In the context of recognized focality of HMB45 immunostains and the association of decreased HMB45 and Melan-A stain in deeper dermal regions of primary melanomas, we cannot rule out some degree of measurement error that might increase our false-negative rate through our strategy of having sampled only 1 1 single 0.6mm histospot from each index lesion. observed in 160 (49.7%) melanomas. Intensity of endogenous melanin pigment did not confound immunolabeling. Among primaries, associations with clinicopathologic parameters revealed a significant relationship only between HMB45 and microsatellitosis (with 5/7 of these lesions arising from the back or shoulder 32. Interestingly, 5/6 of our metastatic triple negatives arose Cyproheptadine hydrochloride in cutaneous or subcutaneous soft tissue however our lesions derived from a more diverse set of primary tumor locations including the face, leg, and abdomen as well as the back. Application of next-generation sequencing to the exomes of S100-negative melanomas might be useful for identifying underlying molecular changes associated with both overall lack of S100 antigenicity and concordant lack of S100, HMB45 and Melan-A/MART-1 immunoreactivity. While the marginal distributions for S100, HMB45 and Melan-A positivity and their associations with recognized melanoma clinicopathologic parameters are well-established 9, the literature describing their joint distributions is much more sparse. While MAA expression discordance can be expected in light of the well-documented differing sensitivities of the commonly used MAAs 9, there are very few published studies that describe the prevalence and clinical significance of MAA-concordant and discordant lesions. The largest of these studies evaluated overlapping HMB45/Melan-A immunoreactivity Cyproheptadine hydrochloride Cyproheptadine hydrochloride in 65 melanoma metastases and 10 cutaneous primaries 14. While the authors explored the distribution across all pair-wise antigen combinations, correlations with clinicopathologic criteria other than histologic subtype were not reported 14. Among their sample of 30 melanomas, Xu et al. observed some HMB45/Melan-A discordance with positive Melan-A expression occurring only in 9/14 S100+/HMB45- spindled or epithelioid melanomas 15. In their study of 17 S100-negative melanomas, Aisner et al. noted only concordance between HMB45 and Melan-A expression 32. To the best of our knowledge, our study of 322 assayable melanomas represents the largest series to date for which the joint distribution of HMB45 and Melan-A are considered and the only study to consider the relationship between joint HMB45/Melan-A expression and commonly reported clinicopathologic criteria among eligible primary lesions. The majority of our lesions were concordant for HMB45 and Melan-A expression with 53.4% expressing both antigens and 17.1% lacking expression in 25% of the arrayed melanoma. We also did observe melanomas discordant for MAA expression with 31 (9.6%) melanomas expressing HMB45 only and 64 (19.9%) melanomas expressing Melan-A only. Further bivariate analyses among the 121 assayed primary melanomas revealed no significant associations with any of the recognized clinicopathologic criteria and survival analyses revealed virtually overlapping survival curves across the 4 joint distribution categories to suggest that the prognostic impact MAA discordance may be small. While the significantly larger (P<.001) number of discordant lesions that express only Melan-A can be explained by Melan-A's recognized comparatively more diffuse and intense staining of melanomas that persists into the dermal layers of the assayed lesions 14,41 that possibly produced fewer false negatives among our sample of F3 representative 0.6 mm histospots, we cannot exclude the possible role for genetic or epigenetic factors that underlie the development and distribution of the 4 classes of HMB45/Melan-A expression-defined melanomas and possible relationships with levels of MITF expression 42 may yield compelling insight into mechanisms relevant for melanocytic lesion development and progression. While our study includes numerous strengths such as our large sample size, use of TMAs and automated image capture that eliminate the potential for laboratory drift by assaying all samples simultaneously in the same experimental batch and comprehensive specimen annotation to enable robust clinicopathologic associations, we also recognize several limitations with our experimental approach. First, although quantitative immunofluorescence is a well-established, unbiased measure of antigen expression across a broad spectrum of cancers 43,44, the presence of photo-reactive melanin might confound immunofluorescent readouts in melanoma 45. Not only does melanin exhibit broad spectral absorption that decreases monotonically with increasing wavelengths from 300-1100 nm 46,47, melanin has also recently been shown to display autofluorescence with separate excitations in the ranges of 370-470 nm and 785 nm and corresponding emissions at 540 nm and 890-900 nm, respectively 47,48 with the former in close proximity to the emission wavelength of our mask fluorophore (546 nm). While HMB45 expression was independent of melanin distribution with similar proportion of HMB45 negative lesions among all four categories of pigmentation, Melan-A immunostaining was associated with melanin levels where, despite similar proportions of Melan-A negative lesions observed in.
In the context of recognized focality of HMB45 immunostains and the association of decreased HMB45 and Melan-A stain in deeper dermal regions of primary melanomas, we cannot rule out some degree of measurement error that might increase our false-negative rate through our strategy of having sampled only 1 1 single 0
