies and isochromosome formation. Cancer Genet Cytogenet. 1995;80(1):40-46.

(8.) Sisley K, Rennie IG, Parsons MA, etal. Abnormalities ofchromosome3 and 8 in posterior uveal melanoma correlate with prognosis. Genes Chromosomes Cancer. 1997;19(1):22-28.

(9.) Sisley K, Parsons MA, Garnham J, et al. Association of specific chromosome alterations with tumour phenotype in posterior uveal melanoma. Br J Cancer. 2000;82(2):330-338.

(10.) Hoglund M, Gisselsson D, Hansen GB, etal. Dissecting karyotypic patterns in malignant melanomas: temporal clustering of losses and gains in melanoma karyotypic evolution. Int J Cancer. 2004;108(1):57-65.

(11.) Loercher AE, Harbour JW. Molecular genetics of uveal melanoma. Curr Eye Res. 2003;27(2):69-74.

(12.) Singh AD, Damato B, Howard P, Harbour JW. Uveal melanoma: genetic aspects. Ophthalmol Clin North Am. 2005;18(1):85-97, viii.

(13.) Patel KA, Edmondson ND, Talbot F, Parsons MA, Rennie IG, Sisley K. Prediction of prognosis in patients with uveal melanoma using fluorescence in situ hybridisation. Br J Ophthalmol. 2001;85(12):1440-1444.

(14.) Damato B, Duke C, Coupland SE, et al. Cytogenetics of uveal melanoma: a 7-year clinical experience. Ophthalmology. 2007;114(10):1925-1931.

(15.) Royds JA, Sharrard RM, Parsons MA, etal. C-myc oncogene expression in ocular melanomas. Graefes Arch Clin Exp Ophthalmol. 1992;230(4):366-371.

(16.) Parrella P, Caballero OL, Sidransky D, Merbs SL. Detection of c-myc amplification in uveal melanoma by fluorescent in situ hybridization. Invest Ophthalmol Vis Sci. 2001;42(8):1679-1684.

(17.) Chana JS, Wilson GD, Cree IA, et al. c-myc, p53, and Bcl-2 expression and clinical outcome in uveal melanoma. Br J Ophthalmol. 1999;83(1):110-114.

(18.) Singh AD, Kivela T. The collaborative ocular melanoma study. Ophthalmol Clin North Am. 2005;18(1):129-142.

(19.) Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med. 2006;12(8):895-904.

(20.) Nguyen DX, Massague J. Genetic determinants of cancer metastasis. Nat Rev Genet. 2007;8(5):341-352.

(21.) Singh AD, Borden EC. Metastatic uveal melanoma. Ophthalmol Clin North Am. 2005;18(1):143-150.

(22.) Rey JA, Bello MJ, de Campos JM, Ramos MC, Benitez J. Cytogenetic findings in a human malignant melanoma metastatic to the brain. Cancer Genet Cytogenet. 1985;16(2):179-1 83.

(23.) Parada LA, Maranon A, Hallen M, etal. Cytogenetic analyses of secondary liver tumors reveal significant differences in genomic imbalances between primary and metastatic colon carcinomas. Clin Exp Metastasis. 1999;17(6):471-479.

(24.) Aalto Y, Eriksson L, Seregard S, Larsson O, Knuutila S. Concomitant loss of chromosome 3 and whole arm losses and gains of chromosome 1, 6, or 8in metastasizing primary uveal melanoma. Invest Ophthalmol Vis Sci. 2001;42(2): 313-317.

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(26.) Parrella P, Sidransky D, Merbs SL. Allelotype of posterior uveal melanoma: implications for a bifurcated tumor progression pathway. Cancer Res. 1999; 59(13):3032-3037.

(27.) Hausler T, Stang A, Anastassiou G, et al. Loss of heterozygosity of 1p in uveal melanomas with monosomy 3. Int J Cancer. 2005;116(6):909-913.

(28.) Kilic E, Naus NC, van Gils W, et al. Concurrent loss of chromosome arm 1p and chromosome 3 predicts a decreased disease-free survival in uveal melanoma patients. Invest Ophthalmol Vis Sci. 2005;46(7):2253-2257.

(29.) Ulmer A, Schmidt-Kittler O, Fischer J, et al. Immunomagnetic enrichment, genomic characterization, and prognostic impact of circulating melanoma cells. Clin Cancer Res. 2004;10(2):531-537.

Arun D. Singh, MD; Raymond Tubbs, MD; Charles Biscotti, MD; Lynn Schoenfield, MD; Pierre Trizzoi, MD

Accepted for publication April 16, 2009.

From the Department of Ophthalmic Oncology, Cole Eye Institute (Dr Singh), the Departments of Clinical Pathology (Drs Tubbs and Schoenfield), Anatomic Pathology (Dr Biscotti), and Hematology/Oncology (Dr Trizzoi), and the Taussig Cancer Center (Drs Singh and Trizzoi), The Cleveland Clinic Foundation, Cleveland, Ohio.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Arun D. Singh, MD, Cole Eye Institute, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195 (e-mail: singha@ ccf.org).
Table 1. Hepatic Metastatic Lesions: General Demographic Data

       Parameter                Data

Age, mean (range), y        50.3 (25-68)
Sex, No.
  Female                          2
  Male                            8
Location, No.
  Cbd-choroidal                   4
  Choroidal                       6
Size, No.
  Small                           0
  Medium                          9
  Large                           1
Ocular treatment, No.
  Plaque                          8
  Proton beam                     1
  Enucleation                     1
Time to metastasis, No.
  0 mo                            2
  1-12 mo                         2
  13-60 mo                        3
  >61 mo                          3
Status, No.
  Alive                           3
  Dead                            7

Abbreviation: Cbd, ciliary body.

Table 2. Hepatic Metastatic Lesions: Fluorescence In Situ Hybridization
Analysis

                    Chromosome 3

Case No.   CEP3   CEP7   Ratio   MONO 3 (a)

1          1.1    2.3     0.5     Present
2          2.5    2.8     0.9     Absent
3          2.1    2.2       1     Absent
4          1.2    2.1     0.6     Present
5          1.8      2     0.9     Absent
6          1.3    2.2     0.6     Present
7          1.3    2.3     0.6     Present
8                 Unsatisfactory stain
9                 Unsatisfactory stain
10         1.8    2.5     0.7     Present

                     Chromosome 8

Case No.   MYC   CEP8   CEP8 (b)    MYC AMP (c)

1          4.6   3.3    Aneusomic     Absent
2          5.8   3.6    Aneusomic     Absent
3          5.2     3    Aneusomic     Absent
4           5    2.1    Eusomic       Present
5                Insufficient material
6          7.1   3.6    Aneusomic     Present
7          5.4   2.3    Eusomic       Present
8          5.1     2    Eusomic       Present
9          3.2   2.8    Eusomic       Absent
10         7.9   3.6    Aneusomic     Present

Abbreviations: AMP, amplification; CEP, chromosome enumeration
probe; MONO, monosomy.

(a) Monosomy 3 was diagnosed when the CEP3:CEP7 ratio was 0.7 or lower.

(b) CEP8 was reported as aneusomic when the tumor averaged 3
or more of chromosome 8 per cell.

(c) MYC amplification was reported as present when the ratio of
MYC probe to CEP8 probe was 2.0 or higher.

Table 3. Uveal Metastatic Lesions: Published Cytogenetic Studies

                                            Chromosomal
                                            Abnormalities
                       No. of
Source                 Cases    Technique   Monosomy 3

Rey, (22) 1985 (a)        1     G banding   Absent
Parada, (23) 1999         1     G banding   Present
Aalto, (24) 2001          6     CGH         Present (80%)
Present study, 2008      10     FISH        Present (83%)

                       Chromosomal Abnormalities

Source                 Aneusomy 8q      Other

Rey, (22) 1985 (a)     Present          i(6p)
Parada, (23) 1999      Present          Hyperdiploid
Aalto, (24) 2001       Present (100%)   6q loss (83%)
Present study, 2008    Present (100%)   MYC amplification (55%)

Abbreviations: CGH, comparative genomic hybridization; FISH,
fluorescence in situ hybridization; G banding, Giemsa banding.

(a) Brain metastatic lesion. All other studies analyzed hepatic
metastatic lesions.
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