The prognostic value of tumor mitotic rate in children and adolescents with cutaneous melanoma: a retrospective cohort study

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Melanoma is the most common skin cancer in children and adolescents. 1Still, \1% of all melanomas occur in patients \20 years of age. 2 Because of its rarity, the published literature on melanoma in children and adolescents is sparse and treatment is primarily based on adult guidelines.
][5][6][7] Evidence suggests that the mitotic rate is lower in melanomas occurring in children and adolescents than in other age groups. 8][15][16][17][18][19][20] The purpose of this study was to assess the prognostic significance of mitotic rate in clinically localized primary cutaneous melanoma in children and adolescents.Secondary aims were to report the clinicopathologic features in a large cohort of melanoma patients \20 years of age, to compare children with adolescent patients, and to assess the relationship between mitotic rate and tumor thickness in this age group.

Patients
The prospectively collected database of Melanoma Institute Australia (MIA) was queried for this retrospective cohort study.Between 1993 and 2013, 259 melanoma patients \20 years of age were managed at MIA.To be included in the current study, a diagnosis of primary cutaneous melanoma had to have been confirmed by $1 MIA-affiliated pathologists.Borderline lesions, such as atypical Spitz nevi/ tumors, melanocytomas, or atypical melanocytic proliferations, were excluded after pathology review (n = 27).Patients were also excluded if they had melanoma in situ (n = 34), a metastasis from an unknown primary melanoma (n = 5), multiple primary melanomas (n = 5), mucosal melanoma (n = 1), macrometastasis at diagnosis (n = 4), or if an MIA-affiliated pathologist could not review the pathology slides (n = 27).One hundred fifty-six patients fulfilled the inclusion criteria.Institutional review board approval was obtained (Sydney South West Area Health Service institutional ethics review committee protocol no.X15-0454).

Data collection
Patients who present to MIA for management of their melanoma after a diagnosis has been established have their pathology slides reviewed by $1 MIAaffiliated pathologists at the Royal Prince Alfred Hospital in Sydney, Australia.The primary tumor pathologic characteristics are assessed and recorded in a second pathology report (the ''MIA pathology report'') and the histopathology slides are returned to the source pathology laboratory.The data used in this study were extracted from MIA pathology reports.In cases with missing data and when the histopathology slides were still available, the cases were rereviewed any missing data were recorded.
Data on demographics, primary tumor characteristics, sentinel node (SN) status, recurrence, treatment, and follow-up were obtained.Patients were stratified by age into 2 groups: \12 years of age (children) and 12 to 19 years of age (adolescents).Twelve years of age was selected to represent the onset of puberty. 21

Mitotic rate
Tumor mitotic rate was measured according to the recommendations of the 1982 International Pathology Workshop. 22Mitoses were recognized by the presence of extensions of chromatin extending from a condensed chromatin mass.The number of mitoses was counted in a 1-mm 2 area (approximately 5 high power fields).The count started in the dermal area of the tumor with the greatest density of mitoses (the ''hot spot'') and continued in immediately adjacent, nonoverlapping fields. 22,23

Statistical analysis
Baseline characteristics were summarized using median (interquartile range) for continuous variables and proportions for categorical variables.Characteristics of childhood and adolescent patients were compared using the  Tumor mitotic rate should be assessed and reported in all childhood and adolescent cases of melanoma to aid prognostic stratification and treatment planning.survival (MSS) was calculated as the time from initial diagnosis until melanoma-related death.Patients who died from nonmelanoma causes or those still alive at last follow-up were censored.Recurrencefree survival (RFS) was defined as the time from diagnosis until recurrence or death.Censoring occurred at the end of follow-up.Univariable and multivariable analyses using Cox proportional hazard models were used to assess the prognostic value of covariates for RFS and MSS.Mitotic rate was the variable of interest in this study.Other known prognostic factors in adult melanoma, such as gender, age, primary tumor site, Breslow thickness, ulceration, and SN status were investigated in a univariable analysis. 5,8,24,25Given the number of patients who developed recurrence (n = 28), only the 2 covariates with P \ .20 from the univariable analysis and with \10% missing values were included in the multivariable model.The proportional hazards assumption was checked for the included variables.
P values were 2-sided and P \.05 was considered statistically significant.Statistical analyses were performed with SPSS software (version 25; IBM SPSS, Chicago, IL).

Patient and tumor characteristics
Baseline characteristics of the 156 patients are shown in Table I.The median age was 17.5 years (range 1-19 years).Thirteen patients (8%) were children at the time of diagnosis, while 143 (92%) were adolescents.Melanomas were most often thin (median Breslow thickness 1.0 mm), nonulcerated (65%), and located on the trunk (34%).The mitotic rate was $1/mm 2 in 104 patients (67%) and correlated with increasing Breslow thickness (Fig 1).
Sentinel node biopsy (SNB) specimens were obtained in 61 patients, with 23 (38%) having a positive SN.Of the 77 patients with tumors [1 mm thick, 48 (62%) underwent SNB.Nineteen SNpositive patients (83%) underwent completion lymph node dissection.Additional nodal metastases were found in 4 of these patients (21%).None of the 4 SN-positive patients who did not have completion lymph node dissection developed a recurrence.

Recurrence and survival
The median follow-up time was 61 months (interquartile range 10-111 months).Melanoma recurrence occurred in 28 patients (18%), and 16 patients (10%) died.Regional lymph nodes were the most common site of first recurrence (19 patients), while 5 patients had their first recurrence at a distant site.All patients whose first recurrence was in a regional node had a negative SN.The time between diagnosis of the primary melanoma and first recurrence ranged from 3 months to 13 years.Five patients (31%) had a recurrence after [5 years.MSS at 5 years was 91% (95% confidence interval [CI] 86-96%) and 10-year MSS was 88% (95% CI 81-95%).Five-year RFS was 84% (95% CI 77-90%) and 10-year RFS was 77% (95% CI 67-86%).Table II shows the characteristics of the 16 patients who died.One patient was 10 years old when her melanoma was diagnosed, while the other patients were adolescents.MSS and RFS were not significantly different between the 2 age groups (P = .83and P = .54,respectively).Mitoses were present in the primary melanomas of 14 patients (88%) and 2 patients (13%) had melanomas with a Breslow thickness \1 mm.Ten patients received chemotherapy, while 3 patients received targeted therapy or immunotherapy.Ipenburg et al 913

Prognostic factors
On univariable analysis, Breslow thickness (P = .001),mitotic rate (P \ .001),and melanoma subtype (P = .04)were found to be significantly associated with RFS.Gender, age, ulceration, primary tumor site, and SN status were not significantly associated with RFS. Figure 2 shows the RFS curves according to mitotic rate.On multivariable analysis including mitotic rate and Breslow thickness, mitotic rate correlated independently with RFS (hazard ratio = 1.2 [95% CI 1.1-1.3]),while Breslow thickness did not (HR = 1.1 [95% CI 0.9-1.2]).The univariable analysis indicated a significantly increased risk of melanoma-related death with increasing mitotic rate (P = .001).The other covariates were not significantly associated with MSS (Table III).Multivariable analysis could not be performed for MSS because of an insufficient number of events (16 melanoma-related deaths).Supplemental Table I (available online at DOI 10.17632/s7f9jsz9yj.1)shows the univariable and multivariable analysis of RFS and MSS of adolescents.

DISCUSSION
This single institution cohort study shows that tumor mitotic rate is the most important independent prognostic factor for RFS in children and adolescents with clinically localized melanoma, with a marginally stronger influence than tumor thickness.Having accurate information about the mitotic rate of the primary melanoma could improve prognostic stratification and treatment planning for individual patients in these age groups.It is important that this parameter is evaluated and recorded in all melanoma pathology reports.
4][5][6][7] Although mitotic rate was an essential part of the 7th edition of the American Joint Committee on Cancer melanoma staging system, it has been scarcely studied in childhood and adolescent melanoma. 25The rarity of melanoma in these patients, with an annual incidence rate of around 5 per million individuals, is probably one of the main reasons for the lack of studies. 26Larger childhood and adolescent melanoma studies generally use data from the National Cancer Database or the Surveillance, Epidemiology, and End Results database. 2,13,15Although valuable, these databases have several limitations.For instance, central pathology review is lacking, recurrence rates are not available, and details of key tumor characteristics such as Breslow thickness, ulceration and mitotic rate are frequently necessary.
Breslow thickness is the strongest prognostic feature in primary cutaneous melanoma in adult patients. 27Interestingly, Breslow thickness was not a significant predictor for melanoma-specific survival in our study of childhood and adolescent patients.A similar finding was also reported in a study based on the National Cancer Database. 15Another large multicenter study showed that primary tumor site and gender were independent prognostic factors for MSS, while mitotic rate and Breslow thickness were not. 8However, 2 previous studies did show that Breslow thickness was an independent predictor of recurrence. 12,28On univariable analysis, MSS was significantly worse with increasing mitotic rate.Unfortunately, multivariable analysis could not be performed for MSS because of an insufficient number of events (16 melanoma-related deaths). 29In line with our results, 3 previous melanoma studies in young patients showed that the presence of mitoses was associated with an increased risk of metastasis on univariable  analysis.However, when adjusted for other prognostic factors, this association was not seen, possibly because of the small sample sizes or the number of missing values in these studies. 10,12,28No significant effect on overall survival has been found. 9,1113]18 The primary tumor location was also different for the 2 groups, with head and neck sites being more common in children and the trunk being the most frequent location in adolescents. 13,15atients with melanoma who are in their late teens are sometimes inappropriately classified as children.Our results confirm that melanoma behaves differently in children and adolescents but MSS and RFS were similar.In contrast, a previous study reported better survival for children. 30This may reflect the fact that cases reported as borderline tumors, such as atypical Spitz tumors, were specifically excluded in our study, whereas these may have been classified as melanoma in other studies. 31etastatic disease was identified in 38% of the patients who underwent SNB in our study.3][34] Contrary to previous studies, RFS and MSS were not significantly different for SNpositive and SN-negative patients in our study. 14,18,20aradoxically, young patients have a higher incidence of SN metastasis but a more favorable survival than adults. 8,13,32The reasons for this remain unclear but superior function of the immune system in younger patients has been proposed as a possible explanation. 33In childhood and adolescence, melanomas frequently resemble benign lesions, which makes them hard to diagnose both clinically and pathologically. 34Almost 50% of the melanomas in young adults do not fulfill the classic melanoma ABCD criteria. 35ecent genomic analysis showed that melanomas in adolescents and young adults harbor mutation patterns that differ from those in older patients. 36ive-year MSS was 91% in our study and 5-year RFS was 84%.Several previous studies reported comparable survival rates with 5-year MSS ranging from 89% to 97% and 5-year RFS ranging from 68% to 90%. 9,11,18,37,38Of the 15 patients who died of melanoma and in whom mitotic rate was assessed, 10 had a tumor mitotic rate of \6/mm 2 .Five of 28 patients with recurrence (31%) experienced that recurrence after [5 years.As in adults, children and adolescents remain at risk of recurrence even after $10 years. 20,39Childhood and adolescent patients are also twice as likely to develop a subsequent melanoma compared with adult patients. 40his emphasizes the importance of continuing follow-up of patients who develop melanoma when they are young for longer than the usual 5year period recommended in the melanoma management guidelines of some countries. 41he strengths of our study include the relatively large cohort of patients.In addition, pathology slides of all patients were reviewed by experienced pathologists, increasing the reliability of the diagnosis and of histologic and staging data.There are also several limitations affecting the study.Because of the moderate number of events, multivariable analysis could not be performed for MSS and only mitotic rate and Breslow thickness could be included in the multivariable analysis for RFS.Supplemental Table II (available online available at DOI 10.17632/ s7f9jsz9yj.1)shows the unstable multivariable analysis of RFS and MSS including Breslow thickness, mitotic rate, and ulceration.Although all cases were reviewed by an MIA-affiliated pathologist, some histologic parameters were missing.The pathology slides of some patients were not available for reassessment.Other limitations are the retrospective design, the arbitrary age cutoff that was used to separate children and adolescents, referral bias, and the short follow-up of some patients.
In summary, our study indicates that mitotic rate is an important prognostic feature for RFS in children and adolescents who develop melanoma, and it is therefore essential that this parameter be assessed and reported in the primary tumors of all young melanoma patients.Although mitotic rate was the only independent predictor of RFS, a larger study is required to confirm these results.By extrapolating the number of recurrences in our study, approximately 500 children and adolescent patients would be needed to assess the prognostic value of the other prognostic factors that are common in adults.A collaborative study involving multiple melanoma centers would be needed. d Abbreviations used:CI:confidence interval HR: hazard ratio MIA: Melanoma Institute Australia MSS: melanoma-specific survival RFS: recurrence-free survival SN:sentinel node SNB: sentinel node biopsy

Fig 2 .
Fig 2. Recurrence-free survival of patients with melanoma according to mitotic rate.
Pearson x 2 or Fisher exact test for categorical features and the ManneWhitney U test for continuous variables.Melanoma-specific

Table I .
Clinicopathologic characteristics *Comparison of children and adolescent patients.

Table II .
Characteristics of patients who died of melanoma

Table III .
Univariable and multivariable analysis of recurrence-free survival and melanoma-specific survival CI, Confidence interval; HR, hazard ratio.