Digital spatial proteomic profiling reveals immune checkpoints as biomarkers in lymphoid aggregates and tumor microenvironment of desmoplastic melanoma.

BACKGROUND: Desmoplastic melanoma (DM) is a rare melanoma subtype characterized by dense fibrous stroma, a propensity for local recurrence, and a high response rate to programmed cell death protein 1 (PD-1) blockade. Occult sentinel lymph node positivity is significantly lower in both pure and mixed DM than in conventional melanoma, underscoring the need for better prognostic biomarkers to inform therapeutic strategies. METHODS: We assembled a tissue microarray comprising various cores of tumor, stroma, and lymphoid aggregates from 45 patients with histologically confirmed DM diagnosed between 1989 and 2018. Using a panel of 62 validated immune-oncology markers, we performed digital spatial profiling using the NanoString GeoMx platform and quantified expression in three tissue compartments defined by fluorescence colocalization (tumor (S100+/PMEL+/SYTO+), leukocytes (CD45+/SYTO+), and non-immune stroma (S100-/PMEL-/CD45-/SYTO+)). RESULTS: We observed higher expression of immune checkpoints (lymphocyte-activation gene 3 [LAG-3] and cytotoxic T-lymphocyte associated protein-4 [CTLA-4]) and cancer-associated fibroblast (CAF) markers (smooth muscle actin (SMA)) in the tumor compartments of pure DMs than mixed DMs. When comparing lymphoid aggregates (LA) to non-LA tumor cores, LAs were more enriched with CD20+B cells, but non-LA intratumoral leukocytes were more enriched with macrophage/monocytic markers (CD163, CD68, CD14) and had higher LAG-3 and CTLA-4 expression levels. Higher intratumoral PD-1 and LA-based LAG-3 expression appear to be associated with worse survival. CONCLUSIONS: Our proteomic analysis reveals an intra-tumoral population of SMA+CAFs enriched in pure DM. Additionally, increased expressions of immune checkpoints (LAG-3 and PD-1) in LA and within tumor were associated with poorer prognosis. These findings might have therapeutic implications and help guide treatment selection in addition to informing potential prognostic significance.

Pitfalls of PRAME Immunohistochemistry in a Large Series of Melanocytic and Nonmelanocytic Lesions With Literature Review.

ABSTRACT: Preferentially expressed antigen in melanoma (PRAME) immunohistochemistry is currently used to facilitate distinction of benign and malignant melanocytic proliferations. We hypothesized that evaluation of 1 institution's experience with PRAME labeling in a large number of consecutive cases might elucidate additional strengths and potential pitfalls and reveal base rates of positivity versus negativity in 1 academic practice. Pathology reports for all specimens on which PRAME labeling was performed at our institution between January 2021 and May 2022 were retrieved from our database. Eighty percent of conventional malignant melanomas were labeled diffusely positive with PRAME; there were no significant differences in mean age, sex, site, Breslow depth, ulceration status, or American Joint Committee on Cancer pathological tumor stage when comparing diffusely PRAME-positive malignant melanomas with those that lack diffuse labeling. Although no banal melanocytic nevi were labeled with PRAME, 13% of dysplastic nevi were diffusely PRAME positive, with junctional proliferations, severe atypia, male gender, and older age being associated with PRAME positivity. As some but not all ambiguous melanocytic lesions in which malignancy could not be excluded based on morphology alone were diffusely PRAME positive, PRAME's accuracy in predicting malignancy remains unclear to the authors; further study is needed to assess the precision to which PRAME immunohistochemistry can separate benign borderline lesions from their malignant counterparts. Among nonmelanocytic lesions, some poorly differentiated tumors, including atypical fibroxanthomas, can be PRAME positive. This series underscores the importance of clinicopathologic correlation and shows that diffuse PRAME positivity is highest in conventional malignant melanomas (∼80%, or 8 of 10 lesions), is seen in about half of challenging borderline lesions at our institution, and can be observed in lesions diagnosed as dysplastic nevi by our group (∼10% or 1 in 10 lesions), as well as in rare poorly differentiated malignancies.

Merkel Cell Carcinoma: Changing Practice Patterns and Impact on Recurrence-Free and Overall Survival at a Single Institution and Nationally.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. Our report describes the evolution of management and characteristics associated with recurrence, disease-specific survival (DSS) and overall survival (OS) in the treatment of MCC. METHODS: A single institution retrospective review of MCC and SEER data to determine factors associated with RFS, DSS, and OS using a multivariable Cox regression on inverse-probability weighted cohorts. RESULTS: One hundred fifty-nine patients were identified with a median age of 75. Of these, 96% were Caucasian and 60% male. Fifty-eight out of 159 (36%) of all patients were deceased with 21/58 (36%) dead from MCC with a median follow-up of 3.1 years. Institutionally, trends over time demonstrated an increased use of immunotherapy with a concomitant decrease in chemotherapy and decreased use of radiotherapy alone. Institutionally and nationally, there has been increased surgical nodal staging. Institutionally, factors associated with shorter DSS included advanced age, active cigarette smoker (p = 0.002), cT2 disease (p = 0.007), and MCC with unknown primary (p < 0.001). Institutionally, factors associated with shorter OS included ages ≥ 75 years (p < 0.001), an immunocompromised state (p < 0.001), truncal primary site (p = 0.002), and cT2 disease (HR 9.59, p < 0.001). CONCLUSION: Changing practice patterns in MCC management have been driven by the adoption of immunotherapy. Our study highlights that competing risks of mortality in MCC patients likely prevents OS from being an accurate surrogate outcome measure to understand factors associated with DSS.

Predictive accuracy of elevated mitotic rate on lymph node positivity and recurrence in thin melanomas.

Background: Mitotic rate (MR) is considered an important prognostic factor for melanoma but is not currently used for staging because its nuanced effect is not yet well-delineated. We sought to determine if T category-specific MR is predictive of sentinel lymph node (SLN) positivity, recurrence, and melanoma-specific mortality (MSM). Methods: A retrospective review of patients with primary cutaneous melanoma from 1994 to 2020 at a single academic center was performed. Patient demographics and tumor characteristics were recorded. MR was considered elevated for each AJCC8-defined T category if it was ≥2 mitoses/mm2 for T1, ≥4 mitoses/mm2 for T2, ≥6 mitoses/mm2 for T3, or ≥7 mitoses/mm2 for T4. Statistical analysis was performed to assess the predictive accuracy of MR on selected outcomes while controlling for ulceration. Results: Data from 2,984 patients with complete records were analyzed. Along with Breslow thickness and ulceration, elevated MR was associated with higher risk of MSM (HR 1.816, P=0.0001). There was no difference among patients with ulcerated T1 or T2 tumors regardless of MR, but those with non-ulcerated T1 or T2 tumors and elevated MR were more likely to have positive SLNs (P<0.0001 and P=0.0043, respectively) and recurrence (P=0.0007 and P=0.0004, respectively) compared to counterparts with low MR. There were no notable differences for T3 or T4 tumors based on MR. Conclusions: Elevated MR is associated with SLN positivity and recurrence in thin melanomas, independent of ulceration. SLN biopsy should therefore be strongly considered for patients with non-ulcerated lesions <0.8 mm thick if the MR is ≥2 mitoses/mm2.

Chronic UV radiation-induced RORγt+ IL-22-producing lymphoid cells are associated with mutant KC clonal expansion.

Chronic ultraviolet (UV) radiation exposure is the greatest risk factor for cutaneous squamous cell carcinoma (cSCC) development, and compromised immunity accelerates this risk. Having previously identified that epidermal Langerhans cells (LC) facilitate the expansion of UV-induced mutant keratinocytes (KC), we sought to more fully elucidate the immune pathways critical to cutaneous carcinogenesis and to identify potential targets of intervention. Herein, we reveal that chronic UV induces and LC enhance a local immune shift toward RORγt+ interleukin (IL)-22/IL-17A-producing cells that occurs in the presence or absence of T cells while identifying a distinct RORγt+ Sca-1+ CD103+ ICOS+ CD2+/- CCR6+ intracellular CD3+ cutaneous innate lymphoid cell type-3 (ILC3) population (uvILC3) that is associated with UV-induced mutant KC growth. We further show that mutant KC clone size is markedly reduced in the absence of RORγt+ lymphocytes or IL-22, both observed in association with expanding KC clones, and find that topical application of a RORγ/γt inhibitor during chronic UV exposure reduces local expression of IL-22 and IL-17A while markedly limiting mutant p53 KC clonal expansion. We implicate upstream Toll-like receptor signaling in driving this immune response to chronic UV exposure, as MyD88/Trif double-deficient mice also show substantially reduced p53 island number and size. These data elucidate key immune components of chronic UV-induced cutaneous carcinogenesis that might represent targets for skin cancer prevention.

A melanoma patient with macrophage-cancer cell hybrids in the primary tumor, a lymph node metastasis and a brain metastasis.

In 1911 it was proposed that cancer might result from fusion and hybridization between macrophages and cancer cells. Using immunohistochemistry it was determined that essentially all solid tumors expressed macrophage-like molecules on their cell surface. More recently we have used forensic (STR) genetics that allows one to detect DNA from more than one individual in the same sample. By studying biopsies from individuals receiving allogeneic stem cell transplants and later developed solid tumor metastases, we were able to detect both donor and patient DNA sequences suggesting that hybrids were present. Previously we found hybrids in biopsies of a renal cell carcinoma, a melanoma in a brain metastasis and a melanoma in a primary tumor with lymph node metastases. Here we have traced hybrids from a primary melanoma to an axillary lymph node to a brain metastasis. This is the first time that the entire metastatic process has been documented.

Delayed Localized Hypersensitivity Reactions to the Moderna COVID-19 Vaccine: A Case Series.

Importance: In response to the coronavirus disease 2019 (COVID-19) pandemic, 2 mRNA vaccines (Pfizer-BioNTech and Moderna) received emergency use authorization from the US Food and Drug Administration in December 2020. Some patients in the US have developed delayed localized cutaneous vaccine reactions that have been dubbed "COVID arm." Objective: To describe the course of localized cutaneous injection-site reactions to the Moderna COVID-19 vaccine, subsequent reactions to the second vaccine dose, and to characterize the findings of histopathologic examination of the reaction. Design, Setting, and Participants: This retrospective case series study was performed at Yale New Haven Hospital, a tertiary medical center in New Haven, Connecticut, with 16 patients referred with localized cutaneous injection-site reactions from January 20 through February 12, 2021. Main Outcomes and Measures: We collected each patient's demographic information, a brief relevant medical history, clinical course, and treatment (if any); and considered the findings of a histopathologic examination of 1 skin biopsy specimen. Results: Of 16 patients (median [range] age, 38 [25-89] years; 13 [81%] women), 14 patients self-identified as White and 2 as Asian. The delayed localized cutaneous reactions developed in a median (range) of 7 (2-12) days after receiving the Moderna COVID-19 vaccine. These reactions occurred at or near the injection site and were described as pruritic, painful, and edematous pink plaques. None of the participants had received the Pfizer-BioNTech vaccine. Results of a skin biopsy specimen demonstrated a mild predominantly perivascular mixed infiltrate with lymphocytes and eosinophils, consistent with a dermal hypersensitivity reaction. Of participants who had a reaction to first vaccine dose (15 of 16 patients), most (11 patients) developed a similar localized injection-site reaction to the second vaccine dose; most (10 patients) also developed the second reaction sooner as compared with the first-dose reaction. Conclusions and Relevance: Clinical and histopathologic findings of this case series study indicate that the localized injection-site reactions to the Moderna COVID-19 vaccine are a delayed hypersensitivity reaction. These reactions may occur sooner after the second dose, but they are self-limited and not associated with serious vaccine adverse effects. In contrast to immediate hypersensitivity reactions (eg, anaphylaxis, urticaria), these delayed reactions (dubbed "COVID arm") are not a contraindication to subsequent vaccination.

NCCN Guidelines® Insights: Melanoma: Cutaneous, Version 2.2021.

Over the past few years, the NCCN Guidelines for Melanoma: Cutaneous have been expanded to include pathways for treatment of microscopic satellitosis (added in v2.2020), and the following Principles sections: Molecular Testing (added in v2.2019), Systemic Therapy Considerations (added in v2.2020), and Brain Metastases Management (added in v3.2020). The v1.2021 update included additional modifications of these sections and notable revisions to Principles of: Pathology, Surgical Margins for Wide Excision of Primary Melanoma, Sentinel Lymph Node Biopsy, Completion/Therapeutic Lymph Node Dissection, and Radiation Therapy. These NCCN Guidelines Insights discuss the important changes to pathology and surgery recommendations, as well as additions to systemic therapy options for patients with advanced disease.

Dermatofibrosarcoma protuberans in pregnancy: a case series and review of the literature.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous sarcoma, which has been reported in pregnancy. This case series reports the clinical and histopathological findings of DFSP in pregnancy. METHODS: Eighteen cases of DFSP, including six unreported cases and 12 cases from the literature, were identified. Age, anatomic location, tumor size, changes in tumor characteristics during pregnancy, histopathological features, and treatment were recorded. Follow-up data, when available, were noted. RESULTS: The average age of the cohort was 30.6 years (range 19-38). Ten tumors (55.6%) were located on the trunk, four (22.2%) on the head and neck, three (16.7%) on the extremities, and one (5.6%) in the genitalia. Most tumors demonstrated features of conventional DFSP (12/18, 66.7%), while the remaining were identified as DFSP with fibrosarcomatous (FS) change (3/18, 16.7%), atrophic DFSP (2/18, 11.1%), and myxoid DFSP (1/18, 5.6%). Treatment was reported in 17 cases, at least nine of which were treated postpartum. Ten patients were treated with excision, while seven underwent Mohs micrographic surgery. Three patients recurred on follow-up, one with local recurrence and two with distant metastasis. CONCLUSIONS: DFSP can undergo enlargement or change in size or color in pregnancy, possibly mediated by hormones. While the majority of cases in this series represented conventional DFSP, unusual clinical and histopathological variants were also present. Treatment in most cases can be safely delayed until after delivery, but recurrent or very large tumors may require treatment prepartum. Close monitoring for recurrence or metastasis is advised.

Cutaneous Involvement in Plasma Cell Myeloma.

OBJECTIVES: Plasma cell myeloma (PCM) involving skin is rare and occurs in 1% to 4% of patients with PCM. We evaluated the clinicopathologic features, cytogenetic findings and clinical follow-up in a series of PCM cases with cutaneous involvement. METHODS: Cases of PCM with cutaneous involvement were retrospectively reviewed with clinical data. RESULTS: Skin involvement in PCM occurred in older individuals (mean, 75 years) and was more frequent in men (7/10 patients). All cases showed bone marrow involvement preceding the cutaneous lesions. Histopathologically, the infiltrate was plasmacytic (n = 5) or primitive or plasmablastic (n = 4), and 1 case showed predominantly lymphoplasmacytic features with cyclin D1 immunoreactivity and CCND1 gene rearrangement. Concurrent amyloid deposition was seen in one biopsy, and another case demonstrated coexisting squamous cell carcinoma. The most common immunophenotype was CD138+, CD20-, and CD56+ with light chain restriction. Cytogenetic analysis (available for 7 cases) showed multiple hyperdiploid abnormalities. Follow-up was available for 8 cases (mean, 42 months; range, 11-156 months) and showed short-term disease-related death in 7 of 8 patients. CONCLUSIONS: Cutaneous involvement in PCM demonstrates a diverse cytomorphologic spectrum with plasmacytic, plasmablastic, or lymphoplasmacytic features and may show concurrent amyloid deposition or neoplasms such as squamous cell carcinoma. Cutaneous involvement typically occurs late in the course of the disease and likely portends poor outcome.

Acral Lentiginous Melanoma: Do Surgical Approach and Sentinel Lymph Node Biopsy Matter?

Management of acral lentiginous melanoma (ALM) remains controversial. Traditionally, ALM was managed with digit amputation (DA), resulting in significant morbidity, but recent evidence has advocated for digit sparing management. Furthermore, the significance of nodal metastasis for ALM is not well reported. The aims of this study were to determine if surgical approach for primary ALM impacts outcomes and to evaluate the predictive value of nodal status for ALM. METHODS: Patients with localized ALM diagnosed from 1982 to 2017 were retrospectively identified. Clinicopathologic characteristics were correlated with surgical approach, nodal metastasis, overall survival, and recurrence-free survival. RESULTS: There were 47 patients with ALM. Median age was 59 years, and median thickness was 3 mm. 51% of patients underwent wide local excision (WLE), 27.9% underwent DA, and 20.9% underwent partial digit amputation (PDA). ALM on the hand versus foot (OR: 12.7, 95%, confidence interval (CI), 2.0-80.1; P = 0.007) and subungual versus nonsubungual location (OR: 28.0, 95% confidence interval, 2.7-295.7; P = 0.006) were significantly associated with surgical approach (DA and PDA versus WLE). There were no significant differences in overall survival or recurrence-free survival between DA, PDA, or WLE cases (P = 0.481 and P = 0.778, respectively). There were no significant differences in overall survival or recurrence-free survival based on nodal status (P = 0.562 and P = 0.136, respectively). CONCLUSIONS: No significant differences in overall survival or recurrence-free survival were seen between ALM patients treated with DA, PDA, and WLE. Given these results, PDA or WLE may be options in select patients with digital ALM; however, careful consideration must be taken when deciding on the surgical approach.

Recent Advances in Studies of Skin Color and Skin Cancer.

The relationship between skin color and skin cancer is well established: the less melanin in one's skin the greater the risk for developing skin cancer. This review is in two parts. First, we summarize the current understanding of the cutaneous pigmentary system and trace melanin from its synthesis in the pigment cell melanosomes through its transfer to keratinocytes. We also present new methods for reducing melanin content in hyper-pigmented areas of skin such as solar lentigenes, melasma, and post-inflammatory hyperpigmentation. Second, we present evidence that at least one mechanism for the development of metastatic melanoma and other solid tumors is fusion and hybridization of leucocytes such as macrophages with primary tumor cells. In this scenario, hybrid cells express both the chemotactic motility of the leucocyte and the de-regulated cell division of the tumor cell, causing the cells to migrate a deadly journey to lymph nodes, distant organs, and tissues.

NCCN Guidelines Insights: Uveal Melanoma, Version 1.2019.

The NCCN Guidelines for Uveal Melanoma include recommendations for staging, treatment, and follow-up of patients diagnosed with uveal melanoma of the choroid or ciliary body. In addition, because distinguishing between uveal melanoma and benign uveal nevi is in some cases difficult, these guidelines also contain recommendations for workup of patients with suspicious pigmented uveal lesions, to clarify the tests needed to distinguish between those who should have further workup and treatment for uveal melanoma versus those with uncertain diagnosis and low risk who should to be followed and later reevaluated. These NCCN Guidelines Insights describe recommendations for treatment of newly diagnosed nonmetastatic uveal melanoma in patients who have already undergone a complete workup.

Janus kinase inhibition induces disease remission in cutaneous sarcoidosis and granuloma annulare.

BACKGROUND: Sarcoidosis and granuloma annulare (GA) are cutaneous granulomatous disorders that can be difficult to treat. There is evidence of underlying Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway activation in sarcoidosis, suggesting that JAK inhibition might be effective. OBJECTIVE: To evaluate treatment with tofacitinib, a JAK inhibitor, in patients with recalcitrant sarcoidosis and GA. METHODS: A prospective evaluation of tofacitinib in 4 consecutive patients with recalcitrant cutaneous sarcoidosis (n = 3) and generalized GA (n = 1) was conducted. Immunohistochemical analysis of skin biopsy specimens from other patients with sarcoidosis (n = 21) and GA (n = 17) was performed to characterize patterns of JAK-STAT pathway activation. RESULTS: Tofacitinib resulted in a mean improvement in the baseline Cutaneous Sarcoidosis Activity and Morphology Instrument and Granuloma Annulare Scoring Index scores of 96% (standard deviation, 2%). Histologic resolution of disease was documented in all patients (3 out of 3) who had skin biopsies while receiving therapy. Constitutive STAT1 and STAT3 activation was observed in both sarcoidosis and GA, albeit in different patterns. Signal regulatory protein α may explain the differences in JAK-STAT signaling between sarcoidosis and GA. LIMITATIONS: The study is limited by the small number of participants. CONCLUSIONS: Tofacitinib resulted in dramatic improvement in 4 patients with cutaneous sarcoidosis and GA. Larger studies are underway to better understand this effect.

Human keratinocyte carcinomas have distinct differences in their tumor-associated macrophages.

Cutaneous squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) have different clinical behaviors, despite both being keratinocyte carcinomas mainly caused by ultraviolet radiation. Whether these distinct features are associated with tumor-associated macrophages (TAMs) is largely unknown. The main goal of this study was to conduct a comprehensive analysis of density and polarization states of TAMs in SCCs versus BCCs. The role of lactic acid in TAM polarization in SCC versus BCC was examined. We found that SCCs have a higher density of CD68 + TAMs compared to BCCs. TAMs in SCCs express higher levels of TAM-associated markers (arginase-1, MMP9, CD40 and CD127) than those in BCCs. Interestingly, differential expression of TAM-associated markers between SCCs and BCCs was reproduced in human monocytic THP-1 cells stimulated with SCC- or BCC-conditioned media. Analysis of soluble factor(s) in these tumors further revealed that SCCs have a significantly higher concentration of lactic acid than BCCs, and lactic acid was sufficient to upregulate TAM markers. Our results demonstrate that TAMs in SCCs versus BCCs differ in density and polarization states, which can be determined by soluble factors including tumor-derived lactic acid. These differences in TAMs may contribute to the distinct clinical behaviors of SCCs versus BCCs. This work was supported by grants from the National Institutes of Health and the Doris Duke Charitable Foundation. RESEARCH IN CONTEXT: Few studies have studied tumor-associated macrophages in the context of SCC versus BCC. It has been demonstrated that macrophages mobilize to the epidermis after being exposed to ultraviolet-B radiation and produce interleukin-10 (IL-10). It has also been shown that the production of IL-10 results in the evasion of T cell-mediated immunity in BCCs and SCCs. However, the relationship between TAMs and the clinical behaviors of SCCs and BCCs remains largely unclear. Our study shows that despite their similar origins, human cutaneous SCCs and BCCs are considerably different in their TAMs. To our knowledge, these results provide the first evidence of differential TAM density and polarization in SCCs versus BCCs, which may contribute to their characteristic clinical behaviors. Future studies are necessary to elucidate the mechanisms by which TAMs influence these cancers with the goal of developing therapies tailored to each type of malignancy.

Cutaneous Melanoma, Version 2.2019, NCCN Clinical Practice Guidelines in Oncology.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Cutaneous melanoma have been significantly revised over the past few years in response to emerging data on immune checkpoint inhibitor therapies and BRAF-targeted therapy. This article summarizes the data and rationale supporting extensive changes to the recommendations for systemic therapy as adjuvant treatment of resected disease and as treatment of unresectable or distant metastatic disease.

Detection of Borrelia in Ixodes scapularis ticks by silver stain, immunohistochemical and direct immunofluorescent methods.

BACKGROUND: Lyme disease (LD) is one of the most common tick-borne diseases caused by several Borrelia species of spirochetes. Ixodes scapularis is responsible for the transmission of LD in the northeastern United States. The rate of infection varies with the duration of tick attachment to the host; however, this information may be unknown. In skin biopsies, it is often difficult to identify spirochetes. Testing of ticks is not routinely performed. Treatment is established by clinical presentation. OBJECTIVE: To test paraffin-embedded I. scapularis ticks for Borrelia by different methods. MATERIALS/METHODS: We examined 20 paraffin-embedded ticks by silver stain, immunohistochemical (IHC) and direct immunofluorescent (DIF) methods and compared the percentage of positivity with DIF results from the Connecticut Agricultural Experiment Station. RESULTS: The results were similar by DIF, which proved to be the most sensitive method, followed by the silver stain and IHC. CONCLUSION: We found that the identification of spirochetes in paraffin-embedded ticks was less difficult than in tissue with a comparable turnaround time to that of a routine biopsy. Timely identification of Borrelia in ticks may influence the clinical management of the patients.