Significance of Aneuploidy in Melanoma of the Extremity

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1 Significance of Aneuploidy in elanoma of the xtremity. W. van Oven,.D.,*. C. Baas,.D.,t J. W. Oosferhuis,.D., h.d.,* H. Schraffordf Koops,.D., h.d.,t and A. Dameiring, B.S.* 09 Tumor nuclear DNA content was determined by flow cytometry in routinely prepared paraffin blocks from 2 primary malignant melanomas of the extremities. Twelve of the tumors were aneuploid, and 3 were euploid. n this series the presence of aneuploidy appeared to have no prognostic value. Cancer 992; 70:093. Key words: melanoma, DNA flow cytometry, ploidy, aneuploidy. The prognosis for patients with primary cutaneous malignant melanoma depends on clinical stage, localization of the tumor, and treatment and can be correlated with a variety of morphologic parameters, among which the most predictive factor seems to be the depth of invasion (Breslow thickness).' However, more and stronger prognostic factors would be welcome and could be helpful in assessing appropriate therapy because there is still uncertainty about the adequate margin of excision and the benefit of regional chemotherapy by isolated regional perfusion. low cytometric DNA analysis appears to have significant value for establishing prognosis in many solid tumors.' n this study the cellular DNA content of a series of melanomas is assessed by means of flow cytometry of paraffinembedded tissue. The results are compared with survival and histologic features of the tumors. atients and ethods Twentynine patients were selected who were treated for melanoma of the extremities between 973 and stage, treatment, tumor thickness, and availability of paraffin blocks. Data of 2 of 29 cases were evaluable (Table ). The series included patients with longterm survival (3 patients) and patients who died as a result of their cancer (2 patients). All patients had malignant melanoma of the extremity; 2 patients had a malignant melanoma of the lower extremity and patients had melanoma of the upper extremity. All patients had Stage A disease according to the. D. Anderson classification. n all cases, treatment consisted of local excision, axillary or iliac node dissection, and hyperthermic isolated perfusion with melphalan. Thirteen patients were female and 2 patients were male. The mean age was 2 years. ollowup for survivors was at least 30 months. The thinnest lesion was a melanoma with a Breslow thickness of at least. mm; thinner lesions were not included to avoid measurements on tissue not representative of the lesion. All histologic sections of the lesions were analyzed by an experienced pathologist (J.W.O.) who had no knowledge of the flow cytometry results. The sections were scored on the following parameters (see Table ): Breslow thickness, Clark level, shape, presence of ulceration, presence of superficial spreading component, number of mitoses, presence of atypical mitoses, cell type, amount of lymphocytic infiltration, and presence of angoinvasion. Nuclear suspensions were made according to methods described by Hedley et with minor modifications. Briefly, 0pmthick sections were cut from representative parts of the paraffin blocks, selected with the use of adjacent hematoxylin and eosinstained his 98 at the Of oncology Of the tologic slides. These were dewaxed in xylene, r&y University Hospital of Groningen. drated in decreasing concentrations of alcohol, and sus The selection of patients was based on survival pended in pepsin. The resultant naked nuclei were (short or long), localization of primary tumor, clinical stained with propidium iodide after RNase digestion, rom the Departments of *athology and tsurgical Oncology, University Hospital of Groningen, Groningen, The Netherlands. Address for reprints:. W. van Oven,.D., Department of athology, University of Groningen, Oostersingel63,973 Z Groningen, The Netherlands. Accepted for publication October, 99. filtered through nylon gauze, and run through an ACS 0 flow cytometer (Becton Dickinson, ttenleur, The Netherlands). The leftmost peak in the histograms was considered to represent the diploid GO/G phase nuclei of host or tumor cells. A lesion was considered to contain

2 Table. Clinical Data, Histologic eatures, and loidy of 2 Cases of elanoma of the xtremity Breslow Clark Superficial atient thickness Age classifica Spreading Lymphocytic no. (mm) Sex (vr)* Site Staee tion Shaue Ulceration elanoma itosest Cell twe infiltration Aneioinvasion loidv 00wu~ >. >.7 37 U 3 L 3 U L 0 L 26 L 60 U 29 L 36 L 8 L L 60 L 60 L 3 L 3 L 23 L L 67 L 8 U 7 L L L 30 L 29 L 3 LS > 3 '3 0 2i + f + + k /S f f S /S S /S * /S /S A A A A A A A A A A A A ND (0 yr) DOD after 3 mo ND (2 yr) ND (0 yr) DOD after 8 mo ND (6 yr) DOD after 30 mo DOD after mo DOD after 0 mo DOD after 62 mo ND (30 mo) ND (8 y') DOD after 6 mo DOD after 29 mo ND ( yr) ND (9 yr) ND (6 yr) DOD after mo ND (0 yr) ND (9 yr) DOD after 20 mo DOD after 0 mo DOD after 8 mo ND ( yr) ND 2 vr) U: upper extremity; L: lower extremity; LS: lower extremity subungual; convex or plateau; : polypoid; flat; : epithelioid, /S: mixed epithelioid and spindle; S: spindle; : none or little; +: much; +: moderate; : euploid; A aneuploid; ND: no evidence of disease; DOD: dead of disease. * Age at time of presentation. t Number of mitoses/five highpower fields. resence of atypical mitotic figures.

3 Aneuploidy in elanomaslvan Oven et al igure. xamples of aneuploid DNA histograms. Vertical axis: number of counted events; horizontal axis: channel number, representing the relative DNA content. (Top left) Aneuploid DNA histogram with one aneuploid stem cell line. (Top right) ultiploid DNA histogram with more than one aneudoid stem cell line. (Bottom) Aneuploid DNA histogram with "neardiploid'.' tumor stem cell line. The shoulder on the peak is the result of the presence of two cell populations with DNA indices close to each other. 0 l l l l "WJ' \ L an aneuploid stem cell line if more than one GO/G peak could be identified or when a clear shoulder was present on a single GO/Gl peak or on the G2/ peak. The DNA index for aneuploid populations was calculated as the ratio of the channel of the aneuploid peak divided by the channel of the diploid peak. The coefficient of variation was calculated for the euploid GO/G peak by use of the half maximum peak height. or statistical analysis of data, the chisquare test was used. Results our of the 29 tumors originally processed were excluded from additional evaluation because the quality of the DNA histograms was not acceptable. The poor quality of the histograms probably resulted from fixation of these specimens in Bouin solution. All other lesions had interpretable DNA histograms, with coefficients of variation varying between and. Nine lesions showed an aneuploid pattern with one or more peaks outside the diploid or tetraploid region (ig. la). The DNA indices of these aneuploid peaks varied between.3 and 2.6. One of these histograms showed a multiploid pattern (ig. lb). Three other lesions showed aneuploidy in the form of shoulders being adjacent to GO/G or G2/ peaks (ig. lc). t was concluded that in these lesions aneuploid cell populations were present with DNA indices close to.0 or 2.0. The other 3 histograms showed a euploid pattern, with only one large peak and variable smaller peaks with DNA indices of approximately 2.0, representing G2/ phase tumor and host cells. There was no significant correlation between ploidy and survival ( > 0.0). Of the 2 patients who died of widespread metastatic melanoma, had an aneuploid tumor and 8 a diploid tumor. Of the 3 tumors from survivors (followup of 30 months to 2 years), 8 were aneuploid and the other euploid. There was no significant correlation between the ploidy pattern and tumor thickness when melanomas larger than 3 mm

4 2 CANCR July 2, 2992, Volume 70, No. were compared with those of smaller size. A correlation also was not found between ploidy and sex or site, or between ploidy and the morphologic features of ulceration, mitoses (more than five per five highpower fields versus less than five per five highpower fields), cell type (epithelioid versus combined epithelioid/spindle), lymphocytic infiltration, or presence of superficial spreading component. Although not significant, of the seven tumors with a spindle cell component or composed of spindle cells only, six appeared to contain aneuploid stem cell lines. ive of these tumors had a favorable clinical course and two had an unfavorable course. Discussion One of the advantages of using paraffinembedded tissue for DNA flow cytometry as compared with fresh tissue is that the nature of the tissue analyzed can be verified easily by review of adjacent histologic sections. This is especially important in melanomas because the small size of the tumors and the frequent admixture of inflammatory cells can be responsible for overrepresentation of host cells, obscuring an aneuploid tumor cell population. However, increased amounts of cellular debris and a high baseline noise may prevent the recognition of small aneuploid peaks, and peridiploid or peritetraploid peaks can become invisible because of the higher coefficients of ~ariation.~,~ Therefore, absence of aneuploidy is difficult to prove, despite the presentation of a euploid DNA histogram. To avoid overrepresentation of normal cells, the representative part of the paraffin block was selected carefully with the use of adjacent histologic sections and by excluding thin lesions. The results of the current study do not confirm the findings of some other authors who found relations between aneuploidy and recurrence and between aneuploidy and other unfavorable prognostic factors. Using fresh tissue, Sondergaard et ale6 found heteroploidy in 26 of 3 primary melanomas. Aneuploid cell populations were present in 7 of the tumors. A correlation was found between heteroploidy and thickness of the tumor. urthermore, a correlation was found between heteroploidy and a high mitotic activity and significant nuclear pleomorphism. Von Roenn et al.7 found aneuploidy in 3 of 3 primary melanomas, using paraffin blocks. Twenty melanomas had a thickness of more than. mm; from this group, 9 contained aneuploid cell populations. This percentage is in agreement with findings from the current investigation. n their series, there appeared to be a significant correlation between the presence of aneuploidy and tumor thickness. Also, there appeared to be a correlation with the rate of recurrence. Another study from the same laboratory reported the presence of aneuploidy in 3 of 62 Stage melanomas, 98 with a thickness greater than. mm.' n contrast to the results obtained in the current study, aneuploidy was correlated significantly with tumor thickness and ulceration. Aneuploid tumors smaller than. mm and more than 3 mm thick were associated with a higher recurrence rate and decreased diseasefree interval. The findings in the current study are in accordance with those reported by Zaloudik et al.,9 who found no correlation between DNA ploidy and clinical course in 0 Stage melanomas larger than 2.0 mm. Aneuploidy was found in 3 of the tumors. The total lack of a relationship between aneuploidy and survival in melanomas in this study is disappointing. One has to realize that all patients in the current series have been treated by regional perfusion of the extremity, which may have influenced the results. The absence of a relationship between aneuploidy and lesion thickness is noticeable. Because it is conceivable that aneuploid stem cell lines develop as a result of clonal evolution," such a relationship could be expected to exist; as time passes and the tumor grows, the chance should increase that a detectable aneuploid subline has formed. A second reason to expect such a relationship is a possible tendency for aneuploid tumors to grow faster than euploid tumors. aybe the exclusion of small lesions in this series has been of influence. The absence of a correlation between aneuploidy and recurrence in combination with the absence of a correlation between aneuploidy and tumor thickness favors the possibility that the biologic potential of melanomas is totally independent of ploidy, with aneuploid tumors not growing faster and not giving rise to early metastases. Without indicating that such a relationship does not exist, however, the results of the current investigation permit at least the conclusion that aneuploid melanomas need not be followed by an unfavorable course of disease. t must be explored whether the high incidence of aneuploidy in spindle cell lesions is helpful in the sometimes difficult differential diagnosis between spindle cell melanoma and spindle cell nevi, in particular Spitz nevi. References. Breslow A. Thickness, crosssectional areas and depth of invasion in the prognosis of cutaneous melanoma. Ann Surg 970; 72: riedlander L, Hedley DW, Taylor W. Clinical and biological

5 Aneuploidy in elanomas/van Oven et al. 3 significance of aneuploidy in human tumors. ] Clin athol 98; Hedley DW, riedlander L, Taylor W, Rugg CA, usgrove A. ethods for analysis of cellular DNA content of paraffinembedded pathological material using flow cytometry. ] Histochem Cyfochem 983; 3:333.. Stephenson RA, Gay H, air WR, elamed R. ffect of section thickness on quality of flow cytometric DNA content determinations in paraffinembedded tissues. Cytometry 986; 7:.. rierson H. low cytometric analysis of ploidy in solid neoplasms: comparison of fresh tissues with formalinfixed paraffin embedded specimens. Hum athol 988; 9: Sondergaard K, Larsen JK, oller U, Christensen J, Houjensen K. DNA ploidycharacteristics of human malignant melanoma analysed by flow cytometry and compared with histology and clinical course. Virchows Arch [B] 983; Von Roenn JH, Kheir S, Wolter J, Coon JS. Significance of DNA abnormalities in primary malignant melanoma and nevi: a retrospective flow cytometric study. Cancer Res 986; 6: Kheir S, Bines SD, Von Roenn JH, Soong S, Urist, Coon JS. rognostic significance of DNA aneuploidy in stage cutaneous melanoma. Ann Surg 988; 207:6. 9. Zaloudik J, oore, Ghosh AK, echl 2, Rejthar A. DNA content and HC class antigen expression in malignant melanoma: clinical course. Clin athol 988; : Nowell C. The clonal evolution of tumor cell populations: acquired genetic lability permits stepwise selection of variant sublines and underlies tumor progression. Science 976; 9:238.