CD68+ and CD163+ macrophages were significantly more abundant in malignant lesions compared with benign nevi (P<0.001) (Fig. 3). CD68+ macrophage number was also higher in deep melanomas (Breslow’s depth >4 mm) and in LNMs compared with dysplastic nevi or in-situ melanomas (P<0.001). Similarly, the number of CD163+ macrophages was higher in LNMs compared with dysplastic nevi (P=0.030).
TAMs were also evaluated from invasive melanomas and LNMs separately from tumor nests and stroma using a semiquantitative method. The proportions of CD68+ and CD163+ macrophages in tumor nests were higher in deep melanomas and LNMs compared with thin melanomas (P<0.001 for CD68 and 0.001 for CD163). There were no statistically significant differences in the stromal macrophage content between the malignant lesions (Fig. 4a−d).
Correlation of tumor-associated macrophages with clinicopathological parameters
Using hotspot analysis, high CD68+ macrophage number correlated positively with ulceration (P=0.006), recurrence rate (P=0.040), and distal recurrence (P=0.002). Males had higher amounts of CD68+ TAMs more often than females (P=0.004) (Table 2). Using semiquantitative analysis, high amounts of CD68+ macrophages in tumor nests correlated positively with the recurrence rate (P=0.006) and also separately with locoregional (P=0.021) and distal recurrence (P=0.001) (Supplemental digital content 4, http://links.lww.com/MR/A85, Table showing associations of CD68+ macrophage proportions in tumor cell nests and stroma, analyzed by the semiquantitative method, to clinicopathological parameters).
Low CD163+ macrophage proportion in stroma, analyzed by the semiquantitative method, was associated with the aggressive nodular growth pattern (P=0.037), the presence of microsatellites (P=0.038), recurrence rate (P=0.005), and locoregional recurrence (P=0.035). Males had low stromal CD163+ macrophage proportions more often than females (P=0.034) (Supplemental digital content 5, http://links.lww.com/MR/A86, Table showing associations of CD163+ macrophage proportions in tumor cell nests and stroma, analyzed by a semiquantitative method, to clinicopathological parameters).
Low stromal CD163+ macrophage proportion correlates with decreased survival in primary melanomas
During the follow-up, 49 patients suffered a relapse. At the end of the follow-up, 37 patients were alive and 61 were deceased. In the univariate survival analysis carried out for the whole patient group, high CD68+ macrophage count analyzed by hotspot analysis correlated with decreased disease-specific survival (DSS) (P=0.032). High CD68+ macrophage proportion in tumor nests was associated with poor DSS and poor **relapse-free survival (RFS) (P=0.016 and 0.005, respectively), whereas low CD163+ macrophage proportion in stroma correlated with poor RFS (P=0.010). TAMs did not correlate with prognosis in pT1 and pT4 groups only.
Multivariate survival analyses were carried out separately for all malignant lesions (pT1, pT4, and pN1) and for primary melanomas only (pT1 and pT4). For all malignant lesions, T and N classification (pT1, pT4, pN1) was used as a covariate. TAMs had no independent prognostic significance in the multivariate analysis carried out for the whole patient group.
For primary melanomas, the covariates used were Breslow’s depth, ulceration, and stromal CD163+ macrophage proportion. Significant factors correlating with OS were Breslow’s depth [P=0.006, hazard ratio (HR): 1.063, 95% confidence interval (CI): 1.018−1.111], ulceration (P=0.021, HR: 3.020, 95% CI: 1.177−1.747), and low stromal CD163+ macrophage proportion (P=0.025, HR: 0.392, 95% CI: 0.173−0.890). Factors associated with DSS were Breslow’s depth (P=0.012, HR: 1.068, 95% CI: 1.015−1.123) and low stromal CD163+ macrophage proportion (P=0.017, HR: 0.153, 95% CI: 0.033−0.715), whereas ulceration (P=0.013, HR: 5.143, 95% CI: 1.419−18.642) and low stromal CD163+ macrophage proportion (P=0.014, HR: 0.171, 95% CI: 0.042−0.696) were associated with RFS (Fig. 4f−h).
In the present study, both CD68+ and CD163+ TAMs were significantly more abundant in malignant melanocytic lesions compared with benign nevi. Assessment of TAMs separately from different tumor compartments showed that the number of TAMs were also higher in the tumor cell nests of deeply invasive melanomas compared with thin melanomas. High CD68+ macrophage content in the tumor cell nests and low CD163+ macrophage proportion in the stroma were associated with recurrence. These findings indicate that the presence of TAMs is associated with tumor progression in CM, but the role of TAMs seems to depend on their microanatomical localization in the tumor.
Although only a few dysplastic nevi act as a precursor for melanoma, they are biological intermediates between common nevi and melanoma and can provide an insight into melanoma tumorigenesis 16. In this study, we did not find a significant difference in the CD68+ macrophage content between the benign and dysplastic nevi and in-situ melanomas. However, the numbers of CD163+ TAMs were higher in dysplastic compared with benign nevi, which may indicate an association between immunosuppression and cell dysplasia. Significantly higher amounts of both CD68+ and CD163+ TAMs in malignant compared with benign melanocytic lesions suggest that TAMs promote tumorigenesis.
This study has shown that TAMs in tumor cell nests are more abundant in deeply invasive compared with superficially spreading melanomas. This is logical as TAMs are known to stimulate tumor invasion and angiogenesis by secreting factors such as matrix metalloproteinases, urokinase-type plasminogen activator, vascular endothelial growth factor, and adrenomedullin 5. The present finding, that the number of TAMs are elevated in more advanced tumors, is in line with a previous study showing that melanomas in the vertical growth phase contain more peritumoral and intratumoral CD68+ TAMs compared with melanomas in the radial growth phase 17. It has also been reported that intratumoral CD68+ and CD163+ macrophages are more abundant in metastatic compared with nonmetastatic melanomas 18, and some studies have shown a correlation between macrophage number and Breslow’s depth, tumor stage, and poor prognosis 11,19–21. Interestingly, there are also studies that failed to find a correlation between the macrophage content and tumor stage or survival 12–14. These controversial findings may be partly because of variable compositions of the study materials in terms of the melanocytic lesions involved. In addition, the different assessment methods may have led to variations in the results. To reduce the source of error produced by the assessment method, we used both the hotspot analysis and a semiquantitative method, which yielded comparable results.
According to our results, high CD68+ macrophage number in tumor cell nests correlates positively with both locoregional and distal recurrence. In line with our finding, Tham et al.22 reported that macrophage depletion reduced postsurgical tumor recurrence and metastatic growth in a murine melanoma model. They showed that macrophage density was associated with the growth of postsurgical tumor and metastases and suggested that surgical excision combined with macrophage depletion might be an effective approach to reduce postoperative tumor recurrence 22. Our results support this finding as CD68+ TAMs seem to be associated positively with recurrence also in human CM.
Recently, Falleni et al.23 reported that CD68+/MRP18-4+ M1 macrophage and CD68+/CD163+ M2 macrophage density were associated inversely with poor prognostic factors and patient survival independent of their intratumoral distribution. These findings differ from our results showing that low stromal CD163+ macrophage proportions predict poor OS, DSS, and RFS in primary melanomas. This discrepancy may be partly explained by the different compositions of the study materials. Falleni et al.23 used melanomas representing Breslow depths 1.01 to less than or equal to 4 mm (53 cases) in addition to melanomas with Breslow depths less than 1 mm (34 cases) and more than 4 mm (7 cases). Our material contained 36 cases of superficial melanomas (<1 mm invasive) and 42 cases of deep melanomas (>4 mm invasive). Thus, melanomas from Breslow depths 1.01 to less than or equal to 4 mm were absent in our material. At present, we can only speculate on the relevance of these differences, which warrants further studies.
In line with our findings, Algars et al.24 have reported that the prognostic significance of TAMs depends on their microanatomical location and tumor stage in colorectal carcinoma. Although we found low stromal CD163+ macrophage numbers correlating with worse outcome in CM, it is not likely that the effect is direct. This may be related to diminutive immune responses against the tumor, or can indicate Th1-polarization of the TME and the dominance of pro-inflammatory M1 macrophages. Earlier reports show that a high density of stromal M1 macrophages in melanoma correlates positively with Breslow’s thickness and Clark level 23. Thus, it is possible that low CD163+ macrophage density in the stroma is associated with abundance of M1 macrophages, which correlates with advanced tumor stage in CM. This hypothesis could also offer an explanation for our results showing low stromal CD163+ macrophage numbers that correlate with decreased survival in CM.
Ulceration and male sex are well-known poor prognostic factors in CM, but the underlying biology is poorly understood 25,26. According to this study, high CD68+ TAM number is associated with both ulceration and male sex. All ulcerated tumors (23 cases) were greater than 4 mm thick melanomas. Nonulcerated tumors included 33 cases of less than 1 mm thick and 12 cases of greater than 4 mm melanomas. Ulcerated tumors contained higher amounts of CD68+ TAMS than non-ulcerated tumors in both groups of all primary melanomas (pT1 and pT4) and of pT4 melanomas only (data not shown). Interestingly, it is known that cutaneous wounds heal slower in elderly men than women 27, and Lai et al.28 found that androgen receptor (AR) expression on macrophages suppresses wound healing. Thus, mechanisms explaining the association of TAMs with ulceration and sex disparities may include macrophage AR expression. In CM, AR expression has been shown to promote metastasis and is associated positively with a poor prognosis 29.
The abundance of melanin pigment has been shown to correlate inversely with the OS and DFS in patients with stage III and IV melanoma 30. Melanogenesis can affect melanoma progression by regulating epidermal homeostasis, for example, by enhancing immunosuppression and creating mutagenic TME 31. However, it has been shown that melanin granules inhibit melanoma cells migration in vitro32. Although the role of melanin pigmentation and melanogenesis in CM is not fully established, tumor melanin content should be considered while interpreting the present results. In our study, CD163+ TAMs were analyzed only from melanin-negative tumors, whereas analyses of CD68+ TAMs also included melanin-positive tumors. To ensure comparability between the results of CD68+ and CD163+ TAMs, the statistical analyses of CD68+ TAMs were repeated separately for melanin-negative tumors only (data not shown). All the reported results were found in this group also. In addition, some new correlations were discovered, and they were in line with the reported results, which indicates that the present results of CD68+ and CD163+ TAMs are comparable. The only exception in the results of the melanin-negative group, compared with the reported results, was the inverse correlation of high CD68+ TAM number with sentinel LNM.
In conclusion, high numbers of TAMs, especially in the tumor cell nests, seem to enhance CM progression. As a highly versatile cell type, TAMs are known to play distinct roles in different TMEs 33. In particular, the role of anti-inflammatory M2 macrophages still remains controversial, and more detailed in-vivo and in-vitro studies are needed to clarify the effects of macrophage subpopulations on CM. However, because of the association of CD68+ TAMs with CM progression and tumor recurrence, TAMs seem to present a plausible target for therapies in the future.
The authors thank Eija Rahunen for her excellent technical assistance and Andrey Bykachev for the collection of clinicopathological data. Financial support for this work was provided by the Sigrid Juselius Foundation (S.P.-S.), Special Government Funding of Kuopio University Hospital (K.T.-K.), The Northern Savo Cancer Fund, and The Paavo Koistinen Foundation (S.S.). The sponsors had no involvement in study design.
S.S. analyzed samples with the hot spot and the semiquantitative method, carried out the statistical analyses, and wrote the manuscript. H.S. participated in designing the study protocol and revised and commented on manuscript. R.S. provided the study material, analyzed the standard histopathological parameters, and commented on the manuscript. K.T-K. commented on the manuscript. B.H-G. analyzed the samples with the hot spot method. M.V. collected the clinicopathological data. P.A. designed the study protocol, helped with statistical analyses, and commented on the manuscript. S.P-S. designed the study protocol and revised and commented on the manuscript.
Conflicts of interest
There are no conflicts of interest.
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CD163; CD68; macrophages; melanoma; tumor associated macrophages
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