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The role of R21 expression in differential diagnosis of melanocytic lesions

1 Department of Pathology, Gaziantep Nizip State Hospital, Gaziantep, Turkey
2 Department of Pathology, Faculty of Medicine, Eskisehir Osmangazi University, Eskisehir, Turkey

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Date of Submission24-Nov-2021
Date of Decision13-May-2022
Date of Acceptance06-May-2022
Date of Web Publication03-Feb-2023


Background: Cyclic adenosine monophosphate (cAMP) is an intracellular signal transmitter involved in the regulation of melanocyte growth, proliferation, and melanogenesis. R21 is a monoclonal antibody against the soluble adenylyl cyclase (sAC) protein. Various nuclear and cytoplasmic R21 expression patterns in melanocytic lesions have been previously reported. Pan-nuclear staining was defined as specific for melanoma and was found supportive in the assessment of surgical margins. Aims: The aim of this study is to evaluate the different expression patterns of R21 immunostain and investigate its effectiveness in the differential diagnosis of cutaneous malignant and benign melanocytic lesions. Settings and Design: Fifty invasive cutaneous melanoma and 50 benign melanocytic proliferation were included in the study. Material and Methods: Paraffin blocks that best reflected tumor morphology were studied via immunohistochemical staining for R21. For all patterns, the cases showing staining in 25% or more tumor cells were considered as positive. Statistical analysis used: Yates' Chi-square, Pearson Chi-square exact test, Spearman correlation were used. Results and Conclusions: Dot-like Golgi staining was characteristic for nevi (12/50) and seen only in one melanoma. Pan-nuclear staining was striking for melanoma (36/50). This pattern was observed in 2 dysplastic and 3 common melanocytic nevi too. None of the Spitz nevi expressed R21 in pan-nuclear pattern. For the diagnosis of melanoma, sensitivity and specificity of the pan-nuclear expression were 72% and 90%, respectively. Positive and negative predictive values were found as 87% and 76%. R21, a second-generation immunohistochemical marker, can be used in the differential diagnosis of benign and malignant melanocytic lesions.

Keywords: Differential diagnosis, melanoma, R21, soluble adenylyl cyclase (sAC)

How to cite this URL:
Turcan D, Paşaoğlu &, Arik D. The role of R21 expression in differential diagnosis of melanocytic lesions. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Nov 30]. Available from:

   Introduction Top

Cutaneous melanocytic lesions have various morphological features and different biological behavior. Although microscopic evaluation is the gold standard in diagnosis, morphological features may not always be sufficient for accurate classification. It is sometimes difficult to differentiate malignant melanoma from benign melanocytic proliferations. In particular, it might be more challenging to separate cutaneous melanoma from Spitz nevi or dysplastic melanocytic nevi.[1],[2],[3] In this case, it may be necessary to use additional diagnostic techniques, such as immunohistochemical (IHC) evaluation. Although, in the literature, several immune markers have been suggested, differential diagnostic challenges persist, and additional markers are needed.[4],[5]

Cyclic adenosine monophosphate (cAMP) is a ubiquitous signaling molecule involved in cellular growth and apoptosis. In mammalian cells, there are 2 types of protein adenylyl cyclase, the enzyme that synthesizes cAMP from adenosine triphosphate. The first type, encoded by the 9 transmembrane adenylyl cyclase gene, is structurally localized in the plasma membrane and is regulated by extracellular signals through the activation of G protein-coupled receptors.[6] The second type is encoded by the soluble adenylyl cyclase (sAC) gene. Unlike transmembrane adenylyl cyclase, sAC is not permanently localized in the plasma membrane, but is found in the cytoplasm, mitochondria, centrioles, and nucleus.[6],[7] In skin, it is identified in melanocytes, keratinocytes, eccrine ductal cells, and cutaneous nerves. It has been shown that the localization of sAC in keratinocytes is not fixed.[8] Studies have shown that sAC migrates from cytosol to the nucleus when keratinocytes and melanocytes transform from benign cells to malignant cells, such as squamous cell skin carcinoma and melanoma.[7],[8] These observations suggest that sAC may play a dynamic role in the transition from benign growth to cancer. Additionally, in another study, there is significant sAC overexpression in prostate carcinoma compared to benign prostate tissue.[9] These data also point to a possible role of sAC in proliferation control. R21 is a mouse monoclonal antibody directed against amino acids 203–216 of human sAC. We aimed to investigate R21 expression patterns and its role in the differential diagnosis of cutaneous malignant and benign melanocytic lesions.

   Material and Methods Top

All retrieved cases were diagnosed between 2010 and 2016 in our department. All cases of surgically resected primary invasive cutaneous melanomas [24 nodular melanoma (NM), 9 superficial spreading melanoma (SSM), 9 lentigo maligna melanoma (LMM), 7 acral lentiginous melanoma (ALM), and 1 unclassified melanoma] and Spitz nevi (12) cases were included. Nineteen common melanocytic nevi and 19 dysplastic melanocytic nevi were randomly selected.

Hematoxylin and Eosin stained slides were re-examined to confirm the diagnosis, and representative paraffin blocks were selected for IHC evaluation. Age and gender of the cases, and tumor localizations were evaluated. Breslow depth, anatomic (Clark) levels, and pathologic tumor stages (pT) were also noted for melanomas.

Paraffin blocks that best reflect tumor morphology were studied via IHC staining for R21 antibody (mouse monoclonal, 1:750 dilution, CEP Biotech, NY, USA). The paraffin blocks were cut into 4 micrometer-thick sections, and the slides were deparaffinized. Then, immunoperoxidase staining was completed using an automated slide staining system (Ventana BenchMark XT, Tucson, AZ, USA) in accordance with the manufacturer's instructions. Chromogenic diaminobenzidine (DAB) was used for signal detection, and the cells were counterstained with Harris hematoxylin. The negative controls were incubated with the same concentration of immunoglobulin (IgG1; Dako, Ely, United Kingdom) instead of the primary antibody. The positive control was LMM specimen.

The sAC immunostaining algorithm and positivity cut-off established by Magro et al.[8] was used in evaluation. The algorithm has two main patterns: Cytoplasmic and nuclear. Both include 3 patterns. With regard to cytoplasmic staining, the following patterns were assessed: (1) Golgi pattern with a discrete dot-like morphologic structure, (2) Golgi pattern showing a broad granular pattern, and (3) diffuse cytoplasmic pattern that was increased relative to the background staining found within the cytoplasm of nonlesional cells (i.e., keratinocytes within the epidermis). In respect of nuclear staining, the following patterns were observed: (1) nucleolar pattern, (2) focal granular staining within the nucleus, and (3) pan-nuclear pattern, where 80% or more of the nucleus diameter demonstrated a homogeneous pattern of staining, granular pattern of staining, or both. The slides were evaluated together by 2 pathologists and scored with consensus. For all patterns, the cases showing staining in 25% or more tumor cells were considered as positive.

Statistical analysis

Statistical analysis was carried out using the IBM SPSS statistics version 21. Yates' Chi-square, Pearson Chi-square exact test, and Pearson Chi-square tests were considered statistically significant if P < 0.05. Spearman correlation was used for correlation analysis. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were also evaluated.

   Results Top

Twenty-nine of 50 invasive cutaneous melanoma cases were female and 21 were male. The mean age was 61.1 years. Half of the cases were Clark level IV. Seven cases were Clark level II, 14 cases were Clark level III, and 4 cases were Clark level V. The Breslow index ranged from 0.38 mm to 28 mm (average = 5.21 mm). Tumor localization was determined: 34% in the head and neck region, 32% in the lower limb, 24% in the trunk, and 10% in the upper limb. The most common pathologic tumor stage was pT4 (20 cases). Four cases were pT1, 10 cases were pT2, and 16 cases were pT3.

Of 50 benign melanocytic proliferations, 29 were female and 21 were male. The mean age was 26.1 years. Tumor localizations were head and neck (38%), trunk (36%), lower limb (20%), and upper limb (6%).

The most common R21 positivity was observed in the pan-nuclear pattern [Figure 1] and [Figure 2] in the cases of invasive cutaneous melanoma. All LMM cases (9/9), most of SSM (6/9) and NM (17/24), and more than half of ALM (4/7) were positive. There was no difference in staining in the superficial and deep areas of the tumor. Diffuse cytoplasmic positivity [Figure 2] was the second common pattern (5 NM, 4 LMM, 3 SSM, 2 ALM). Pan-nuclear positivity was also present in 12 of these 14 cases. The Golgi expression pattern was not observed except 1 NMM case, which showed dot-like Golgi expression. None of the cases were positive for broad Golgi expression. It was seen focally (not exceeding 25%) in only one NMM [Figure 3]. Ten cases were negative for all patterns in the melanoma subgroups (no staining exceeding 25%).
Figure 1: R21 immunostaining in pan-nuclear pattern in malignant melanocytes. Nuclear staining observed in some of the normal keratinocytes in the epidermis (arrowhead) (×200)

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Figure 2: R21 immunostaining in pan-nuclear pattern and accompanying cytoplasmic positivity. Nuclear staining observed in some of the normal keratinocytes in the epidermis (arrowhead) (×200)

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Figure 3: Golgi pattern, showing a broad granular pattern (×400)

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In 50 cases of nevi, 29 of them were evaluated as negative (no staining exceeding 25%). Positive cases (12 cases) mostly showed staining in the dot-like Golgi pattern [Figure 4]. Nucleolar and granular nuclear positivity was observed in 4 cases. Five cases were positive as pan-nuclear pattern. Diffuse cytoplasmic pattern was not observed even less than 25% of lesional cells. Staining in the broad Golgi pattern did not exceed 25% in any case. There was staining in 6 cases (3 dysplastic melanocytic nevi, 2 Spitz nevi, 1 common melanocytic nevi) that did not reach the positive cut-off limit.
Figure 4: Cytoplasmic background staining of R21 in skin adnexa (arrow). Dot-like Golgi pattern, characterized by small dot-like Golgi positivity (arrowhead) around the nucleus, was most common pattern in nevi cases (×400)

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Pale background staining was found within the cytoplasm of epidermal keratinocytes, skin adnexa, and nevus cells [Figure 4]. Besides this background staining, nuclear staining was observed in some keratinocytes (<10%) [Figure 1] and [Figure 2]. Positive cases are listed in [Table 1] according to the sAC immunostaining algorithm.
Table 1: Soluble adenylyl cyclase immunostaining positivity*

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Statistically R21 was pan-nuclear positive in a considerably higher proportion of melanoma than melanocytic nevi (P < 0.001). Sensitivity was 72%, specificity was 90%, PPV was 87%, and NPV was 76% for the diagnosis of melanoma. The dot-like Golgi pattern was detected in a considerably higher proportion of melanocytic nevi than melanoma (P < 0.001).

There was no statistically significant difference in pan-nuclear positivity between melanoma subtypes (P = 0.133). And pan-nuclear R21 expression was not associated with Breslow depth, Clark level, pT stage, patient's age or tumor localization (P > 0.05).

   Discussion Top

Most cutaneous melanomas contain driver mutations that activate the mitogen-activated protein kinase (MAPK) pathway. The classical MAPK pathway consists of the signaling proteins RAS, RAF, MEK, and ERK, which sequentially transmit proliferative signals from the cell surface receptor tyrosine kinase (RTK). c-KIT is an RTK found in melanocytes and when it binds to its ligand, stem cell factor (SCF), small GTPase activates RAS. RAS activates the RAF kinase (MAP3K) family. Members of the RAF kinase family, including BRAF and CRAF, then activate MEK (MAP2K), which activates ERK. ERK phosphorylates and activates numerous downstream targets, including MITF, and contributes to the regulation of numerous cellular functions in melanocytes. Multiple levels of interconnection between the cAMP and MAPK pathways have been described. Cross-talk between the MAPK pathway and the cAMP signal indicates a possible role of cAMP signaling in melanoma formation. However, the role of cAMP in melanoma is not fully understood, and studies examining the effect of cAMP signaling in the context of melanoma often report conflicting results.[10]

In mammals, ten different isoforms of adenylyl cyclase have been identified. All of them are transmembrane enzymes except for adenylyl cyclase 10, which is a soluble isoform also known as sAC.[6],[11],[12] sAC protein was first identified in cytosolic extracts of mammalian testis, then it was demonstrated in many mammalian tissues including skin.[13],[14],[15] sAC is mainly regulated by bicarbonate ions and is responsible for the production of cAMP from adenosine triphosphate. cAMP is an intracellular signaling messenger and has an essential role in regulating melanocyte growth, proliferation, and melanogenesis.[15],[16],[17] R21 is a mouse monoclonal antibody directed against amino acids 203–216 of human sAC protein.[7],[18]

Recently, it has been shown by R21 immunostain that sAC is extensively expressed in many components of the skin, including melanocytes, keratinocytes, eccrine ductal cells, and cutaneous nerves.[7] It can be found in intracellular subdivisions of the cell such as mitochondria, cytosol, nucleus, and centrioles.[15],[16],[18] sAC plays a dual role in cell death, which depends on its localization. The cytosolic pool of sAC promotes mitochondrial apoptosis by triggering Bax binding in a PKA-dependent manner.[19] Alternatively, recent studies have shown that sAC is localized within the mitochondria and controls the activity of certain proteins in the respiratory chain, thereby playing a life-sustaining role. When there is a malignant transition in melonocytes or keratinocytes, sAC accumulates in the nucleus.[7],[8]

Differential diagnosis of melanocytic lesions is one of the most critical diagnostic challenges in dermatopathology. Conventional light microscopy is the gold standard in the classification of these lesions, but clear differential diagnosis may not always be possible. In cases where microscopic features are not sufficient to make a definitive diagnosis, the use of IHC markers may be considered, but none of these markers used so far are sufficiently specific or sensitive.[1],[2],[3],[4],[5] As a result of our study, we suggest that R21 is useful in the differential diagnosis.

Magro et al.[8] investigated various expression patterns of sAC in the cell in various melanocytic lesions. In most cases of melanoma, they observed pan-nuclear expression of the tumor cells. In contrast, nuclear expression was low in benign melanocytic lesions. The expression was mostly in a dot-like Golgi expression pattern. Researchers reported that the pan-nuclear staining pattern was specific for melanoma. Li et al.[20] studied on different expression patterns identified and concluded that the nuclear staining pattern had a diagnostic value for ALM. Numerous case reports support the hypothesis that R21 may be a useful marker for melanocytic lesions.[21],[22],[23]

In our study, we investigated the usefulness of R21 expression to differentiate cutaneous melanomas from various melanocytic lesions such as Spitz nevi, common melanocytic nevi, and dysplastic melanocytic nevi. sAC immunostaining algorithm defined by Magro et al.[8] was used. The positivity of the cases was evaluated by adhering to the 25% threshold for all defined patterns. Striking positives were observed in pan-nuclear pattern and dot-like Golgi pattern. Diffuse cytoplasmic positivity was a second expression pattern accompanying pan-nuclear positivity in many of the cases (12/14). Pan-nuclear R21 positivity was detected in 72% of cutaneous melanomas and 10% in melanocytic nevi [Table 1]. It was noteworthy that none of the Spitz nevi were positive. The pan-nuclear expression statistics we obtained in melanomas (sensitivity 72%, specificity 90%, PPV 87%, NPV 76%) were parallel to the results obtained by Magro et al.[8] (sensitivity 75%, specificity 94%, PPV 92%, NPV 81%). Pan-nuclear staining is an important supportive finding in the diagnosis of melanoma. It will help accurate diagnosis in difficult cases. Dot-like Golgi pattern positivity excludes the diagnosis of melanoma. It can be challenging to detect discrete dot-like Golgi pattern, especially when considering the presence of background non-lesional cells staining. Therefore, pan-nuclear pattern has a robust nature and is easy to evaluate. Evaluating these two patterns together, namely, 25% or more tumor cells having pan-nuclear expression or dot-like Golgi expression less than 25%, will increase the sensitivity for melanoma to 85%. This combination makes specificity 57%, PPV 66%, and NPV 79%.

In our study, some cases were evaluated as negative for R21. In some of these cases, staining was below the 25% cut-off value. In some cases, there was no staining. There are very few negative cases in the literature. It is reported that each case shows at least one of the patterns.[8] The reason for this difference may be the use of different antibody clones or different laboratory conditions.

In the previous study, it was observed that ALMs and LMMs showed more common pan-nuclear R21 staining compared to SSMs and NMs.[8] In our study, while all LMM cases showed pan-nuclear R21 positivity, the expression difference between cutaneous melanoma subtypes was not statistically significant (P = 0.133). The restriction of the study is limited number of cases in most of the subtypes.

It has been shown that approximately 10% of keratinocytes have positive nuclei for sAC in normal skin.[7] Consistent with this finding, nuclear keratinocyte staining was also observed in this study. Interestingly, this staining was more pronounced in the epidermis on the surface of the malignant melanoma lesion.

The aim of this study was to evaluate the different expression patterns of the R21 immunostain and to investigate its effectiveness in the differential diagnosis of cutaneous malignant and benign melanocytic lesions. The use of the pan-nuclear model in the differential diagnosis is quite appropriate due to its ease of evaluation and statistical sensitivity and specificity. However, it is important to be aware of other staining patterns as well as pan-nuclear staining to make the correct assessment. While most cases of cutaneous melanoma were pan-nuclear positive, only a few melanocytic nevi showed this positivity. In addition, most Spitz nevi were completely negative for all patterns, and none showed pan-nuclear positivity. In line with the recent literature,[8],[17],[20],[24] our data also showed R21 is a second-generation IHC marker, which is overexpressed in the nuclei of melanoma cells but not in native melanocytes. We concluded that R21 may have utility in the correct categorization of melanocytic lesions.


This article was written from the Medical Specialization Thesis of Dr. Didem Turcan, which was completed under the supervision of Dr. Özgül Paşaoğlu at the Pathology Department of the Eskisehir Osmangazi University Faculty of Medicine. We thank Dr. Jonathan Zippin (Cornell University) for kindly providing R21 antibody.

Financial support and sponsorship

Eskisehir Osmangazi University Scientific Research Projects Coordination Unit supported this study (grant number 201611048).

Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Didem Turcan,
Department of Pathology, Gaziantep Nizip State Hospital, Gaziantep
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_1146_21


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

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