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Original Study

Use of the Ciliation Index to Distinguish Invasive Melanoma From Associated Conventional Melanocytic Nevi

Lang, Ursula E. MD, PhD*,†; Love, Nicholas R. PhD*; Cheung, Christine BS*; McCalmont, Timothy H. MD; Kim, Jinah MD, PhD*,‡

Author Information
The American Journal of Dermatopathology: January 2020 - Volume 42 - Issue 1 - p 11-15
doi: 10.1097/DAD.0000000000001459
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Abstract

INTRODUCTION

Melanocytic tumor diagnosis comprises one of the most challenging areas in pathology due in part to morphologic heterogeneity. Histopathologic features range from bland-appearing nevoid melanoma to variants with highly pleomorphic morphology. When the architectural and cytologic features are beyond threshold criteria, consensus on the diagnosis may be relatively uniform. However, when more than 1 morphology is present within a single lesion, the Breslow thickness may be in question and accurately distinguishing melanoma from an associated dermal nevus can be extremely challenging, leading to the use of ancillary stains and expert consultation. No immunohistochemical stain can reliably distinguish benign versus malignant and instead must be interpreted in the context of all the available information, both clinically and histopathologically.

Primary cilia (PC) are cell surface microtubular organelles that are often compared with antennae due to their involvement in sensing and transducing various extracellular signals to initiate cellular signaling.1 They are composed of the extracellular portion termed the axoneme and the intracellular base termed the basal body (formed from the mother centriole), which anchors the cilium to the cell surface. The PC senses the extracellular environment through resident receptor and effector molecules, which include Hedgehog, canonical and noncanonical Wnt and Notch signaling.2–5 It is therefore not surprising that ciliary dysfunction can lead to altered cell cycle dynamics, loss of response to growth signals, loss of cell polarity control, and altered interaction with the extracellular milieu, as characterized in classic ciliopathies.6 In fact, studies have shown the loss of PC in pancreatic cancer, renal cell carcinoma, breast cancer, cholangiocarcinoma, and melanoma.7–11

As the centrioles that would otherwise anchor the PC are diverted for use in the cell cycle, the lack of a cell surface PC has close correlations with cell cycle progression. However, studies have shown that an increased proliferation rate does not completely explain the close to complete lack of PC in melanoma. Likewise, inducing cell cycle arrest in melanoma cell lines when compared with control melanocytes does not result in complete recovery of PC formation.12 Therefore, we hypothesize that the cumulative changes in melanoma cells, regardless of the precise unique set of genetic changes, culminate in inhibiting or disrupting PC formation. To further our understanding of the status of PC in melanocytic lesions, we collected a set of combined melanocytic lesions in which a nevus component is identified next to an invasive melanoma. In these cases, the ciliation index of the nevus portion can be used as an internal control for the relative loss of PC formation in the melanoma portion.

MATERIALS AND METHODS

Clinical Samples

Tissue samples were procured from the archives of the University of California, San Francisco, and all cases were reviewed, and histopathologic diagnosis was reverified (J.K., T.H.M., and U.E.L.).

Microscopy and Analysis

Immunostaining and microscopy was performed, as previously described.8 Briefly, 4-μm-thick formalin-fixed paraffin-embedded sections were deparaffinized in xylene, rehydrated through graded ethanol dilutions, and subjected to antigen retrieval according to standard procedures. These specimens were costained with antibodies against SOX10 to detect melanocytes,13 acetylated alpha-Tubulin to detect the modified tubulins enriched in the ciliary axoneme,14 and gamma-Tubulin to detect the basal bodies of cilia15 (gamma-Tubulin also marks the centrosome when it is not elongating a ciliary axoneme). To assess the abundance of PC in SOX10-positive melanocytes, we defined an elongated acetylated alpha-Tubulin–positive structure in association with a gamma-Tubulin–positive structure to be a PC (as previously described).16 The counting was performed using a Keyence BZ-X710 (Keyence Corporation of America, Itasca, IL) fluorescence microscope and its accompanying software at ×60 magnification.

Antibodies and Reagents

Immunofluorescence staining was performed using the following antibodies: mouse anti-SOX10 IgG1 (catalog #API 3099; Biocare, Pacheco, CA), rabbit anti–gamma-Tubulin (catalog #T5192; Sigma, Burlington, MA), and mouse anti–acetylated-alpha-Tubulin IgG2b (catalog #T6793; Sigma) antibodies. The following secondary antibodies were used: Alexa Fluor 594 donkey anti-mouse IgG1 (catalog #A-21203; Life Technologies, Carlsbad, CA), Alexa Fluor 647 chicken anti-rabbit (catalog #A-21443; Life Technologies), and Alexa Fluor 488 goat anti-mouse IgG2b (catalog #A-21141; Life Technologies). Primary antibodies were used at 1:2000 and secondary antibodies at 1:300 dilution. Slides were mounted using ProLong Gold Antifade Mountant (catalog #P36930; Life Technologies) and stored at room temperature.

Statistical Analysis

Results were obtained using Microsoft Excel software and expressed as mean ± SE. P values (Student 2-tailed, unpaired t test). Statistical significance is considered for data with P ≤ 0.05.

RESULTS

Clinical, Histopathologic, and Immunohistochemical Findings

Invasive melanomas with associated melanocytic nevi from 10 patients were studied. Patients had a median age of 61 years (range 35–78 years). The lesions were located on the back, arm, abdomen, and shoulder. There were 4 women and 6 men (Table 1). Characteristic architectural and cytomorphologic features were used to distinguish the invasive melanoma component from the dermal nevus within the lesions (Figs. 1A, B). In addition, HMB-45 staining was performed and demonstrated strong staining within the melanoma portion and loss of staining within the nevus in all cases (Table 2 and Fig. 1C). The nevus portion in all samples retained p16 expression, whereas the melanoma portion showed either a partial or a total loss of staining in 9/10 samples (Fig. 1D). Also, a combined Melan-A/Ki-67 stain was performed and showed a mild increase in the number of positive melanocytes within the melanoma portion (Fig. 1E, arrow-heads show positive cells) and no staining in the nevus portion.

TABLE 1
TABLE 1:
Demographics, Clinical Data, and Ciliation Indices
FIGURE 1
FIGURE 1:
Melanoma with associated dermal nevus (case #3). A and B, H&E staining of invasive melanoma with associated dermal nevus (×20 magnification; black box in (A) is cropped for part (B)). C, Immunostaining for HMB-45 shows strong staining in the melanoma portion and no staining in the underlying nevus. D, Immunostaining for p16 shows retention of p16 staining in both the melanoma and nevus portions. E, Immunostaining for combined Melan-A and Ki-67 shows a relatively mild increased rate of proliferation within the melanoma compared with the nevus. The positive cells are highlighted black arrowheads.
TABLE 2
TABLE 2:
Histologic Features and Immunohistochemistry Results

Primary Cilia Status

An average of 192 melanocytes (range: 150–250) were counted per nevus region, and an average of 174 melanocytes (range: 150–250) were counted per melanoma region. A positive melanocyte was defined by the presence of centrioles (dot-like structures) along with an elongated ciliary axoneme (hair-like structures). Immunofluorescence results revealed that a mean of 4% ciliation (SD: 7%) in the melanoma component, whereas the associated nevus component had an average of 59% ciliation (SD: 17%). There was a significant decrease in the number of PC in the melanoma versus nevus components in each of these cases (P < 0.00001) (Table 1 and Figs. 2 and 3).

FIGURE 2
FIGURE 2:
Reduction of PC on melanocytes in melanoma compared with associated dermal nevus. A, ×10 magnification of case #3 with the region containing both melanoma and associated nevus identified in boxed area. Immunofluorescence staining identifies melanocytes with the nuclear marker, SOX10 (blue). Acetylated alpha-Tubulin highlights the PC (green), and gamma-Tubulin highlights the centrioles (red). B, ×40 magnification of the boxed region in image A. The nevus portion is highlighted by a box with dotted line and the melanoma portion is highlighted by a box with solid line. C, ×100 magnification of the nevus portion (dotted line box in image in B) with white arrowhead highlighting a primary cilia. D, ×100 magnification of melanoma portion (solid line box in image B) with black arrowhead highlighting the centriole lacking the ciliary axoneme.
FIGURE 3
FIGURE 3:
PC are significantly reduced in the melanoma portion compared with adjacent nevi. A, The melanocytes in 4 ×60 fields for each sample (with a minimum of 150 cells total) were examined for absence or presence of the primary cilium. An average of 192 melanocytes (range: 150–250) per nevus and an average of 174 melanocytes (range: 15–250) per melanoma were counted. The average ciliation index per nevus was 59% (SD: 17%) and for the melanoma was 4% (SD: 7%). B, Each case is graphed individually with paired melanoma and adjacent nevus.

Although we counted the number of PC containing melanocytes in each component to be quantitative, qualitative assessment was interpreted as being “high” in the nevus portion and “low” in the melanoma portion for each case. There was fairly uniform ciliation in each component of these lesions; therefore, random areas were selected for counting cells. These results are similar to previous reports of melanoma having very low percentage ciliation.8 A benign congenital melanocytic nevus was used as a positive control with a 98% ciliation index.

DISCUSSION

Previous work has demonstrated that PC are lost in melanoma in situ, primary invasive melanoma, and metastatic melanoma, whereas retained in melanocytic nevi.8 Our current study demonstrates an additional utility of using the relative PC loss within combined melanocytic lesions to distinguish benign from malignant. This technique requires minimal tissue in the form of 1 unstained formalin-fixed paraffin embedded slide, and the turnaround time can be as little as 1–2 days. However, the lack of automation and requirement of an immunofluorescent microscope for the actual analysis may be restrictive for use in some laboratories. As our study is limited to 10 cases, a larger cohort for validation is also still necessary.

We performed immunohistochemical stains for HMB-45 and p16 and found that the nevus component demonstrated retention of p16 staining in all cases, and the melanoma portion showed either partial or total loss in 9/10 cases. HMB-45 stained strongly in the melanoma and was lost in the nevus component. These additional stains further support the original diagnosis made by histopathologic examination. The 1 case (#3) that showed retention of p16 in both melanoma and nevus highlights the limits of usefulness that a single immunohistochemical stain has in any diagnosis. A combined Melan-A/Ki-67 stain demonstrated a mild increase in the proliferative index in the melanoma portion compared with the nevus but did not come close to the relative change in the ciliation index between the 2 components.

Compared with the benign congenital melanocytic nevus used as the control with a 98% ciliation index, the average 59% ciliation within the nevoid component of combined cases raises the question of where on the spectrum of malignant potential these associated nevi belong. One could hypothesize that these associated nevi are in a more permissive state of additional transformation due to partial dysregulation of the PC. As the PC is involved in many biological processes including cell cycle, structural influences of the cytoskeleton, cellular proteostasis, and signal transduction, it is equally possible that alterations to any of these pathways could have effects on PC status. Although analyzing the genetic landscape of these combined lesions was beyond the scope of this project, it will be a critical next step in further understanding and characterizing the various factors that lead to melanoma progression.

Although most melanomas are believed to arise de novo and not in association with a precursor nevus, our ability to reliably distinguish an existing precursor within a malignancy generally depends on there being distinct morphologic or architectural differences between the 2 components. Although the cases used in this study demonstrated morphologically distinct regions of melanoma versus nevus with immunohistochemical staining patterns further supporting the diagnosis, this is not always the case. Using the presence or absence of an additional morphologic feature, such as the PC, to further analyze a given lesion may be helpful in challenging cases. The study of the PC in this context is one example of a biologic state the melanocyte may have reached after integrating genetic mutations, epigenetic changes, posttranslational modification, and signaling from the extracellular environment.

The underlying mechanisms for PC loss are undoubtedly multifactorial, reflecting the complexities of a dynamic cellular process, and it is currently unclear whether the loss of PC plays a role in cancer progression or is a consequence of transformation. Ultimately, these findings raise limitless questions, which must be addressed experimentally to further define our understanding of how melanocytes and their respective PC organelle coexist. Until that point, we can still leverage the potential of the ciliation index as a novel marker of malignant potential, to be used within the context of the overall clinical and microscopic features.

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Keywords:

primary cilia; combined lesions; melanocytic; melanoma

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