Variation of monosomy 3 status within uveal melanoma.
Abstract: * Context.--Determining the most significant prognostic variables in uveal melanoma is important for stratifying-patients for metastasis surveillance and possible initiation of chemotherapy or immunotherapy. Monosomy 3, one such variable, can be determined using fluorescence in situ hybridization, either on enucleated samples, fine-needle aspiration biopsy, or tumor sample obtained by vitrector.

Objective.--To evaluate possible regional discordance in chromosome 3 by sites likely to be sampled by different biopsy methods.

Design.--Eighteen consecutive patients with uveal melanoma who underwent primary enucleation were studied. Representative paraffin blocks were selected based on review of hematoxylin-eosin stained sections, and the apex and base of each tumor was demarcated. Unstained paraffin sections, 4 in thickness, were prepared, and fluorescence in situ hybridization, looking for monosomy 3, was performed. The chromosomal analysis was also correlated with histologic evaluation for melanoma cell type (spindle vs epithelioid cell), ciliary body involvement, presence of positive periodic acid-Schiff vascular mimicry pat terns, scleral or extrascleral spread and size. One case was excluded because of necrosis.

Results.--Ten of the 17 remaining cases (59%) demonstrated monosomy 3 (in either the base or both base and apex of the tumor) with 7 cases (41%) showing disomy. Seven cases (70%) with monosomy 3 demonstrated this in both the apex and the base locations, whereas 3 cases (30%) showed monosomy in one location only (always at the base). Fourteen of the 17 cases (82%) revealed concordance in chromosome 3-monosomy 3 (7 of 14, 50%) or chromosome 3-disomy 3 (7 of 14, 50%). All 3 discordant cases demonstrated the monosomy 3 at the base with disomy at the apex. Lack of concordance between the base and apex did not correlate with melanoma cell type.

Conclusions.--Prognostic variables are important in management of neoplasms, and this study points out that the site of tissue biopsy for prognostication in uveal melanoma could affect the results obtained, at least for the presence of monosomy 3.

(Arch Pathol Lab Med. 2009;133:1219-1222)
Article Type: Report
Subject: Immunotherapy (Methods)
Chemotherapy (Methods)
Metastasis (Diagnosis)
Metastasis (Care and treatment)
Melanoma (Diagnosis)
Melanoma (Care and treatment)
Cancer (Chemotherapy)
Cancer (Methods)
Authors: Schoenfield, Lynn
Pettay, James
Tubbs, Raymond R.
Singh, Arun D.
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
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230247105
Full Text: Uveal malignant melanoma is the most common primary intraocular malignancy in the adult population, with an annual incidence of approximately 4.3 cases per million per year in the United States, a rate which has essentially been stable during the past 50 years. (1) It comprises approximately 5% of all melanomas, and 85% of ocular melanomas are uveal in origin. (2) Mortality, principally from liver metastasis, has historically been approximately 50%; this has not changed significantly in recent years despite advances in early diagnosis and local treatment. (3) Determining the most significant prognostic variables has been elusive but important for stratifying patients for metastasis surveillance and possible initiation of chemotherapy or immunotherapy. (4)

Traditionally, several features, such as large size, epithelioid cell type, presence of positive periodic acid-Schiff (PAS) vascular mimicry patterns (or extracellular matrix patterns), and ciliary body involvement have been noted to correlate with poor prognosis. (5-8) More recently, studies have turned to cytogenetic and molecular genetic analyses. Chromosomal changes of prognostic significance have been found and include monosomy 3 and alterations of 8q and 6p. (9-15) Studies (16,17) have reported heterogeneity for monosomy 3 within uveal melanoma but did not look at it from a geographic point of view, which could have clinical relevance.

Monosomy 3 can be determined using fluorescence in situ hybridization (FISH), either on enucleated samples, fine-needle aspiration biopsy, or tumor sample obtained by vitrector.18 Our hypothesis was that, just as there is histologic variability, there may also be variability in the genetic alterations within a given tumor. Therefore, we examined enucleated globes with uveal melanoma for the presence of monosomy 3 or disomy 3 in both the apex and base locations of the tumor, looking for possible discordant patterns in the chromosome 3 number.

MATERIALS AND METHODS

Eighteen consecutive patients with uveal melanoma, who underwent primary enucleation, were studied. One case was excluded because of extensive tumor necrosis, which interfered with the FISH analysis. Institutional review board approval was obtained. Representative paraffin blocks were selected based on review of sections stained with hematoxylin-eosin, and the apex and base of each tumor was demarcated. Unstained paraffin sections, 4 [micro]m in thickness, were prepared and applied to electrostatically charged slides. Using standard techniques, FISH was performed with directly labeled pericentromeric chromosome enumeration probes (CEP3 and CEP4) labeled with SpectrumOrange and SpectrumGreen, respectively (Abbott Molecular, Abbott Park, Illinois). Sections were counterstained with 4',6-diamidino-2-phenylindole Antifade. Twenty nuclei in the preselected apex and base areas were counted; only nuclei with at least 2 reference CEP4 signals were counted. Cells in different areas of the base and apex were used in counting. The CEP3:CEP4 ratio was then calculated and the loss of CEP3:monosomy 3 was determined to be present if the CEP3:CEP4 ratio was <0.7. Care was taken to examine cells with obviously malignant nuclei to avoid the possibility of negative results for monosomy 3 from looking at pigmented macrophages. Pigmentation of some cells did not interfere with the chromosomal analyses.

The cases were all examined by light microscopy with both hematoxylin-eosin and PAS stains. The tumor cell type was determined in both the apex and the base. In addition, ciliary body involvement, spread into the sclera or extraocular spread, presence of PAS+ vascular mimicry patterns, and tumor size were also noted.

RESULTS

The patients ranged in age from 26 to 91 (mean, 64 years), and 10 were men (59%) and 7 (41%) were women (Table). At 33 months follow-up, 9 patients were alive (53%) and 8 patients had died (47%). Six of the 8 patients (75%) who had died demonstrated monosomy 3, and 2 (25%) showed disomy. Of the 9 living patients, 5 (56%) had tumors with disomy, and 4 (44%) had tumors with monosomy 3.

Ten of the 17 cases (59%) demonstrated monosomy 3 (in either the base or both the base and the apex of the tumor) with 7 cases (41%) showing disomy. Seven cases (70%) with monosomy 3 demonstrated this in both apex and base locations, whereas 3 cases (30%) showed monosomy in one location only (always at the base, with disomy at the apex; Figure 1). Fourteen of the 17 cases (82%) revealed concordance in chromosome 3-monosomy 3 (7 of 14, 50%) or disomy 3 (7 of 14, 50%). Figure 2 shows a cell with monosomy 3.

Histologically, six cases (35%) showed variation in the cell type between the apex and the base. These cases all showed either spindle B or epithelioid cell type at one location and a mixed pattern at the other location. Lack of monosomy 3 concordance between the base and apex did not correlate with cell type because 2 of the cases had epithelioid cells or were mixed epithelioid and spindle cells, and 1 had spindle B cells only.

Ciliary body involvement was found in 8 of 17 cases (47%). Five of these 8 cases (63%) showed monosomy 3 (all with concordance in apex and base). Nine of the 17 cases (53%) showed tumor within the sclera or involving extraocular tissue; and of these, 5 cases (56%) demonstrated monosomy 3, including all 3 cases with a discordant pattern for monosomy 3. Positive periodic acid-Schiff vascular mimicry patterns were seen in 13 of 17 cases (76%), and 8 of these cases (62%) showed monosomy 3, including all 3 cases showing a discordant pattern for monosomy 3. Tumors with monosomy 3 in either one location or both had an ultrasound size range of 9 to 20 mm (mean, 16 mm), compared with a range of 15 to 18 mm (mean, 16 mm) for the tumors with disomy for chromosome 3.

COMMENT

Mortality from uveal melanoma, due to liver metastasis, has historically been approximately 50%, and this number has not changed significantly in recent years despite advances in early diagnosis and treatment. (3) Traditionally, several features, such as large size, epithelioid cell type, presence of PAS+ vascular mimicry patterns (or extracellular matrix patterns), and ciliary body involvement have been noted to correlate with poor prognosis. (6-8) More recently, studies (9-15) have turned to cytogenetic and molecular genetic analyses. Chromosomal changes of prognostic significance have been found and include monosomy 3 and alterations of 8q and 6p. Prescher et al (10) noted a reduction in probability of 5-year survival from approximately 100% to less than 50% in those cases demonstrating monosomy 3. White et al11 found that the poor outcome associated with abnormalities in chromosomes 3 and 8 required that both chromosomal abnormalities occur together. Scholes et al (19) found monosomy 3 to correlate with a poor prognosis after treatment and further noted that this chromosomal change showed a greater association with metastatic disease than did epithelioid histology, PAS + loops, ciliary body involvement, or large basal tumor diameter. Gene expression profiling has identified 2 classes of uveal melanomas that correlate strongly with prognosis. Class II correlates with liver metastasis and poor prognosis. (20,21)

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Unlike most other tumors, uveal melanomas are usually treated without a histopathologic diagnosis. Small- and medium-sized tumors are often treated with eye-saving modalities, such as brachytherapy. Recently, however, in some centers, ocular oncologists have begun to acquire tumoral tissue either by fine-needle aspiration or vitrector, and these samples can be used for FISH analysis for monosomy 3. (18) The possibility that a limited sample from one area of the tumor might not be representative and provide the essential prognostic information is the basis of this study. Our hypothesis was that just as there is histologic variability in cell type in many uveal melanomas, there may also be variability in the genetic alterations within a given tumor. Therefore, we examined 17 cases of enucleated uveal melanoma by FISH for monosomy 3, both in the apex of the tumor and in the base, to see if some tumors had a discordant pattern. We also correlated these findings with tumor cell type, ciliary body involvement, presence of PAS+ networks, largest basal diameter, and extraocular spread. Finally, monosomy 3 was correlated with clinical outcome, which was available in all 17 cases.

Ten of the 17 cases (59%) that we examined showed monosomy 3, and 3 of these 10 cases (30%) showed a disparate pattern between the apex and the base. Thus, we conclude that uveal melanoma may display tumor heterogeneity regarding monosomy 3 status within the base and the apex of the tumor. In contrast to our observations in 17 cases, a recent publication (22) found focal or diffuse heterogeneity of monosomy 3, but the authors did not detect geographic differences between the apex and the base of the tumors. For this part of their study, they examined 16 cases (similar to our study), and whereas they found heterogeneity, it is not clear why they did not demonstrate geographic differences between the apex and the base cell population. Still, they conclude that multiple samples should be obtained from different areas of the tumor to reduce the possibility of missing monosomy 3 and thus high-risk patients because of the heterogeneity.

Our study demonstrates that fine-needle aspiration biopsy samples obtained from the tumor base (scleral approach) and the apex (transvitreal approach) may yield disparate results. In the 3 discordant cases (18%), monosomy 3 was present at the base with an intact pattern at the apex. Our preliminary data suggest that a scleral approach would be the preferred route for obtaining tumor tissue for prognostication in uveal melanoma. Studies on a larger number of patients with counting of more cells per case for FISH are underway to validate these initial results.

Accepted for publication March 20, 2009.

References

(1.) Singh AD, Topham A. Incidence of uveal melanoma in the United States: 1973-1997. Ophthalmology. 2003;1 10:956-961.

(2.) Singh AD, Bergman L, Seregard S. Uveal melanoma: epidemiologic-aspects. Ophthalmol Clin North Am. 2005;18:75-84, viii.

(3.) Diener-West M, Hawkins BS, Markowitz JA, Schachat AP. A review of mortality from choroidal melanoma, II: a meta-analysis of 5-year mortality rates following enucleation, 1 966 through 1988. Arch Ophthalmol. 1992;1 10(2):245 250.

(4.) Damato BE, Taktak A. Uveal malignant melanoma: mortality. In: Singh AD, Damato BE, Pe'er J, Murphree AL, Perry JD, eds. Clinical Ophthalmic Oncology. Philadelphia, PA: Saunders-Elsevier; 2007:277-282.

(5.) Singh AD, Shields CL, Shields JA. Prognostic factors in uveal melanoma. Melanoma Res. 2001;11(3):1-9.

(6.) McLean IW, Foster WD, Zimmerman LE. Uveal melanoma: location, size, cell type, and enucleation as risk factors in metastasis. Hum Pathol. 1982;13(2): 123-132.

(7.) Coleman K, Baak JP, Van Diest P, Mullaney J, Farrell M, Fenton M. Prognostic factors following enucleation of 111 uveal melanomas. Br J Ophthalmol. 1993;77(11):688-692.

(8.) Folberg R, Rummelt V, Parys-Van Ginderdeuren R, et al. The prognostic value of tumor blood vessel morphology in primary uveal melanoma. Ophthalmology. 1 993;100(9):1389-1398.

(9.) Sisley K, Rennie IG, Parsons MA, et al. Abnormalities of chromosome 3 and 8 in posterior uveal melanoma correlate with prognosis. Genes Chromosomes Cancer. 1 997;1 9(1):22-28.

(10.) Prescher G, Bornfeld N, Hirche H, Horsthemke B, Jockel KH, Becher R. Prognostic implications of monosomy 3 in uveal melanoma. Lancet. 1996; 347(9010):1222-1225.

(11.) White VA, Chambers JD, Courtright PD, Chang WY, Horsman DE. Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma. Cancer. 1998;83(2):354-359.

(12.) 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.

(13.) 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.

(14.) Kilic E, van Gils W, Lodder E, et al. Clinical and cytogenetic analyses in uveal melanoma. Invest Ophthalmol Vis Sci. 2006;47(9):3703-3707.

(15.) Singh AD, Boghosian-Sell L, Wary KK, et al. Cytogenetic findings in primary uveal melanoma. Cancer Genet Cytogenet. 1994;72(2):109-115.

(16.) Sandinha T, Farquharson M, McKay I, Roberts F. Correlation of heterogeneity for chromosome 3 copy number with cell type in choroidal melanoma of mixed-cell type. Invest Ophthalmol Vis Sci. 2006;47(12):5177-5180.

(17.) Maat W, Jordanova ES, van Zelderen-Bhola SL, et al. The heterogeneous distribution of monosomy 3 in uveal melanomas: implications for prognostication based on fine-needle aspiration biopsies. Arch Pathol Lab Med. 2007;131(1):91 96.

(18.) Sisley K, Nichols C, Parsons MA, Farr R, Rees RC, Rennie IG. Clinical applications of chromosome analysis, from fine needle aspiration biopsies, of posterior uveal melanomas. Eye. 1998;12(pt2):203-207.

(19.) Scholes AG, Damato BE, Nunn J, HiscottP, Grierson I, Field JK. Monosomy 3 in uveal melanoma: correlation with clinical and histologic predictors of survival. Invest Ophthalmol Vis Sci. 2003;44(3):1008-1011.

(20.) Onken MD, Worley LA, Ehlers JP, Harbour JW. Novel molecular classification of uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res. 2004;64:7205-7209.

(21.) Tschentscher F, Husing J, Holter T, et al. Tumor classification based on gene expression profiling shows that uveal melanomas with and without monosomy 3 represent two distinct entities. Cancer Res. 2003;63(10):2578-2584.

(22.) Mensink HW, Vaarwater J, Kilic E, et al. Chromosome 3 intratumor heterogeneity in uveal melanoma. Invest Ophthalmol Vis Sci. 2009;50(2):500-504.

Lynn Schoenfield, MD; James Pettay, BS, MT(ASCP); Raymond R. Tubbs, DO; Arun D. Singh, MD

From the Departments of Anatomic Pathology (Dr Schoenfield) and Molecular Pathology (Mr Pettay and Dr Tubbs), and the Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio (Dr Singh).

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

Reprints: Lynn Schoenfield, MD, Department of Anatomic Pathology, Pathology and Laboratory Medicine Institute, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195 (e-mail: schoenl@ccf.org.)
Clinical, Histopathologic, and Cytogenetic (Monosomy 3) Parameters
of the Study Group

                                                      Cell Type
Case                  Tumor Size
No.    Age, y/Sex   Base/Height, mm    Location     Apex      Base

 1       82/F           18/12         Cbd-choroid   Spindle   Spindle
 2       73/M           18/5          Choroid       Spindle   Mixed
 3       54/M           15/12         Choroid       Epith     Mixed
 4       57/M           15/12         Choroid       Spindle   Spindle
 5       83/F           16/13         Cbd-choroid   Mixed     Mixed
 6       83/M           17/10         Cbd-choroid   Mixed     Mixed
 7       61/M           10/8          Choroid       Spindle   Spindle
 8       50/F           18/8          Choroid       Spindle   Mixed
 9       72/F           16/4          Choroid       Epith     Mixed
10       26/M           18/10         Cbd-choroid   Mixed     Mixed
11       75/F           18/9          Cbd-choroid   Epith     Epith
12       53/M           20/10         Cbd-choroid   Mixed     Mixed
13       60/M           19/11         Cbd-choroid   Mixed     Mixed
14       62/M           19/8          Cbd-choroid   Mixed     Epith
15       54/M           9/4           Choroid       Epith     Mixed
16       91/F           16/11         Choroid       Mixed     Mixed
17       59/F           12/3          Choroid       Spindle   Spindle

                                       Monosomy 3
Case      [PAS.sup.+]     Scleral
No.    Vascular Mimicry   Invasion   Apex      Base

 1             +              +      Absent    Absent
 2             +              +      Absent    Absent
 3             -              -      Absent    Absent
 4             +              -      Absent    Absent
 5             +              -      Absent    Absent
 6             -              +      Absent    Absent
 7             +              +      Absent    Absent
 8             -              -      Present   Present
 9             -              +      Present   Present
10             +              +      Present   Present
11             +              -      Present   Present
12             +              -      Present   Present
13             +              -      Present   Present
14             +              -      Present   Present
15             +              +      Absent    Present
16             +              +      Absent    Present
17             +              +      Absent    Present

Abbreviations: Cbd, ciliary body; Epith, epithelioid;
[PAS.sup.+],  periodic acid-Schiff positive.
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