Exploring the Prognostic Role of Ki67 Proliferative Index in Merkel Cell Carcinoma of the Skin: Clinico-Pathologic Analysis of 84 Cases and Review of the Literature

Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma of the skin, but its incidence has largely increased over the last 20 years, probably depending on the increasing effects of risk factors such as advanced age, UV exposure, and systemic immunosuppression [1]. The reported annual incidence varies between 0.1 and 0.88 per 100,000 people, with differences among geographical regions: higher rates have been observed in Australia and New Zealand and the lowest in Eastern France and Scotland [2]. MCC is more frequently observed in elderly white male patients (mean age at diagnosis of 73.6 years), although younger patients, mostly if immunosuppressed due to organ transplantation, can also be affected. MCC incidence is also increased in patients with other malignancies including melanoma, multiple myeloma, and non-Hodgkin lymphoma, especially chronic lymphocytic leukemia [2].

MCC is an aggressive cancer, and several attempts have been made to identify clinico-pathologic factors useful to stratify patients in different prognostic categories. From an etiological point of view, MCC can be divided into two main groups with apparent different behaviors on the basis of the presence or absence of an associated infection by Merkel cell polyomavirus (MCPyV). MCC unassociated with MCPyV infection seems to show a worse prognosis [3]. However, although several prognostic markers have been proposed, the exact prediction of the individual outcome remains difficult to be determined.

Ki67 proliferative index is a well-known prognostic marker for both well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs) of the digestive system, and is currently routinely evaluated in their diagnostic work-up [4]. Although its prognostic value has also been recognized in other neuroendocrine neoplasms (NENs), such as those located in the pituitary, parathyroid, and lung [5‐8], Ki67 proliferative index has been poorly investigated in cutaneous MCC; thus, its role remains to be clarified.

In the present retrospective study, we explored the prognostic role of Ki67 proliferative index in a large series of MCCs integrating our results with literature findings. In addition, we investigated other potential clinico-pathologic prognosticators, with the aim to identify useful parameters to stratify MCC patients in different risk categories.

MCC of the skin is a rare neuroendocrine carcinoma with a reported 5-year overall survival (OS) ranging from 30 to 60% [12‐14], and our findings are in line with this, showing a 5-year OS of 52%, worse than the 5-year DSS (66%). This probably reflects the characteristics of our cohort, where most of patients were elderly (mean age 76 years) with other comorbidities and fatal events for unrelated causes. A similar feature was observed by Jemec et al., whose series of MCCs showed an OS worse than DSS after a mean follow-up time of 68.2 months [15]. Several different clinico-pathological factors negatively influencing patients’ survival have been proposed for cutaneous MCC and included age, male gender, clinical evidence of lymph node metastasis, depth of tumor invasion, infiltrative growth pattern, loss of RB function, lack of MCPyV immunoreactivity, and p63 expression [16‐19]. More recently, promoter methylation of the immune checkpoint receptor CD279 (PD-1) and intratumor lymphocyte subtypes have been suggested as additional prognostic factors [20, 21].

Ki67 proliferative index represents one of the most important biological markers routinely used as prognosticators in NENs. Its role was first demonstrated in pancreatic NETs [22, 23] and then in other digestive NETs [4] as well as in NETs of other sites (e.g., in the pituitary gland) [5, 6]. Recent data also suggest a prognostic role of Ki67 proliferative index in lung NENs and in non-epithelial NENs, like olfactory neuroblastoma [8, 24]. In addition to NETs, Ki67 proliferative index also plays a prognostic role in NECs, as observed in the digestive system; indeed, NECs with a Ki67 index > 55% show worse prognosis than NECs with Ki67 index < 55%, despite a better initial response of the former to chemotherapy [10, 11]. For all these reasons, the evaluation of Ki67 is a required (digestive system) or strongly recommended (other sites) parameter in the diagnostic work-up of NENs.

Despite the large available literature on the prognostic role of Ki67 index in NENs, the available data on MCC are heterogeneous, mainly due to the different methods used to assess Ki67 proliferative index and the relatively small number of cases studied in each series. Although some studies suggested an association between Ki67 index and a more aggressive clinical behavior [19, 25, 26], others did not confirm this data [15, 27‐32] (Table 4). Although Ki67 is a continuous variable, the current stratification of patients is generally related to specific Ki67 cut-offs, which are able to separate patients in different prognostic categories. However, standardized method of evaluation such as that proposed by the WHO [4, 9] is mandatory. Since MCC may be considered as the cutaneous counterpart of visceral NECs and since no specific prognostic Ki67 cut-off has been identified for MCC, we decided to use the only available and efficient Ki67 threshold (55%), although it refers to digestive NECs [10, 11]. This choice was also supported by the Youden index analysis, which identified best cut-point Ki67 values at 52.5% and 57.5%. We observed an association between Ki67 proliferative index higher than 55% and tumor recurrence, in line with other published findings [27, 29, 35, 36, 39]. In addition, we demonstrated, at univariate analysis, the relationship between high Ki67 proliferative index and DSS, a finding not confirmed by previous studies (Table 4). Indeed, in three studies, only the association between Ki67 proliferative index and OS, but not DSS, was observed [19, 25, 26]. These discrepancies may be due to the different methods used to evaluate the Ki67 proliferative index and to the different cut-offs established for the survival analysis. Regarding the first point, we are the first to use the WHO recommendations by counting the number of positive cells in at least 500 tumor cells in hot spot areas on camera-captured printed images [4, 9]. As discussed above, the choice of 55% Ki67 cut-off was based on the analogy with digestive NECs and on the Youden index analysis. Our results on MCCs are in line with those observed in digestive NECs that showed different survivals, when separated in two groups based on Ki67 proliferative index (< 55% vs > 55%), [11]. However, although Ki67 proliferative index resulted significantly associated with prognosis at univariate analysis, together with tumor stage IV, MCPyV lack, and p63 expression [16‐19], it was not an independent predictor at the multivariate analysis, where survival resulted independently influenced by p63 expression and tumor stage, only.

The prognostic role of p63 expression is in line with previous findings [19], and it may be related to its function as tumor suppressor as demonstrated in p63 knockout mice [43]. However, it is worth noting that different p63 isoforms exist and may have distinct effects on oncogenesis. Since the anti-p63 antibody used in this study does not distinguish among different isoforms, we are not able to identify the p63 isoform having the major pathogenetic role. Asioli et al. have demonstrated that TAp63a and DNp63a were the most frequently expressed isoforms (42%) in low-stage MCCs, suggesting that an early anomalous regulation of their expression might determine an aggressive phenotype [44].

In contrast to proliferation markers, a possible prognostic role of factors influencing apoptosis has been poorly investigated in MCCs. Indeed, although apoptotic cells were detected in MCCs using TUNEL, DNA ladder and immunohistochemistry for Fas (Apo-1/CD95) [45], the prognostic role of apoptotic index and of proteins and mediators involved in the apoptotic machinery (Bcl-2, p53, surviving, and CXCR4) remains to be elucidated [30].

In conclusion, this study, which integrates original findings with literature data, is the first one analyzing the prognostic role of Ki67 proliferative index in MCC using a standardized methods of Ki67 evaluation. Nevertheless, although Ki67 proliferative index was associated with recurrent disease and shorter patients’ survival at univariate analysis, it did not prove to independently influence prognosis, which in our relatively large series mainly depended on high tumor stage and p63 expression.