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Marginal and joint distributions of S100, HMB-45, and Melan-A across a large series of cutaneous melanomas.

Cutaneous malignant melanoma, with an estimated 70230 new cases and 8790 deaths expected in the United States during 2011, (1) continues to be a major public health concern. In particular, localized melanomas greater than 1.00 mm in thickness (stage II), present a therapeutic challenge for clinicians, as their prognosis is uncertain. While 10-year melanoma-specific mortality among patients with stage II disease after a curative resection approaches 50%, (2) the adverse risk profile of available adjuvant chemotherapy supports its administration only among those with thick (>4.0 mm), ulcerated melanomas (stage IIC). (3) The need for melanoma prognostic models capable of identifying those patients with the highest risk of recurrence at the time of diagnosis is well established (4) and efforts to identify additional prognostic biomarkers are ongoing.

Melanoma-associated antigens (MAAs), owing to their high sensitivity for cells of melanocytic origin, are routinely used in the clinic to discriminate melanomas from other neoplastic lesions. (5) Of the MAAs most commonly used in the clinic, S100 shows greater sensitivity, staining approximately 95% of assayed lesions and, despite a specificity of 75% to 87%, is considered the gold standard for immunohistochemical discrimination of melanocytic cells. (6) Both S100 monoclonal and polyclonal antibodies stain primary and metastatic melanomas with equal efficiency, and labeling is noted in both the cytoplasm and nucleus of positively staining specimens. (7) Furthermore, among positively staining melanomas, S100 immunostain is more intense in melanomas with a higher Ki-67 fraction. (8) The observed reduced specificity results from S100 expression in tumors of glial origin as well as in chondrocytes, adipocytes, dendritic cells, and tumors derived from these tissues. (6,9,10) Consequently, when morphology and S100 staining do not completely inform the diagnosis, such as occurs in unusual morphologic variants of melanoma (11,12) or in the setting of melanoma mimics, (6) staining is conducted with additional MAAs such as gp100, Melan-A/MART-1, or tyrosinase. While the sensitivity of these antibodies rarely exceeds 90%, their specificity for melanoma is typically greater than 95%6,9 and contributes to the differential diagnosis.

Although the immunohistochemical evaluation of melanocytic lesions during the diagnostic process typically involves simultaneous assay of multiple MAAs on serial tumor sections, (13) and the marginal

distributions for S100, HMB-45, and Melan-A are well-established, (9) their joint distributions are only described for selected case series. The most robust of these data are reported by Jungbluth et al (14) who have noted overlapping HMB-45 and Melan-A immunoreactivity among 53 of 65 melanoma metastases and 7 of 10 cutaneous primary tumors; exclusive Melan-A staining in 8 of 65 metastases and 1 of 10 primary tumors; and nonreactivity with both reagents in 11 metastases and 2 primary tumors. While the authors (14) further commented that dual-negative lesions included the assayed spindle and desmoplastic variants, S100 immunoreactivity was not reported for this series. Xu et al (15) evaluated a series of 30 S100+ melanomas, reporting the presence of 8 HMB-45+/ Melan-A+, 9 HMB-45-/Melan-A+, and 13 HMB-45-/ Melan-A- lesions, the latter including 8 desmoplastic melanomas. Busam et al (16) reported Melan-A immunoreactivity for 26 of 26 S100+ epithelioid melanomas but only for 4 of 14 S100+/HMB45- spindled or desmoplastic melanomas. Kucher et al, (17) reporting on a retrospective series of 40 sentinel lymph node biopsy specimens positive for S100-immunoreactive melanoma, noted 34 of 40 as HMB-45+/ Melan-A+, 1 as HMB-45+/Melan-A-, and 5 as HMB-45-/ Melan-A-. While other groups have published articles in which the methods include immunostaining of multiple MAAs on serial sections of melanoma lesions, the reported results are limited to either the marginal distributions of the assayed antibodies or highlighting of certain expression categories (eg, negative for all assayed antibodies) and do not offer the full joint distributions of the assayed MAAs. (18-21) Taken together, while most melanomas express multiple MAAs and, in particular, are positive for both HMB-45 and Melan-A, there are still few data describing the prevalence and clinicopathologic correlates across joint MAA expression categories such as HMB-45/Melan-A-discordant lesions.

To address this gap, we characterized the joint distributions of S100, HMB-45, and Melan-A expression across a large series of 121 cutaneous melanoma primary tumors and 201 melanoma metastases. We define subcategories of melanomas according to their independent, individually considered (ie, "marginal"), and simultaneously combined (ie, "joint") distributions across the 3 assayed antigens; we also describe associations of these subcategories with established melanoma clinicopathologic criteria and, among primary melanomas, with melanoma-specific survival. We not only consider those melanomas discordant for HMB-45 and Melan-A expression, but also describe the clinicopathologic characteristics and HMB-45/Melan-A expression patterns for our sample of 27 S100-negative melanomas, one of the largest such series reported to date.


Patient Tumor Samples and Tissue Microarray Construction

Data from 3 nonoverlapping series of melanoma patients were included in the analysis (Yale Human Investigations Committee protocol No. 8219), which consisted of 212 primary and 237 metastatic cutaneous melanomas surgically removed at Yale-New Haven Hospital during 1959-1994, and 104 additional metastatic melanomas surgically removed during 1995-2005, for which the formalin-fixed, paraffin-embedded (FFPE) tissue blocks were not exhausted during the diagnostic process and for which clinical information was available. Demographic data, clinical course, and follow-up through March 1, 2011, were obtained through comprehensive review of the participant's medical record, the archives of the Connecticut Tumor Registry, the Social Security Death Index, and the State of Connecticut Vital Records. Incomplete medical records resulted in missing clinical and demographic annotations.

A tissue microarray (TMA) representing single 0.6-mm-diameter cores from each of the eligible specimens was constructed by using the standard method. (22) For internal quality control, 4 primary melanomas and 61 metastases were cored in duplicate. Duplicate cores of FFPE pellets constructed from each of 15 melanoma cell lines (23,24) were added as additional controls. Histopathologic annotation was conducted by pathologic review of included cases as previously described, (25) with incomplete data fields arising from cases with missing tissue blocks/slides, which prevented correct readjudication of the case. Assessment of melanin levels in each histospot was conducted by a single observer (W.R.B.) on a hematoxylin-eosin (H&E)-stained cut of the TMA and graded on a semiquantitative, 4-level scale with 0 representing no observable melanin and 3 indicating deep pigment in greater than 75% of the melanocytes.

Immunohistochemical Staining and Automated Image Capture

Two serial sections of the TMA were dewaxed in 2 exchanges of xylene and rehydrated with an ethanol gradient. Following antigen retrieval at supra-atmospheric pressure in boiling 6.5 mM sodium citrate (pH 6.0) for 10 minutes, endogenous peroxidase activity was blocked with 0.75% hydrogen peroxide, and nonspecific antigens were neutralized with 0.3% bovine serum albumin. Each of prediluted, neat HMB-45 or anti-Melan-A A103 mouse monoclonal antibodies (Biogenex, Fremont, California) were multiplexed with rabbit anti-S100 polyclonal antibody (1:200; Dako, Carpinteria, California), added to the TMA slide, and incubated overnight at 4[degrees]C. Secondary antibodies, AlexaFluor-546-conjugated goat anti-rabbit (1:100; Life Technologies, Carlsbad, California) diluted into anti-mouse Envision (neat, Dako), were then added for 1 hour at room temperature, followed by a 10-minute incubation with Cy5-tyramide (Perkin-Elmer Life Sciences, Waltham, Massachusetts) to label the anti-mouse Envision. Slides were coverslipped by using Prolong Gold with 4',6-diamidino-2-phenylindole (DAPI, Life Technologies), the latter to visualize nuclei. Negative controls were obtained through omission of the primary antibody.

Automated image acquisition was done as described previously. (23,26) Briefly, sets of monochromatic, high-resolution (1024 X 1024 pixel, 0.5 [micro]m) images were captured for each histospot in each of the DAPI, AlexaFluor-546, and Cy5 fluorescent channels by using a modified, computer-controlled epifluorescence microscope (Olympus BX-51 with xy stage and z controller, Olympus America Inc, Center Valley, Pennsylvania) illuminated by a high-pressure mercury bulb (Photonics Solutions, Edinburgh, United Kingdom) coupled with a high-resolution monochromatic camera (PCO-Tech, Romulus, Michigan).

Immunostaining Evaluation and Statistical Analysis

Histospots containing less than 3% of tumor tissue were excluded from further analysis. Photomicrographs representing S100, HMB-45, and Melan-A immunostaining were visually compared to a referent H&E section for melanoma-specific immunostaining by 2 independent observers (H.V., B.E.G.R.). A case was scored "positive" for the selected antigen if at least 25% of the tumor cells, as defined by the corresponding area on the referent H&E section, displayed immunofluorescence. For cases represented by more than 1 histospot, the case was designated as "positive" if all histospots demonstrated at least 25% immunoreactivity. Cases with immunofluorescence covering less than 25% of the tumor region were classified as "negative." Discrepancies in staining evaluation were resolved through consultation with a third investigator (K.A.S.). Marginal, as well as pairwise and 3-way joint distributions across the assayed melanoma antigens, were determined by using standard univariate statistics. Bivariate analyses comparing marginal and joint immunostaining patterns with individual clinicopathologic parameters were evaluated by using [chi square] analysis or analysis of variance, as appropriate. Associations with melanoma-specific survival were performed by using Kaplan-Meier product-limit and Cox proportional hazards survival analyses. All statistical analyses were conducted with the StatView statistical package (SAS Institute, Cary, North Carolina).


Marginal and Joint Distributions of S100, HMB-45, and Melan-A

To assess the marginal and joint distributions of S100, HMB-45, and Melan-A/MART-1 in our large series of cutaneous melanomas, we stained 2 serial sections of our TMA, multiplexing either HMB-45 or the anti-Melan-A/ MART-1 A103 mouse monoclonal antibody with the Dako anti-S100 rabbit polyclonal antibody on a single slide. The 322 surgical specimens (121 primary tumors and 201 metastases) with evaluable data across all 3 markers were included in this analysis. S100 staining that persisted over at least 25% of the tumor area was detected in 295 of 322 of the included histospots (91.6%), with immunofluorescent signal noted in both the nucleus and cytoplasm of positive-staining specimens. By contrast, the remaining 27 melanomas were S100 negative, with evidence of S100 expression in 5% or fewer of the spotted cells, consistent with the clinical definition of S100 negativity; focal S100 expression was not observed in our included histospots. Similarly, HMB-45 stained at least 25% of the tumor area in 203 of 322 melanomas (63.0%) (73 primary tumors and 130 metastases), and Melan-A/MART-1 stained 236 melanomas (73.3%) (84 primary tumors and 152 metastases). For HMB-45 and Melan-A, focal expression in fewer than 25% of the assayed melanocytes was scored as a negative histospot.

A total of 27 melanomas, including 21 of 201 metastases and 6 of 121 primary tumors (10.4% versus 5.0%, P = .10) were S100 negative. The clinicopathologic characteristics of the S100-negative melanomas are displayed in Table 1. Seventeen (63.0%) of the S100-negative melanomas developed in male patients (P = .61). S100 negativity was observed across a broad spectrum of histopathologic subtypes among the primary tumors and in locoregional as well as visceral disease among the metastases. Other than a higher but nonsignificant count of pan-MAA-negative melanomas noted among the metastases (5 of 201 [2.5%] versus 1 of 121 [0.8%]); Fisher exact, P = .42), these melanomas were unremarkable with respect to clinicopathologic criteria including growth pattern, overall morphology, and pigmentation. Review of their associated H&E sections revealed melanomas with unremarkable histology. S100-negative primary melanomas displayed solid-nested growth, whereas S100-negative metastases were all solid, with cell morphology that included epithelioid, spindled, or hybrid morphology, typical of the disease (Figure 1, A through F).

Next, we considered the joint distributions across all 3 antigens (Table 2, Figure 2, A through P). Cellular immunoreactivity of 25% or greater for all 3 antigens assayed was observed in only 160 melanomas (49.7%). By contrast, 156 melanomas expressed only 1 or 2 of the assayed antigens, and all combinations of the 3 assayed antigens were observed among our data set. Additional commonly occurring categories included S100+/HMB-45-/ Melan-A+ melanomas (n = 61; 18.9%) and melanomas expressing only S100 (n = 49; 15.2%). HMB-45 or Melan-A immunoreactivity was also observed among the S100-negative melanomas, with each antigen present in a similar number of assayed lesions (18 versus 15). Yet, 10 S100-negative melanomas demonstrated discordant staining patterns between HMB-45 and Melan-A/MART-1. Finally, 1 primary melanoma and 5 metastatic melanomas did not react with any assayed antibody. The 2-way cross-tabular joint analysis of HMB-45 and Melan-A expression identified 172 (53.4%) melanomas positive for both antigens, 55 (17.1%) negative for both antigens, and 95 (29.5%) discordant for the assayed MAAs, with similar distributions noted among primary tumors and metastases (P = .65). Among melanomas discordant across HMB-45 and Melan-A expression, a significantly larger number were Melan-A+ (n = 64) versus HMB-45+ (n = 31; P < .001).

We also assessed the association between antigen marginal and joint distributions and the semiquantitative melanin pigment score. Whereas HMB-45 staining was independent of pigmentation (P = .13), Melan-A staining was associated with pigmentation, as a larger percentage of moderately or highly pigmented melanomas were Melan-A negative (melanin = 2 or 3; n = 17 of 42, 40.5%) when compared to mildly or unpigmented lesions (melanin = 1 or 0; n = 62 of 260, 23.8%) (P = .01, Table 3). Of the 42 moderately or highly pigmented melanomas with S100 immunostaining data, only 5 were S100 negative, whereas, by contrast, 22 S100-negative melanomas arose in unpigmented lesions. The association between joint HMB-45/ Melan-A distribution and degree of pigmentation yielded a highly significant result (P = .007; Table 3), with a smaller percentage of HMB-45+/Melan-A+ lesions (35.7% versus 51.7%-60.7%) and a larger percentage of doubly negative lesions (28.6% versus 9.8%-17.7%) among the highly pigmented lesions.

For the subset of primary melanomas (n = 121), we examined the associations between HMB-45 and Melan-A marginal and joint expression and other clinicopathologic features as well as melanoma-specific survival. Among single-marker bivariate comparisons, the only significant association revealed that HMB-45- melanomas were more likely to possess in-transit metastases at the time of diagnosis (P = .02); all of the remaining comparisons yielded null results (Table 4). We also noted no significant differences in the distribution of clinicopathologic criteria across the HMB-45/Melan-A joint expression categories (Table 5). Lack of expression of either HMB-45 (hazard ratio [HR] = 1.20, 95% confidence interval [CI]: 0.65-2.21; P = .56) or Melan-A (HR = 1.09, 95% CI: 0.56-2.12; P = .81) was not associated with melanoma-specific mortality after adjusting for Breslow thickness (mm), stage at diagnosis, and age at diagnosis. Survival analysis across the 4 joint distribution categories yielded overlapping product-limit survival curves on univariate analysis (P > .99; Figure 3) and no significant difference in a multivariable proportional hazards model after adjusting for Breslow thickness and stage at diagnosis (P = .88; Table 6).


The differential expression of S100, HMB-45, and Melan-A/MART-1 has been extensively studied in cutaneous malignant melanoma. Yet, while trends for their marginal distributions are well known, the overlap in their expression is less well characterized. To the best of our knowledge, no study has evaluated the joint distributions of these antigens in samples of greater than 100 melanomas, and no study has considered the distribution of established melanoma clinicopathologic parameters in HMB-45/Melan-A null or discordant lesions compared with the referent HMB-45+/ Melan-A+ tumor. (14-17) To address this issue, we considered the marginal and joint expression of S100, HMB-45, and Melan-A/MART-1 across a large TMA containing representative histospots from 213 primary melanomas and 342 melanoma metastases. Data from the 322 lesions (121 primary tumors and 201 metastases) with evaluable data for all 3 markers were included in our analysis.

With immunofluorescence-based immunohistochemistry to assay our targeted antigens, 91.6% of our samples were S100+, 63.0% were HMB-45+, and 73.3% were Melan-A/ MART-1+, each registering slightly below the recognized sensitivities for each of these respective antigens, (9) with similar rates of immunoreactivity for primary and metastatic lesions. As we selected a cutoff of at least 25% for immunolabeling across eligible melanocytes in each arrayed histospot, our observed decrease in sensitivity might be due to a more stringent criteria than the clinical standard of greater-than-5% positivity, (27) such that lesions with patchy, focal staining would be alternatively classified as negative in our analysis. We also did not observe any association between HMB-45 or Melan-A immunoreactivity with any assayed clinicopathologic factor except for an increase in the lack of HMB-45 immunoreactivity among melanoma primary tumors with known microsatellitosis, which is consistent with the observation of less frequent HMB-45 antigenicity among melanoma metastases. (9,21) These data could suggest that lack of HMB-45 immunoreactivity might be a property of primary melanomas that are more likely to metastasize as opposed to a phenotype acquired after metastasis. We also did not detect an association with melanoma-specific mortality with either HMB-45 (P = .56) or Melan-A (P = .81), among our subset of primary melanomas, after adjusting for Breslow thickness, stage at diagnosis, or age at diagnosis. Although our data are consistent with previously published null results for Melan-A, (28,29) the data for HMB-45 are less straightforward, with reports of significantly improved, (29) significantly impaired, (30) and no association with survival (28) for HMB-45 expression, all reported by using cutoffs of either 50% or 90% staining efficiency. (31)

Interestingly, 27 of our assayed melanomas (8.4%) were S100 negative, representing, to the best of our knowledge, the largest single collection of S100-negative melanomas reported in the literature. To confirm this, we conducted a systematic search of the PubMed (US National Library of Medicine) database through November 18, 2011, by using the following keyword search: ("S100-negative" AND "melanoma"). The search returned 4 articles of which two (32,33) addressed melanoma clinical samples. Nine additional articles were identified through review of cited references,7,8,34-40 including 1 article (35) that contained a meta-analysis of 12 additional articles all published before 1992. Although the meta-analysis published cumulative data describing S100 negativity in 13 of 230 primary tumors and 9 of 166 melanoma metastases, no single article identified in our search reported on more than 20 S100negative melanomas with all but 2 studies (32,36) describing fewer than 5 cases.

S100-negative lesions have been described more frequently among junctional nests on sun-damaged skin [33] and among metastatic lesions, even in the setting of a previously documented S100-positive primary tumor. (32) Lack of S100 expression may occur more frequently among ocular or acral melanomas (32,34) but has been observed across all histologic subtypes. (35) Six of our S100-negative melanomas arose in primary lesions with the remaining 21 occurring in metastases. One study reporting on 11 S100-negative melanoma metastases with matched primary blocks suggested that loss of S100 antigenicity might be acquired along with metastatic competency, as 9 of 11 had S100-positive primary lesions (32); a second study with 12 S100-negative melanomas reported equal rates between primary tumors and metastases.36 While a higher percentage of our assayed metastases (10.4%) were S100 negative than were our primary tumors (5.0%), this difference trended toward but did not achieve statistical significance (P = .10), most likely owing to the small size of our S100-negative sample.

We also did not detect any significant associations between S100 immunoreactivity and other clinicopathologic criteria. Melanomas arising in males were as likely to be S100-negative as were melanomas arising in females. Among primary lesions, S100-negative melanomas occurred across a broad range of Breslow thicknesses and histopathologic subtypes. While 1 study noted a possibly increased rate of S100-negative lesions among acral lentiginous melanomas (34) and our study reports 2 S100-negative lesions arising in acral lentiginous or amelanotic melanomas, most of our S100-negative lesions arose in superficial spreading or nodular melanomas. Among metastases, lack of S100 antigenicity was observed for cutaneous, soft tissue, lymph node, and visceral lesions.

Six of our S100-negative melanomas, 1 primary and 5 metastases, also lacked expression of HMB-45 and Melan-A. This "triple-negative" pattern was also noted among 7 of 17 S100-negative metastases reported by Aisner et a (l,32) with 5 of these 7 lesions arising from the back or shoulder. Interestingly, 5 of 6 of our metastatic "triple negatives" arose 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, HMB-45, and Melan-A/MART-1 immunoreactivity.

While the marginal distributions for S100, HMB-45, and Melan-A positivity and their associations with recognized melanoma clinicopathologic parameters are well established, (9) the literature describing their joint distributions is much sparser. 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 MAA-discordant lesions. The largest of these studies evaluated overlapping HMB-45/Melan-A immunoreactivity in 65 melanoma metastases and 10 cutaneous primary tumors. (14) While the authors (14) explored the distribution across all pairwise antigen combinations, correlations with clinicopathologic criteria other than histologic subtype were not reported. Among their sample of 30 melanomas, Xu et al (15) observed some HMB-45/Melan-A discordance, with positive MelanA expression occurring only in 9 of 14 S100+/HMB45-spindled or epithelioid melanomas. In their study of 17 S100-negative melanomas, Aisner et al (32) noted that all lesions were concordant for HMB-45 and Melan-A, and HMB-45/Melan-A discordant lesions were not observed. To the best of our knowledge, our study of 322 assayable melanomas represents the largest series to date for which the joint distribution of HMB-45 and Melan-A is considered and the only study to consider the relationship between joint HMB-45/Melan-A expression and commonly reported clinicopathologic criteria among eligible primary lesions. Most of our lesions were concordant for HMB-45 and Melan-A expression, with 53.4% expressing both antigens and 17.1% lacking expression in 25% or more of the arrayed melanomas. We also observed melanomas discordant for MAA expression, with 31 melanomas (9.6%) expressing HMB-45 only and 64 melanomas (19.9%) expressing Melan-A only. Further bivariate analyses for 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, suggesting that the prognostic impact of MAA discordance may be small. While the significantly larger number (P < .001) of discordant lesions that express only Melan-A can be explained by Melan-A's comparatively more diffuse and intense staining of melanomas--which persists into the dermal layers of the assayed lesions (14,41) and possibly produced fewer false negatives among our sample of representative 0.6-mm histospots--we cannot exclude the possible role of genetic or epigenetic factors, which underlie the development and distribution of the 4 classes of HMB45/Melan-A expression-defined melanomas; moreover, a possible relationship with the levels of Microphthalmia-associated transcription factor (MITF) expression (42) may yield compelling insight into mechanisms relevant for melanocytic lesion development and progression.

While our study includes numerous strengths, including 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 photoreactive melanin might confound immunofluorescent readouts in melanoma. (45) Melanin not only exhibits broad spectral absorption that decreases monotonically with increasing wavelengths from 300 to 1100 nm, (46,47) but also has recently been shown to display autofluorescence with separate excitations in the ranges of 370 to 470 nm and 785 nm, and corresponding emissions at 540 nm and 890 to 900 nm, respectively, (47,48) with the former in close proximity to the emission wavelength of our mask fluorophore (546 nm). While HMB-45 expression was independent of melanin distribution (with similar proportion of HMB-45-negative lesions among all 4 categories of pigmentation), Melan-A immunostaining was associated with melanin levels; and despite similar proportions of Melan-A-negative lesions observed in each of the unpigmented and the mildly and moderately pigmented melanomas, an excess of Melan-A negative lesions was observed among the highly pigmented melanomas. This effect also translated into a similar significant association among the HMB-45/Melan-A joint distribution categories. Because the association with melanin expression did not extend to all assayed MAAs, we cannot rule out an underlying biologic mechanism that requires further elucidation.

A second limitation of our analysis is that our data were collected from a TMA that represented each melanoma with a single 0.6-mm histospot and were not validated on whole sections. Although current clinical diagnostic standards typically stipulate adjudication of whole sections and define MAA positivity as any immunoreactivity in 5% of cells therein, (27) use of TMAs circumvents the risk for batch-to-batch variation, which could be incurred during a whole-slide approach. However, recent TMA validation experiments have demonstrated that intratumoral heterogeneity can yield significant core-to-core variability of protein expression. The observed variance was driven by both the lability of the target protein and the antibody selected for its measurement such that, in a marker-dependent fashion, as many as 11 independent histospots may be required to adequately capture potential heterogeneity. (49) Specific TMA validation experiments conducted with a small series of antigens with prognostic potential suggest that, for melanoma, a minimum of 3 independent cores are needed to achieve 90% concordance for overall positive staining with whole sections. (50-53) In the context of recognized focality of HMB-45 immunostaining and the association of decreased HMB-45 and Melan-A staining in deeper dermal regions of primary melanomas, we cannot rule out some degree of measurement error, which may have increased our false-negative rate through our strategy of sampling only 1 single 0.6-mm histospot from each index lesion. Future work would include validating our findings across additional, redundant builds of the TMA.

Our experimental design is further limited by the need to sample HMB-45 and Melan-A independently of each other on serial sections of our TMA and then to reconstruct joint distributions through the overlay of corresponding images across the serial sections. Although we forgo some precision by not being able to multiplex both HMB-45 and Melan-A on the same section, our method for assessing joint distribution matches current clinical practice for evaluating occult micrometastases and malignancies of unknown primary tumor in the surgical pathology suite. (27)

In summary, we have comprehensively characterized the marginal and joint distributions, clinicopathologic correlates, and prognostic potential for 3 clinically relevant MAAs in a large series of primary and metastatic melanomas. Our study also describes the largest single series to date of S100-negative melanomas. Future directions include transcriptome profiling and whole-exome sequencing of lesions representative of each joint distribution category, as well as analysis of MITF and other MAA expression to further elucidate discriminating molecular characteristics that define the individual MAA-based melanoma subclasses.

Caption: Figure 1. Histologic characteristics of S100-negative and triple-antigen-negative melanomas. Overall, the lesions show a predominant solid grow pattern, are composed mainly of epithelioid cells, and present mild/focal pigmentation. A, Case 490 (SW0 negative): primary lesion showing atypical intraepidermal lentiginous proliferation of large melanoma cells with suprabasal pagetoid spread (arrows) and extensive dermal infiltration I epithelioid cells with nuclear atypia and focal pigmentation (arrowheads). B, Case 369 (triple-antigen negative): metastatic nodular lesion composed of a dense sheet of atypical epithelioid cells with hyperchromatic nuclei, ample eosinophilic cytoplasm, and lack of pigmentation. C, Case 275 (triple-antigen negative): metastatic melanoma showing a solid proliferation of atypical, predominantly epithelioid cells with highly pleomorphic nuclei, prominent nucleoli, numerous mitotic figures (arrows), and absence of melanic-type pigment. D, Case 161 (S100 negative): solid metastatic proliferation of large atypical cells with epithelioid morphology, prominent nucleoli, nuclear pseudoinclusions (arrows), and focal pigment deposition (arrowheads). E, Case 269 (triple-antigen negative): primary cutaneous lesion showing solid dermal sheets and intraepithelial proliferation of atypical cells with predominant clear cytoplasm and nuclear atypia (arrows). Note focal pigmentation. F, Case 173 (SI00 negative: primary melanoma composed of highly atypical cells with some "rhabdoid" features (arrows), displaying eosinophilic cytoplasm, slight lateral nuclear displacement, and focal nuclear vacuolation (hematoxylin-eosin, original magnifications X200 [A, B, E, and F] and X400 [C and D]).

Caption: Figure 2. Index histospots showing bright field hematoxylin-eosin and immunofluorescence photomicrographs depicting staining patterns of S100 (rabbit polyclonal, Dako, Carpinteria, California), Melan-A (monoclonal A103, Biogenex, Fremont, California), and HMB- 45 (Biogenex) from case examples demonstrating selected melanoma-associated antigen joint distribution patterns. Case 304 (images A through D) expresses all 3 melanoma-associated antigens. Case 18 (images E through H) is S100 negative but expresses both HMB-45 and Melan-A. Case 37 (images I through L) expresses only HMB-45. Case 395 (images M through P) does not express any of the 3 assayed melanoma-associated antigens (original magnifications X100 [A through P]).

Caption: Figure 3. Differential melanoma-specific survival according to category of joint HMB-45/Melan-A expression.


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Mollis Viray, BS; William R. Bradley, BS; Kurt A. Schalper, MD, PhD; David L. Rimm, MD, PhD; Bonnie E. Gould Rothberg, MD, PhD, MPH

Accepted for publication August 29, 2012.

From the Department of Pathology (Ms Viray, Mr Bradley, and Drs Schalper and Rimm) and Section of Medical Oncology, Department of Internal Medicine and Department of Epidemiology and Public Health (Dr Rothberg), Yale School of Medicine, New Haven, Connecticut. Mr Bradley is now with the Tufts University School of Medicine, Boston, Massachusetts.

Funding support is from National Institutes of Health grants K08CA-151645 (B.E.G.R.) and R01-CA-114277 (D.L.R.).

Reprints: Bonnie E. Gould Rothberg, MD, PhD, MPH, Department of Internal Medicine (Medical Oncology), Yale School of Medicine, Yale Cancer Center, WWW216, PO Box 208028, New Haven, CT 06520 (e-mail:

Table 1. Clinicopathologic Characteristics of S100-Negative Melanomas

            Age at
Sample    Diagnosis,         Resection      Histopathologic
ID            y        Sex     Year             Subtype

Primary melanomas
(n = 6;5.0%)

167           67        M      1994      Superficial spreading
173           77        M      1978      Nodular
229           77        F      1992      Acral lentiginous
269 (a)       61        M      1989      Superficial spreading
283           34        M      1978      Superficial spreading
490           71        M      1976      Nodular

Metastatic melanomas
(n = 21; 10.4%)

18            77        M      1969      Cutaneous
37            53        M      1998      Lymph node
57            58        M      1996      Cutaneous
110           77        M      1998      Soft tissue
161           62        F      1998      Cutaneous
217           58        F      1978      Cutaneous
224           43        F      2001      Lymph node
252           81        F      1991      Cutaneous
255           75        M      1991      Visceral (colon)
275 (a)       70        M      1985      Cutaneous
287 (a)       53        F      1999      Cutaneous
299           26        M      1982      Lymph node
311 (a)       68        M      1971      Cutaneous
369 (a)       36        M      1982      Cutaneous
395 (a)       81        F      1974      Visceral (colon)
419           73        M      1976      Visceral (abdomen)
462           51        F      1964      Lymph node
521           64        M      1990      Cutaneous
536           85        M      1980      Cutaneous
551           62        F      1978      Visceral (brain)
563           21        M      1973      Lymph node

Sample    Thickness,      Growth
ID            mm         Pattern        Cell Morphology

Primary melanomas
(n = 6;5.0%)

167          0.92      Solid-nested   Epithelioid
173          7.19      Solid-sheets   Epithelioid
229          1.00      Solid-nested   Epithelioid
269 (a)      0.80      Solid-nested   Epithelioid/spindled
283          3.49      Solid-nested   Epithelioid/spindled
490          1.40      Solid-sheets   Epithelioid

Metastatic melanomas
(n = 21; 10.4%)

18           1.7       Solid          Epithelioid
37           13.0      Solid          Epithelioid
57           5.2       Solid          Epithelioid
110          1.0       Solid          Epithelioid/spindled
161          1.0       Solid          Epithelioid/spindled
217          Not       Solid          Epithelioid/spindled
224          9.0       Solid          Spindled
252          0.7       Solid          Epithelioid
255          1.8       Solid          Epithelioid
275 (a)      2.8       Solid          Spindled
287 (a)      0.5       Solid          Epithelioid
299          10.0      Solid          Spindled
311 (a)      1.0       Solid          Epithelioid
369 (a)      1.5       Solid          Epithelioid
395 (a)      10.8      Solid          Epithelioid
419          3.0       Solid          Epithelioid
462          6.0       Solid          Epithelioid
521          3.0       Solid          Epithelioid
536          Not       Solid          Epithelioid
551          2.5       Solid          Epithelioid/spindled
563          1.0       Solid          Epithelioid

ID           Pigmentation

Primary melanomas
(n = 6;5.0%)

167       2: intense, focal
173       0: absent
229       1: mild, focal
269 (a)   1: moderate, focal
283       1: mild, focal
490       2: moderate, focal

Metastatic melanomas
(n = 21; 10.4%)

18        0: absent
37        2: intense, focal
57        0: absent
110       2: intense, focal
161       1: moderate, focal
217       1: mild, focal
224       3: intense, global
252       0: absent
255       0: absent
275 (a)   0: absent
287 (a)   0: absent
299       0: absent
311 (a)   2: intense, focal
369 (a)   0: absent
395 (a)   0: absent
419       0: absent
462       3: intense, global
521       1: mild, focal
536       0: absent
551       0: absent
563       0: absent

(a) Melanoma showed no immunoreactivity to each of
S100, HMB-45, and Melan-A.

Table 2. Distribution of Joint Immunostaining Across
S100, HMB-45, and Melan-A/MART-1

No. (%)      S100   HMB-45   Melan-A

160 (49.7)   Yes     Yes       Yes
25 (7.8)     Yes     Yes        No
61 (18.9)    Yes      No       Yes
49 (15.2)    Yes      No        No
12 (3.7)      No     Yes       Yes
6 (1.9)       No     Yes        No
3 (0.9)       No      No       Yes
6 (1.9)       No      No        No

Table 3. Independent and Joint HMB-45 and Melan-A Immunoreactivity
According to Degree of Pigmentation

                                                 Mild      Moderate
                               No Pigment,     Pigment,    Pigment,
                                 No. (%)        No. (%)     No. (%)
                                (n = 209)      (n = 51)    (n = 28)

Marginal HMB-45 expression
  HMB-45+                      123 (58.9) (a)  35 (68.6)   22 (78.6)
  HMB-45-                       86 (41.1)      16 (31.4)    6 (21.4)
Marginal Melan-A expression
  Melan-A+                     157 (75.1)      41 (80.4)   20 (71.4)
  Melan-A-                      52 (24.9)      10 (19.6)    8 (28.6)
Joint HMB-45/Melan-A
  HMB-45 +/Melan-A+            108 (51.7)      30 (58.8)   17 (60.7)
  HMB-45+/Melan-A-              15 (7.2)        5 (9.8)     5 (17.9)
  HMB-45-/Melan-A+              49 (23.4)      11 (21.6)    3 (10.7)
  HMB-45-/Melan-A-              37 (17.7)       5 (9.8)     3 (10.7)

                               No. (%)
                              (n = 14)       P Value

Marginal HMB-45 expression
  HMB-45+                     10 (71.4)      .13
  HMB-45-                      4 (28.6)
Marginal Melan-A expression
  Melan-A+                     5 (35.7)      .01 (b)
  Melan-A-                     9 (64.3)
Joint HMB-45/Melan-A
  HMB-45 +/Melan-A+            5 (35.7)      .007 (b)
  HMB-45+/Melan-A-             5 (35.7)
  HMB-45-/Melan-A+             0 (0.0)
  HMB-45-/Melan-A-             4 (28.6)

(a) Percentages may not total to 100% due to rounding.

(b) Significant at P < .05.

Table 4. Individual HMB-45 and Melan-A Immunostaining Associations
With Clinicopathologic Parameters: Primary Melanomas

                            HMB-45 Positive        HMB-45 Negative
Variable                        (n = 73)               (n = 48)

Age at diagnosis,         56.44 [+ or -] 16.54   60.33 [+ or -] 13.88
  mean [+ or -] SD, y
Breslow thickness,         2.85 [+ or -] 2.24     2.48 [+ or -] 2.01
  mean [+ or -] SD, mm
Sex, No. (%)
  M                            37 (50.7) (a)          22 (45.8)
  F                            36 (49.3)              26 (54.2)
Stage at diagnosis,
  No. (%)
  Localized                    57 (85.1)              38 (88.4)
  Regional/distant             10 (14.9)               5 (11.6)
Clark level, No. (%)
  II                            6 (8.2)                1 (2.0)
  III                          24 (32.9)              18 (37.5)
  IV                           32 (43.8)              23 (47.9)
  V                            11 (15.1)               6 (12.5)
Ulceration, No. (%)
  Yes                          24 (32.9)              17 (36.2)
  No                           49 (67.1)              30 (63.8)
  No. (%)
  Yes                          13 (17.8)              16 (34.0)
  No                           60 (82.2)              31 (66.0)
  subtype, No. (%)
  Superficial spreading        45 (61.6)              34 (72.3)
  Nodular                      16 (21.9)               6 (12.8)
  Lentigo maligna               1 (1.4)                1 (2.1)
  Acral lentiginous             6 (8.2)                1 (2.1)
  Other                         5 (6.8)                5 (10.6)
  lymphocytes, No. (%)
  Absent/sparse                18 (24.7)               6 (12.5)
  Nonbrisk                     44 (60.3)              29 (60.4)
  Brisk                        11 (15.1)              13 (27.1)
Anatomic site of x
  lymphocytes, No. (%)
  Head/neck                     7 (9.6)                8 (18.2)
  Trunk                        21 (28.8)              16 (36.4)
  Upper extremity              11 (15.0)               6 (13.6)
  Lower extremity              34 (46.6)              14 (31.8)

                                      Melan-A Positive
Variable                  P Value         (n = 84)

Age at diagnosis,           .18     58.44 [+ or -] 16.24
  mean [+ or -] SD, y
Breslow thickness,          .36      2.75 [+ or -] 2.06
  mean [+ or -] SD, mm
Sex, No. (%)
  M                         .60          39 (46.4)
  F                                      45 (53.6)
Stage at diagnosis,
  No. (%)
  Localized                 .62          66 (85.7)
  Regional/distant                       11 (14.3)
Clark level, No. (%)
  II                        .51           5 (6.0)
  III                                    26 (31.0)
  IV                                     41 (48.8)
  V                                      12 (14.3)
Ulceration, No. (%)
  Yes                       .71          32 (38.1)
  No                                     52 (61.9)
  No. (%)
  Yes                       .04 (b)      19 (22.6)
  No                                     65 (77.4)
  subtype, No. (%)
  Superficial spreading     .38          52 (61.9)
  Nodular                                15 (17.9)
  Lentigo maligna                         2 (2.4)
  Acral lentiginous                       7 (8.3)
  Other                                   8 (9.5)
  lymphocytes, No. (%)
  Absent/sparse             .12          17 (20.2)
  Nonbrisk                               54 (64.3)
  Brisk                                  13 (15.5)
Anatomic site of x
  lymphocytes, No. (%)
  Head/neck                 .31          11 (13.3)
  Trunk                                  22 (26.5)
  Upper extremity                        12 (14.5)
  Lower extremity                        38 (45.8)

                            Melan-A Negative
Variable                        (n = 37)         P Value

Age at diagnosis,         57.00 [+ or -] 14.19     .64
  mean [+ or -] SD, y
Breslow thickness,         2.61 [+ or -] 2.38      .74
  mean [+ or -] SD, mm
Sex, No. (%)
  M                            20 (54.1)           .44
  F                            17 (45.9)
Stage at diagnosis,
  No. (%)
  Localized                    29 (87.8)           .76
  Regional/distant              4 (12.1)
Clark level, No. (%)
  II                            2 (5.4)            .61
  III                          16 (43.2)
  IV                           14 (37.8)
  V                             5 (13.5)
Ulceration, No. (%)
  Yes                           9 (25.0)           .17
  No                           27 (75.0)
  No. (%)
  Yes                          10 (27.8)           .55
  No                           26 (72.2)
  subtype, No. (%)
  Superficial spreading        27 (75.0)           .28
  Nodular                       7 (19.4)
  Lentigo maligna               0 (0.0)
  Acral lentiginous             0 (0.0)
  Other                         2 (5.4)
  lymphocytes, No. (%)
  Absent/sparse                 7 (18.9)           .19
  Nonbrisk                     19 (51.4)
  Brisk                        11 (29.7)
Anatomic site of x
  lymphocytes, No. (%)
  Head/neck                     4 (12.1)           .26
  Trunk                        15 (45.5)
  Upper extremity               5 (15.2)
  Lower extremity              10 (30.3)

(a) Numbers may not sum to total due to missing values;
percentages may not sum to 100% due to rounding.

(b) Significant at P < .05.

Table 5. HMB-45/Melan-A Joint Immunostaining Associations With
Clinicopathologic Parameters: Primary Melanomas

                               HMB-45+/                HMB-45+/
                               Melan-A+                Melan-A-
Variable                       (n = 60)                (n = 13)

Age at diagnosis,        57.15 [+ or -] 15.89    53.23 [+ or -] 19.62
  mean [+ or -] SD, y
Breslow thickness,        2.91 [+ or -] 2.24      2.57 [+ or -] 2.29
  mean [+ or -] SD, mm
Sex, No. (%)
  M                            29 (48.3) (a)            8 (61.5)
  F                            31 (51.7)                5 (38.5)
Stage at diagnosis,
  No. (%)
  Localized                    46 (83.6)               11 (91.7)
  Regional/distant              9 (16.4)                1 (8.3)
Clark level, No. (%)
  II                            4 (6.7)                 2 (15.4)
  III                          19 (31.7)                5 (38.5)
  IV                           28 (46.7)                4 (30.8)
  V                             9 (15.0)                2 (15.4)
Ulceration, No. (%)
  Yes                          20 (33.3)                4 (30.8)
  No                           40 (66.7)                9 (69.2)
  No. (%)
  Yes                          11 (18.3)                2 (15.4)
  No                           49 (81.7)               11 (84.6)
  subtype, No. (%)
  Superficial                  36 (60.0)                9 (69.2)
  Nodular                      12 (20.0)                4 (30.8)
  Lentigo maligna               1 (1.7)                 0 (0.0)
  Acral lentiginous             6 (10.0)                0 (0.0)
  Other                         5 (8.3)                 0 (0.0)
  lymphocytes, No. (%)
  Absent/sparse                16 (26.7)                2 (15.4)
  Nonbrisk                     37 (61.7)                7 (53.8)
  Brisk                         7 (11.7)                4 (30.8)
Anatomic site of
  primary tumor,
  No. (%)
  Head/neck                     6 (10.0)                1 (7.7)
  Trunk                        15 (25.0)                6 (46.2)
  Upper extremity               9 (15.0)                2 (15.4)
  Lower extremity              30 (50.0)                4 (13.8)

                               HMB-45-/                HMB-45-/
                               Melan-A+                Melan-A-
Variable                       (n = 24)                (n = 24)

Age at diagnosis,        61.63 [+ or -] 1 6.98   59.04 [+ or -] 10.09
  mean [+ or -] SD, y
Breslow thickness,        2.34 [+ or -] 1.45      2.63 [+ or -] 2.48
  mean [+ or -] SD, mm
Sex, No. (%)
  M                            10 (41.7)               12 (50.0)
  F                            14 (58.3)               12 (50.0)
Stage at diagnosis,
  No. (%)
  Localized                    20 (90.9)               18 (85.7)
  Regional/distant              2 (9.1)                 3 (14.3)
Clark level, No. (%)
  II                            1 (4.2)                 0 (0.0)
  III                           7 (29.2)               11 (52.4)
  IV                           13 (54.2)               10 (47.6)
  V                             3 (12.5)                0 (0.0)
Ulceration, No. (%)
  Yes                          12 (50.0)               18 (78.3)
  No                           12 (50.0)                5 (21.7)
  No. (%)
  Yes                           8 (33.3)                8 (34.8)
  No                           16 (66.7)               15 (65.2)
  subtype, No. (%)
  Superficial                  16 (66.7)               18 (78.3)
  Nodular                       3 (12.5)                3 (13.0)
  Lentigo maligna               1 (4.2)                 0 (0.0)
  Acral lentiginous             1 (4.2)                 0 (0.0)
  Other                         3 (12.5)                2 (8.7)
  lymphocytes, No. (%)
  Absent/sparse                 1 (4.2)                 5 (20.8)
  Nonbrisk                     17 (70.8)               12 (50.0)
  Brisk                         6 (25.0)                7 (29.2)
Anatomic site of
  primary tumor,
  No. (%)
  Head/neck                     5 (21.7)                3 (14.3)
  Trunk                         7 (30.4)                9 (42.9)
  Upper extremity               3 (13.0)                3 (14.3)
  Lower extremity               8 (34.8)                6 (28.6)

Variable                 P Value

Age at diagnosis,          .43
  mean [+ or -] SD, y
Breslow thickness,         .72
  mean [+ or -] SD, mm
Sex, No. (%)
  M                        .72
Stage at diagnosis,
  No. (%)
  Localized                .79
Clark level, No. (%)
  II                       .72
Ulceration, No. (%)
  Yes                      .23
  No. (%)
  Yes                      .24
  subtype, No. (%)
  Superficial              .62
  Lentigo maligna
  Acral lentiginous
  lymphocytes, No. (%)
  Absent/sparse            .14
Anatomic site of
  primary tumor,
  No. (%)
  Head/neck                .61
  Upper extremity
  Lower extremity

(a) Numbers may not sum to total due to missing values;
percentages may not sum to 100% due to rounding.

Table 6. Joint HMB-45/Melan-A Immunostaining
Melanoma-Specific Survival Hazard Ratios

Variable                              Hazard Ratio (95% CI)   P Value

Breslow thickness, mm                 1.18 (1.04-1.34)        .01 (a)
Stage at diagnosis
  Localized                           1.00                    .01 (a)
  Regional/distant                    2.72 (1.24-5.95)
HMB-45/Melan-A joint distributions
  HMB-45-/Melan-A-                    1.00                    .88
  HMB-45-/Melan-A+                    0.86 (0.34-2.16)
  HMB-45+/Melan-A-                    0.64 (0.20-2.09)
  HMB-45 +/Melan-A+                   0.77 (0.35-1.73)

(a) Significant at P < .05.


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Author:Viray, Mollis; Bradley, William R.; Schalper, Kurt A.; Rimm, David L.; Rothberg, Bonnie E. Gould
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Geographic Code:1USA
Date:Aug 1, 2013
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