Association of miR-146a-5p and miR-21-5p with Prognostic Features in Melanomas.

BACKGROUND: Cutaneous melanoma (CM) is one of the most lethal tumors among skin cancers and its incidence is rising worldwide. Recent data support the role of microRNAs (miRNAs) in melanoma carcinogenesis and their potential use as disease biomarkers. METHODS: We quantified the expression of miR-146a-5p and miR-21-5p in 170 formalin-fixed paraffin embedded (FFPE) samples of CM, namely 116 superficial spreading melanoma (SSM), 26 nodular melanoma (NM), and 28 lentigo maligna melanoma (LMM). We correlated miRNA expression with specific histopathologic features including Breslow thickness (BT), histological subtype, ulceration and regression status, and mitotic index. RESULTS: miR-146a-5p and miR-21-5p were significantly higher in NM compared to SSM and LMM. The positive correlation between miR-146a-5p and miR-21-5p expression and BT was confirmed for both miRNAs in SSM. Considering the ulceration status, we assessed that individual miR-21-5p expression was significantly higher in ulcerated CMs. The increased combined expression of the two miRNAs was strongly associated with ulceration (p = 0.0093) and higher mitotic rate (≥1/mm2) (p = 0.0005). We demonstrated that the combination of two-miRNA expression and prognostic features (BT and ulceration) can better differentiate cutaneous melanoma prognostic groups, considering overall survival and time-to-relapse clinical outcomes. Specifically, miRNA expression can further stratify prognostic groups among patients with BT ≥ 0.8 mm but without ulceration. Our findings provide further insights into the characterization of CM with specific prognostic features. The graphical abstract was created with BioRender.com.

Folliculotropism in Actinic Keratoses in Patients not Responding to Treatments: A Pilot Study.

Dear Editor, Actinic keratoses (AK) have a high prevalence in the general population, with greater rates in Caucasian patients after the fourth and fifth decades of life (37.5-60.0%) (1,2). Standard histopathologic reporting of AKs does not provide information on the presence of atypical keratinocytes extending to the hair follicle, also defined as folliculotropism (FLC). Commonly, atypical cells in AKs do not present FLC, but this feature can be observed in bowenoid AKs with full-thickness epidermal atypia (3,4). FLC has been considered a possible element enhancing the chances of a progression toward invasive SCC (iSCC). Fernandez-Figueras et al. (3) reported that the depth of FLC in AKs was correlated with the invasiveness of associated iSCC. Pandey et al. (5) reported a positive association between AKs with FLC and history of invasive cutaneous cancer or melanoma, more often in men at an older age. The role of FLC in cutaneous melanoma is still debated, but it is considered a parameter that may correlate with treatment response in lentigo maligna and disease progression or recurrences in invasive tumors (6,7). These studies draw particular attention to the potential role of hair bulge compartment stem cells in favoring tumor progression through the expression of adhesion molecules, cytokines, and growth factor receptors (8). Aks are known to have a high recurrence rate after topical treatment (1). The risk of evolution to an iSCC is not completely clear, but it has been estimated to be around 0.6% at 12 months and up to 2.5% at 48 months (1,3,7). Considering the possible progression and the heavy burden of AK treatments, including the economic burden, it is imperative to focus on histopathologic features associated with treatment failure. The aim of this preliminary study was to assess the histopathologic features, specifically FLC, of AK samples from patients considered "non-responders" to specific topical treatments. A secondary endpoint was to assess the clinical/dermoscopic features. Patients were considered "non-responders" if the lesions persisted after two alternated completed cycles of treatments with ingenol mebutate, imiquimod, diclofenac 3%, or 5-fluoruracil. Patients with a positive history of immunosuppression or genetic diseases were excluded. The study was approved by the local Ethics Committee. Slides of AKs diagnosed at the Laboratory of Dermatopathology, University of Bologna, Italy from January 2016 to October 2018 were reviewed by two dermatopathologists (CM, PAF). 155 "non-responder" AKs of five main histopathologic subtypes were included, classified from grade I to III according to the Roewert-Huber classification (9) (Table 1). The proliferative and atrophic histopathologic subtypes of AKs were detected in 33.6% and 30.4% samples, respectively. FLC was observed in 75.3% of the cases, subdivided into two categories, periadnexal (48.9%) and intraadnexal (26.4%). Periadnexal FLC was detected in 31.0% of atrophic and in 50.3% of proliferative AKs, while intraadnexal FLC was found in 48.7% and 29.2%, respectively (Figure 1, a, b). At dermoscopy, most lesions had been classified as grade I or II (38.8% and 45.8%), and only 15.4% as grade III, showing an unexpected non-response to treatment according to the dermoscopic criteria. In contrast, almost half of the AKs were classified as grade III at histology, revealing a discrepancy between the dermoscopic grading and histological findings in a majority of cases (77.4%) (Figure 2, c, d). Furthermore, atrophic and proliferative AKs accounted for 64.0% of total cases, and these are the variants associated with a higher probability of evolution toward an iSCC (10). The clinical/histological discrepancy has already been reported in the literature (9) and may represent a misleading factor for treatment choice and outcomes. We believe that a comparative analysis with dermoscopy and histology should be performed in non-responding AKs, in order to choose the best therapeutic option. In fact, some superficial treatments (such as cryotherapy) may not provide a good response in deep hair follicles (4). We also suggest encouraging greater focus on FLC and its description in pathology reports. This is a preliminary observational study, but it reinforces the need to further larger clinical studies investigating the role of specific histopathologic parameters in AKs, including FLC, that may correlate with treatment outcomes.

"Paradoxical" p16 overexpression in cutaneous melanoma: Molecular and immunohistochemical analysis of a rare phenomenon with a focus on cell cycle regulatory molecules.

BACKGROUND: One of the most relevant genetic alterations in cutaneous melanoma (CM) is the biallelic inactivation/loss-of-heterozygosis (LOH) of cyclin-dependent kinase inhibitor 2 A (CDKN2A), which results in the immunohistochemical loss of p16 frequently found in CM. However, we recently described a rare case of dermal/deep-seated melanoma arising in giant congenital nevus (DDM-GCN) with p16 overexpression combined with p53 loss and tumor protein 53 (TP53) mutation. Herein, we reported a case series of CM with p16 overexpression and analyzed their clinicopathologic features, immunohistochemical expression of the cell cycle regulatory molecules (CCRM: p53, p21, Cyclin D1, Rb), and mutational landscape. METHODS: We retrospectively tested for p16 all cases of CM diagnosed at our institution between January 1st 2019-April 1st 2022. In CM with p16 overexpression, we reported clinicopathologic features, immunohistochemical results for melanocytic markers and CCRM, and mutational landscape investigated with a next-generation sequencing (NGS) panel. In cases with zonal p16 overexpression, the immunohistochemical assessment for melanocytic markers and CCRM, as well as the NGS analysis have been performed in both components {with and without p16 overexpression [p16(+)c and p16(-)]}. RESULTS: Overexpression of p16 was found in 10/2879 (0.35%) CM [5/10 (50%) diffuse and 5/10 (50%) zonal]. We combined the immunohistochemical results for CCRM and molecular data to classify the cases as follows: a) Group 1 with altered expression of at least one CCRM but no TP53 mutations [3/10 (30%), all with Rb altered/lost]; b) Group 2 with altered expression of at least one CCRM and TP53 mutations [4/10 (40%), all with p53 altered]; c) Group 3 with normal expression of CCRM and no TP53 mutations [3/10 (30%), all with mutations in MAPK pathway genes (NRAS and BRAF)]. In CM with zonal p16 overexpression, the histologic appearance of p16(+)c was heterogeneous, whereas combining CCRM profiles and molecular data the cases could be categorized as follows: a) cases with the same CCRM and molecular profiles in both p16(+)c and p16(-)c; b) cases with p16(+)c showing additional genetic mutations and/or modifications of CCRM expression. CONCLUSIONS: p16 overexpression is a rare event, occurring in advanced-stage, clinically- and histologically-heterogeneous CM. These lesions may be classified into three different groups based on CCRM expression and mutational profiles (including TP53 mutation). The analysis of CM with zonal p16 overexpression suggests that, at least in a subset of cases, this phenomenon could represent a sign of "molecular progression" due to the acquisition of additional genetic mutations and/or modifications of the CCRM profile.

Follicular colonization in melanocytic nevi and melanoma: A literature review.

The lentiginous spread of melanocytes into the hair follicle can be observed in a number of benign melanocytic neoplasms such as in nevi but also in sun-induced melanocytic hyperplasia and melanoma. The follicular colonization by melanocytes in melanoma is classified into three distinct patterns: primary follicular melanoma, melanoma with folliculotropism, and invasive melanoma arising from melanoma in situ with folliculotropism. The role of follicular colonization in melanoma pathologic staging is still a matter of debate though the description of the latter has been recommended by the International Collaboration on Cancer Reporting. In this review, we will discuss the role of follicular colonization in melanoma and melanocytic nevi as well as the facts and controversies regarding this topic.

A Comparative Demographic Study of Atypical Spitz Nevi and Malignant Melanoma.

Spitz tumors are a subset of melanocytic neoplasms characterized by epithelioid or spindled melanocytes(1). The benign nature of the "Spitz nevus" has since been clarified, but the debate regarding Spitzoidtumors (STs) is still ongoing. Spitzoid tumors encompass a wide spectrum of cutaneous lesions ranging from benign Spitz nevus (SN) to Spitzoid melanoma (SM), the latter displaying capacity for widespread metastasis and a potentially lethal outcome (2). The term atypical Spitz tumors (ASTs) refers to melanocytic tumors exhibiting the morphological features of SN, as well as some features associated with malignancy, but not sufficient to classify them as SMs. Currently, histopathology is the gold standard for the diagnosis of STs and cutaneous MM. However, the differential diagnosis between benign and malignant melanocytic lesions with spitzoid features remains challenging (3-6). In order to facilitate the work of clinicians and pathologists, we attempted a comparative clinical and demographic study comparing ASTs and MMs of patients referred to two Italian institutes. Patient data were obtained from two different Italian dermatological centers (Melanoma Registry of the Instituto Dermopaticodell'Immacolata IDI-IRCCS Rome, Lazio and the Skin Cancer Unit of Dermatology, Hospital Sant'Orsola-Malpighi, University of Bologna), from January 2007 to December 2017. Histological reports presenting pre-operative queries of both "atypical Spitz nevi" or "malignant melanoma" and a final diagnosis confirming one of the queries were included in the study. The chi-square test or Mann-Whitney U-test were applied to analyze differences between the groups for categorical variables such as sex, diagnosis, and continuous variables (age). The "anatomic site" variable was classified into three categories as follows: the limbs, trunk, and head/neck. A multivariate binary logistic model was used to investigate if the anatomic site was an independent predictor of MM. Age and sex were considered confounding factors. A total of 504 patients (51.8% men; 48.2% women) met the inclusion study criteria (mean age 52 years, SD = 22.8) (Table 1). 373 were cases of MM and 131 were cases of AST. Mean age of MM cases and AST were 61.2 years old (SD = 17.6) and 25.8 years old (SD = 13.8), respectively. Subjects with MM were predominantly men (58.2% versus 33.6%) (P<0.0001) and older (median age 62 years versus 25 years) (P=0.0001) than subjects with AST. The most frequent anatomic site for MM was the trunk (39.7 %), while the lower limb was the most frequent anatomic site for AST (48.1 %) (P<0.0001). Table 2 shows the multivariable analysis used to assess if anatomic site was an independent predictor of cutaneous melanoma. Multivariate analysis confirmed an increased risk for MM in comparison with AST for both localization on the trunk (OR:2.78; 95 %CI: 1.74-4.45) (P<0.0001) and head/neck (OR:3.20; 95% CI: 1.60-6.38) (P=0.0001). After introducing age (model 1, OR: 2.11; 95% CI: 1.08-4.12) (P=0.003) and sex into the model, the only anatomic site that remained statistically significant was the trunk (model 2, OR: 2.03; 95% CI: 1.0.3-3.99) (P=0.04). The results show that if the lesion was located on the trunk, the probability of being a MM was two times higher than that of AST, independent of sex, age, or center. After stratifying for sex, the effect was stronger for women (OR: 2.72; 95% CI: 1.14-6.50). After stratifying for age, the effect was stronger for younger subjects (<40 years) (OR: 2. 59; 95% CI: 1.20-5.60) (P=0.02). In this study, we focused on the clinical-epidemiological data in an attempt to improve the identification of nodular melanocytic lesions by providing clinicians with further information in order to reduce the rate of misdiagnosis and assist in providing critical clinical information to surgeons and pathologists. Consistently with the literature, ASTs were mainly found in young-adult patients (mean age was 25.8 years), in the female sex (66.4%), and were typically located on the lower limbs (48.1%) (3,7-10). MM were found to be slightly more common in male patients (58.2%) in the overall patient group; the mean age at the time of the diagnosis was 61.2 years old, and the majority of lesions were located on the trunk (39.7%). These data were similar to those reported by other authors (11-13). ASTs cases were mainly women and younger than MM cases, and were typically located on the lower limbs (Figure 3 and Figure 4). Nodules located on the trunk resulted in a two times greater risk of MM in comparison with AST. In summary, distinguishing ASTs from MMs is often challenging, and histopathology remains the diagnostic gold standard for melanocytic neoplasms, but a specific clinical framework may help surgeons, pathologists, and clinicians to correctly diagnose and manage these lesions in children and adults.

Cutaneous Melanomas: A Single Center Experience on the Usage of Immunohistochemistry Applied for the Diagnosis.

Cutaneous melanoma (cM) is the deadliest of all primary skin cancers. Its prognosis is strongly influenced by the stage at diagnosis, with early stages having a good prognosis and being potentially treatable with surgery alone; advanced stages display a much worse prognosis, with a high rate of recurrence and metastasis. For this reason, the accurate and early diagnosis of cM is crucial-misdiagnosis may have extremely dangerous consequences for the patient and drastically reduce their chances of survival. Although the histological exam remains the "gold standard" for the diagnosis of cM, a continuously increasing number of immunohistochemical markers that could help in diagnosis, prognostic characterization, and appropriate therapeutical choices are identified every day, with some of them becoming part of routine practice. This review aims to discuss and summarize all the data related to the immunohistochemical analyses that are potentially useful for the diagnosis of cM, thus rendering it easier to appropriately applicate to routine practice. We will discuss these topics, as well as the role of these molecules in the biology of cM and potential impact on diagnosis and treatment, integrating the literature data with the experience of our surgical pathology department.

Female melanoma and estrogen receptors expression: an immunohistochemical pilot study.

Epidemiologic data highlight sex differences in melanoma outcome. A putative role of sex hormones is still under investigation. Very few laboratory investigations have focused on the level of expression of estrogen receptors in melanoma. We evaluated the presence of estrogen receptors alpha (ERα) and beta (ERß) in melanoma specimens from female patients with a previous history of breast carcinoma (BC). Moreover, another group of female patients undergoing ovarian stimulation (OS) were also compared to two control groups matched for age and melanoma staging. The study was performed at the IRCCS Policlinico di Sant'Orsola Hospital's Melanoma Unit from January 2017 to December 2019. The nuclear and cytoplasmatic immunohistochemical staining was evaluated and scored by the percentage of stained tumour cells: 0 (≤20%), 1 (21-50%) or 2 (≥50%). Twenty-eight specimens were analysed. ERß nuclear presence was detected in all cases of women with a history of breast cancer. Cytoplasmatic ERß was clearly expressed with a score of 2 in seven cases. In the respective control group, nuclear and cytoplasmatic ERß expression was much lower. A cytoplasmatic ERα positivity was also detected in almost all cases. In the second group of women who experienced ovarian stimulation for Assisted Reproductive Technology (ART), a lower abundance of nuclear ERs was detected. Conversely, cytoplasmatic ERß and α expression ranged widely. Melanoma of women treated with anti-estrogen therapy is generally more prone to express estrogen receptors compared with women of the same age and CM staging but also compared with women in fertile age with and without a history of OS.

Dysplastic nevi and melanoma: microRNAs tell a divergent story.

BACKGROUND: dysplastic nevi (DN) share some clinical and histological features with melanoma and have been considered intermediate lesions toward malignant transformation. However, scientific evidence of DN representing melanoma precursors is still incomplete, and many observations pointed toward their being a distinct biological entity. The current definition of DN is also confusing and the practical consequence of this uncertainty is the excessive excision of DN with severe atypia. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and whose global expression can classify normal and pathological tissues. OBJECTIVES: given these considerations, we decided to perform a small RNA profiling study in a group of DN and invasive melanomas obtained from the same patient, to assess tumor evolution according to the global microRNA expression. METHODS: we performed a small-RNA sequencing of 6 DN, 2 congenital nevi and 4 cutaneous melanomas obtained from 4 subjects and evaluated the global miRNA expression correlation between samples. RESULTS AND CONCLUSIONS: the hierarchical clustering and principal component analyses of global miRNA expression, independently grouped together DN and their matching congenital nevi and showed a divergence of DN miRNA profile from melanoma. Our study suggests that DN have a peculiar and different miRNA expression profile compared to melanomas developed in the same patient, thus supporting the hypothesis that DN are distinct biological entities and not melanoma precursors.

The EORTC protocol for sentinel lymph node biopsy (SLNB) reveals a high number of nodal nevi and a strong association with nevus-associated melanoma.

BACKGROUND: The diagnosis of nodal nevi (NN) is challenging as they mimic melanoma metastases (MM), with a detection rate mostly ranging between 1% and 11% in sentinel lymph node biopsy (SLNB). Herein, we assessed the incidence of NN and the association with the clinical-pathological features of primary melanoma, adopting the updated European Organisation for Research and Treatment of Cancer (EORTC) protocol for SLNB. METHODS: All cases of paired melanoma and SLNB were retrospectively evaluated (April 2019-May 2020). Appropriate statistical tests were adopted, with significant variables included in the logistic regression model. RESULTS: 81 patients and a total of 186 lymph nodes (LNs) were included. Eleven patients had only NN and 4 had both NN and MM (18.5%); 29 LNs (15.6%) showed at least one NN and 12 (6.5%) showed more than one NN (a total amount of 43 NN was detected). All NN and none MM stained for p16. NN were associated with age < 60 years (p: 0.042), no ulceration (p: 0.025) and nevus-associated melanoma (NAM) (p: 0.018), with this latter being the only predictor at the logistic regression model (p: 0.022). CONCLUSIONS: The updated EORTC protocol shows a high number of NN and highlights a strong association with NAM.

Hematoxylin and eosin or double stain for CD34/SOX10: Which is better for the detection of lymphovascular invasion in cutaneous melanoma?

BACKGROUND: Lymphovascular invasion (LVI) is considered an unfavorable prognostic factor in cutaneous melanoma (CM). However, its detection by hematoxylin and eosin (H&E) is challenging, with discordant data about its association with clinical-pathological features and no previous studies investigating the inter- (IrOA) and intra-observer (IaOA) agreement. Herein, we tested H&E and double staining (DS) for CD34/SOX10 to detect the LVI in a cohort of 92 CMs, evaluating the IrOA, the IaOA, and the association with the other clinical-pathological features. METHODS: Five authors independently evaluated 92 consecutive and retrospectively enrolled cases of CMs. We assessed the IrOA (Fleiss's Kappa/FK and intraclass correlation coefficient/ICC) and the IaOA (Cohen's Kappa/CK) with both H&E and CD34/SOX10. Furthermore, we compared the LVI assessment with the two stains and analyzed the association with other clinical-pathological features [χ2 tests for dichotomous and categorical data; Student t-test (normal distribution) and Mann-Whitney U-test (non-normal distribution) for continuous data]. RESULTS: The IrOA was almost identical with H&E (FK=0.446; ICC=0.805) and CD34/SOX10 (FK=0.454; ICC=0.810); by contrast, the IaOA was higher with H&E for one pathologist (CK: 0.809) and with CD34/SOX10 for the other one (CK: 0.563). Applying previously defined criteria, LVI was detected in 10 (9.2%) and 11 (10.1%) cases with H&E and CD34/SOX10, respectively (p = 1.000). Both H&E and CD34/SOX10 were significantly associated with vertical growth phase (H&E, p: 0.014; CD34/SOX10, p: 0.010), mitosis ≥ 1/mm2 (H&E, p: 0.000; CD34/SOX10, p: 0.004), pT (H&E, p: 0.000; CD34/SOX10, p: 0.001), Breslow thickness (H&E, p: 0.000; CD34/SOX10, p: 0.001), and lymph node and/or distant metastasis (H&E, p: 0.005; CD34/SOX10, p: 0.000); only H&E was associated with ulceration (p: 0.002) and distant metastasis (p: 0.000), conversely, only CD34/SOX10 was associated with lymph node metastasis (p: 0.003). CONCLUSIONS: CD34/SOX10 does not improve the IrOA and the IaOA of the LVI assessment in CM; furthermore, H&E and CD34/SOX10 show a similar profile of association with the other unfavorable clinical-pathological features of CM. As result, CD34/SOX10 could be a redundant diagnostic tool if applied for the prognostic characterization of not-selected CM in a routine diagnostic scenario.