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Mitotic rate and S-phase fraction as prognostic factors in stage I cutaneous malignant melanoma.

Clinical data from 369 patients with clinical stage I cutaneous malignant melanoma treated in Kuopio University Hospital district between 1974 and 1989 with a mean follow-up of 6.4 years were analysed. Clinical parameters, histology, DNA index, S-phase fraction (SPF) and mitotic indices [mitotic activity index (MAI) and volume-corrected mitotic index (M/V index)] were correlated with the outcome of the disease to establish their value as predictors of stage I cutaneous malignant melanoma. In univariate survival analyses, bleeding, gender, tumour thickness, level of invasion according to Clark, TNM category, MAI, M/V index and SPF were the most significant predictors of recurrence-free (RFS) and overall survival. In Cox's multivariate analysis, tumour thickness (P = 0.0021), bleeding (P = 0.0106) and M/V index (P = 0.0058) predicted poor RFS in the 259 patients available for the analysis. Poor overall survival was predicted by MAI (P = 0.0002), bleeding (P = 0.004), SPF (P = 0.009) and male gender (P = 0.034). The present results indicate that mitotic activity index (MAI), volume-corrected mitotic index (M/V index) and S-phase fraction (SPF) are important prognostic factors in addition to the well-established Breslow thickness in stage I cutaneous malignant melanoma.

One method of assessing cell proliferation in routinely fixed histological sections is mitotic counting, which has been related to patient survival in several human malignancies (Eskelinen et al, 1992;Cross and Start, 1996). Univariate (Ramsay et al, 1995;Clemente et al, 1996) and multivariate (Barnhill et al, 1996;Straume and Akslen, 1996) survival analyses have also revealed the value of mitotic rate as a prognosticator in stage I cutaneous melanoma, whereas contradictory results have been reported (Talve et al, 1996). DNA index (DI) has been related to the biological behaviour of human solid malignancies (Friedlander et al, 1984), including stage I cutaneous malignant melanoma (von Roenn et al, 1986;Kheir et al, 1988;Gattuso et al, 1990; Bartkowiak et al, 1991). There are no previous studies on mitotic indices, S-phase fraction and DNA index in stage I cutaneous malignant melanoma using multivariate survival analysis. The present study was conducted to assess the applicability of mitotic rate, SPF and DI in stage I cutaneous malignant melanoma and to analyse their inter-relationship and relation to traditional prognostic factors of cutaneous melanoma as well as to patient survival.

PATIENTS AND METHODS Patients
This retrospective study consisted of 369 patients diagnosed and treated for clinical stage I cutaneous malignant melanoma in the district of Kuopio University Hospital between 1974 and 1989. The patients were selected from 473 consecutive stage I melanoma patients, based on the availability of sufficient material from the primary tumour. The clinical staging of all tumours was done according to UICC (UICC, 1987). Patient records were reviewed and the pertinent clinical data are shown in Table 1. The mean follow-up time of the patients was 6.4 years (range 0.2-18 years). The cause of death was obtained from the patient records and from the files of the Finnish Cancer Registry and General Statistical Office in Finland.

Histological methods
The tumour samples were fixed in buffered formalin (pH 7.0), embedded in paraffin, sectioned at 5 gm and stained with haematoxylin and eosin (HE). The histological diagnosis was confirmed by reviewing one to four original sections of the primary tumour. Tumour thickness according to Breslow (1970) and level of invasion according to Clark et al (1969) were re-examined by the same pathologist (VMK), unaware of the clinical data. Mountain View, CA, USA) using 15 mW excitation at 488 nm. The total emission above 560 nm was recorded, and at least 10 000 98 nuclei from each specimen were analysed. No internal standard 152 was added, as the staining intensity varied from sample to sample. 119 The lowest peak was assigned a DNA index (DI of 1.00), and the DI values of other peaks were calculated using this as a reference.

200
Therefore, possible hypodiploid peaks were identified as diploid 38 and the normal diploid peak as hyperdiploid. Tumours with one 131 peak were considered to be diploid, and those with more than one 66 peak aneuploid. The histograms were interpreted by one of us 46 (SN), unaware of the clinical outcome. 257 A full peak (GO/G1) coefficient of variation (CV) was calculated, and only samples with a CV of less than 10% were accepted 106 for further analysis. This was done because, in samples with a high 263 CV, a near-diploid peak (DI < 1. 1) could remain undetected. The S-phase fraction was calculated either using the Cellfit 21 program of the FACScan flow cytometer or manually by a modi-68 fied rectilinear method (Baisch et al, 1975;Camplejohn et al, 106 1989). The SPF of the stem line with the highest DI was calcu-29 lated. In cases with different SPF values obtained by the automatic and manual methods, the lower value was chosen. Usually the 82 manual method gave a lower result, because it was applied only in 88 tumours in which the automatic method seemed to give too high 120 values. In these cases, there was usually a skewness to the right of 2°the G1 peak or a noticeable G2 peak of the diploid population 29 within the S-phase of the aneuploid population.

Mitotic indices
The mitotic figures were ranked in ten consecutive high-power fields (HPF) where mitotic activity was highest. The mitotic activity index (MAI) is the number of sharply defined mitoses per ten HPF without consideration of the percentage representing tumour tissue. The volume-corrected mitotic index (MIV index) was determined by the method and formula originally introduced by Haapasalo et al (1989a). The MN index expresses the number of mitotic figures mm-2 of neoplastic tissue in the microscope image. Both measurements were done by one investigator (JK) and, thus, interobserver variation was avoided. All mitotic figures were counted using an objective magnification of 40 x (field diameter 490 ,um) with an Olympus Vanox T microscope. In 37 specimens, there was not enough tumour material to measure ten consecutive high-power fields; these were therefore excluded. Thus, mitotic indices from 332 patients were analysed.

Statistical analyses
The SPSS-Win program package was used in a PC computer for basic statistical calculations. The statistical tests used are indicated in the results when appropriate. Correlations of categorical variables were examined by contingency tables, which were further analysed by chi-square tests (Pearson correlation coefficient).
Univariate survival analyses were based on the Kaplan-Meier method (log rank analysis; Kaplan and Meier, 1958). Multivariate survival analysis was done with the SPSS-Cox (Cox, 1972) programme package using a forward stepwise procedure and the L ratio significance test. Overall survival analysis included as an event only the deaths resulting from malignant melanoma. Deaths attributable to post-operative complications within 30 days were excluded. Recurrence-free survival (RFS) was defined as the time elapsed between the primary treatment and the recurrent tumour.
For all statistical tests, a critical significance level of 5% was chosen. In Cox's multivariate analysis, a removal limit of P < 0. 0 was used as an additional inclusion criteria.

RESULTS
There were 191 women (52%) and 178 men (48%) in the cohort. The mean age was 55.5 years (s.d. 15.2) with a range of Prognosis in cutaneous malignant melanoma 1919 19.0-90.0 years. The most common location was the trunk and perineum (47%); 20% were in the lower limbs, 16% in the upper limbs and 17% in the head and neck area.
In the statistical analyses for mitotic rates and SPF, we used the median as the cut-off value (1 for MAI, 7 for MN index and 4% for SPF respectively). The associations between tumour proliferative activity indicators (MAI, MN index and SPF) and conventional clinicopathological parameters are shown in Tables 2 and 3. There was a significant association between high (>4%) SPF and high mitotic frequency measured by MAI (P = 0.002) and MN index (P = 0.001). The association of MAI with SPF is shown in Table 4.
During the follow-up, 106 patients (29%) had a recurrence, 66 patients (18%) died of melanoma and 46 patients (13%) died of other causes. The crude 5-year survival rate of the patients was 78%. The overall 5-year survival rate of the patients was 85%, and the 5-year RFS rate was 76%.
Clinical, histological and quantitative features predicting RFS and overall survival are shown in Table 5 A multivariate Cox analysis was performed on 259 patients with a complete set of data available. It included only the variables that were significant in univariate analysis (gender, bleeding, tumour thickness, Clark level of invasion, MAI, M/V index and SPF). The pT category (consisting of Clark level of invasion and tumour thickness) was excluded from the final model. Tumour thickness (P = 0.0021), bleeding (P = 0.0106) and high MN index (P = 0.0058) predicted poor recurrence-free survival. High MAI (P = 0.0002), bleeding (P = 0.004), high SPF (over 4%; P = 0.009) and male gender (P = 0.034) were statistically significant predictors of poor overall survival (Table 6).
In order to address whether mitotic rates or SPF add significant information to normally available clinical prognosticators, we combined the proliferation markers (MAI, M/V index and SPF) with conventional variables (tumour thickness, bleeding and In overall survival, MAI/bleeding (P = 0.0035) and MAI/SPF (P = 0.0001) were those variables that clearly added prognostic power to the model. In patients with bleeding tumours, the relative risk of melanoma death was 9.05 (CI 2.1-39.7) in the high MAI (> 1) subgroup compared with the risk of patients with nonbleeding tumours in the low MAI (< 1) subgroup. The relative risk of melanoma death for the patients with high MAI (> 1) and high SPF (> 4%) categories was 10.6 (CI 3.2-35.6) compared with the risk of patients with low MAI (< 1) and low SPF (< 4%) categories (other data not shown). In the recurrence-free survival analysis, the MN index/Breslow variable was a better prognosticator (overall P = 0.0002) than either MN index or Breslow thickness. The risk of melanoma recurrence between the patients with (CI 5-293) compared with the risk of patients with tumour thickness < 0.75 mm and MN index < 7 (other data not shown).

DISCUSSION
The proliferative activity of cancer cells has a significant prognostic value in several human malignancies (Quinn and Wright, 1990). Mitotic counting is the most commonly used method of assessing proliferative activity in human tumours. The mitotic activity has an independent prognostic value in many human epithelial tumours (Cross and Start, 1996). In malignant melanoma, mitotic counts have been shown to be of prognostic significance in several studies (Schmoeckel and Braun-Falco, 1978;Salman and Rogers, 1990;Evans et al, 1992;Ramsay et al, 1995;Clemente et al, 1996). The potential value of the mitotic index as a prognostic parameter has been questioned because different authors have obtained varying results (Quinn and Wright, 1990). The variability in fixation, intratumoral heterogeneity, variations in cell size and the criteria for recognition of mitotic figures may all cause interobserver variations (Weidner et al, 1994;Collan et al, 1996;Cross and Start, 1996;Jannink et al, 1996). The reproducibility of the MNV index and MAI have been documented in human tumours (Donhuijsen, 1986;Montironi et al, 1988;Haapasalo et al, 1989b;Lipponen et al, 1990). Jannink et al (1996) found a high degree of intratumour heterogeneity of mitotic activity (MAI and MNV index) in breast cancer. They conclude that multiple blocks should be taken, and the areas with highest proliferation should be selected. According to their results, a correction for the volume percentage of epithelium did not result in remarkable heterogeneity in results between MAI and MN index. As long as the criteria to assess the mitotic activity are strict, the mitotic index will be reproducible and prognostically relevant.
Flow cytometric analysis of nuclear DNA content and SPF is a feasible method for estimating the malignant potential and growth characteristics of malignant tumours (Seckinger et al, 1989;Keshgegian and Cnaan, 1995). DNA aneuploid primary melanomas recur earlier and more frequently than do DNA diploid ones (von Roenn et al, 1986;Kheir et al, 1988). DNA aneuploidy has also been associated with a shorter survival in primary melanoma (Kheir et al, 1988;Lindholm et al, 1989;Gattuso et al, 1990;Bartkowiak et al, 1991). In our study, DNA ploidy had no impact on survival. Differences in tissue processing, the nuclei measured, DNA histogram/cell cycle analysis and intratumoral heterogeneity may explain the above-mentioned divergent results (Kallioniemi, 1988;Bergers et al, 1996). Like DNA ploidy, SPF has a prognostic value in cutaneous stage I (Bartkowiak et al, 1991) and metastatic (Muhonen et al, 1992) melanoma. Our study supports the important prognostic role of SPF in malignant melanoma. High SPF (over 4%) predicted poor recurrence-free and overall survival in univariate analysis and poor overall survival in multivariate analysis. However, in order to use SPF as a marker of cell proliferative activity, we have to consider that SPF varies considerably in different samples from the same tumour (Kallioniemi, 1988). SPF does not indicate the growth rate of the tumour directly, but merely indicates the proportion of cells synthesizing DNA.
Other cell proliferation markers that can be used on routine tissue sections are Ki-67 antigen, proliferative cell nuclear antigen (PCNA) and silver-binding nucleolar organizer region (AgNOR) staining (Cross and Start, 1996).  antigen can be detected with either a polyclonal Ki-67 antibody or a specific monoclonal antibody for Ki-67 epitope (MIB-1; Gerdes et al, 1991). MIB-1 expression correlates with mitotic counts in breast and renal cell carcinoma (Weidner et al, 1994;Cross and Start, 1996;Jochum et al, 1996), but it is liable to the same reproducibility problems as mitotic counts. MIB-1 staining had a prognostic independent value even superior to tumour thickness and mitotic index in primary thick cutaneous melanomas (Ramsay et al, 1995). In addition, immunostaining for Ki-67 antigen is helpful in identifying individuals with thick nodular melanomas who are at risk of metastatic disease (Vogt et al, 1997). PCNA expression in cutaneous melanomas seems to be a marker of tumour progression (Takahashi et al, 1991;Evans et al, 1992), but it may not help in predicting prognosis in these tumours (Reddy et al, 1995). AgNOR counts often correlate with other markers of cell proliferation, but the staining techniques and the counting methods suffer standardization problems (Cross and Start, 1996). So far, AgNOR counting has failed in predicting the prognosis of cutaneous malignant melanoma, and its correlation with other cell proliferation markers in cutaneous melanomas is also controversial (Evans et al, 1992).
In our study, the most important prognostic factors observed in univariate analyses (Table 5)  Follow-up time (months) Figure 5 Overall survival according to SPF in stage cutaneous malignant melanoma (SPF < 4%, n = 155 and SPF > 4%, n = 135; P = 0.0007; X2 = 11.43) invasion according to Clark, TNM category and proliferative activity (M/V index, MAI and S-phase fraction). This observation is in agreement with previous studies (Breslow, 1970;Gattuso et al, 1990;Garbe et al, 1995;Straume and Akslen, 1996). In the multivariate analysis, tumour thickness was the best predictor of RFS followed by M/V index and bleeding. The best predictors of overall survival in order of importance were MAI, bleeding, SPF and male gender.
We found a significant association between high mitotic rate and high SPF, and we suggest that SPF measured by FCM from paraffin blocks can be used to predict the aggressiveness of cutaneous malignant melanoma. However, intratumour heterogeneity of DNA ploidy and SPF may interfere with the results when only one tumour sample is analysed (Lipponen et al, 1991;Bergers et al, 1996).
Recently, Kirkwood et al (1996) reported promising results in treating high-risk resected melanoma patients with adjuvant interferon alfa-2b (IFNx-2b). As adjuvant treatments can be toxic and the overall benefits with node-negative patients can be relatively modest, a question arises: are there any subsets of high-risk nodenegative patients who would benefit from such treatments? In our study, the combinations of MAI with bleeding, MAI with SPF and M/V index with tumour thickness are variables that add significant information to the normal clinical data set available.
To conclude, MAI and MN index are strong predictors of the overall and recurrence-free survival of stage I cutaneous malignant melanoma patients in our material. One advantage of the mitotic indices compared with SPF is that no special equipment is needed.  (11), [1917][1918][1919][1920][1921][1922][1923][1924][1925] However, if available, SPF is also a strong independent prognosticator in stage I cutaneous malignant melanoma. We suggest that tumour proliferation assessed by mitotic rate or SPF, together with conventionally available prognosticators, might be considered as a patient inclusion criteria for further adjuvant treatment trials in node-negative cutaneous malignant melanoma patients.