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Background The definitive diagnosis of melanocytic neoplasia using solely histopathologic evaluation can be challenging. Novel techniques that objectively confirm diagnoses are needed. This study details the development and validation of a melanoma prediction model from spatially resolved multivariate protein expression profiles generated by Imaging Mass Spectrometry (IMS). Methods Three board-certified dermatopathologists blindly evaluated 333 samples. Samples with triply concordant diagnoses were included in this study, divided into a training set (n = 241) and a test set (n = 92). Both the training and test sets included various representative subclasses of unambiguous nevi and melanomas. A prediction model was developed from the training set using a linear support vector machine (SVM) classification model. Results We validated the prediction model on the independent test set of 92 specimens (75 classified correctly, two misclassified, and 15 indeterminate). IMS detects melanoma with a sensitivity of 97.6% and a specificity of 96.4% when evaluating each unique spot. IMS predicts melanoma at the sample level with a sensitivity of 97.3% and a specificity of 97.5%. Indeterminate results were excluded from sensitivity and specificity calculations. Conclusion This study provides evidence that IMS-based proteomics results are highly concordant to diagnostic results obtained by careful histopathologic evaluation from a panel of expert dermatopathologists. This article is protected by copyright. All rights reserved.

T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive neoplasm derived from post-thymic T-cells. Patients are typically middle aged with a slight male predominance who present with a high white blood cell count, hepatosplenomegaly, lymphadenopathy, and other symptoms typically associated with leukemia. Although cutaneous involvement has been reported in up to 30% of cases of T-PLL, to our knowledge, none have presented with a presentation resembling livedoid vasculopathy. In the correct clinical context, an underlying hematolymphoid neoplasm should be included in the differential diagnosis of a patient presenting with livedoid vasculopathy.

Context.— Accurate diagnosis of melanocytic lesions is fundamental for appropriate clinical management. Objective.— To evaluate the degree of discordance, if any, between histopathologic diagnoses of melanocytic lesions at referring institutions and at a tertiary referral cancer center and the potential impact of such discordance on clinical management. Design.— We retrospectively identified all patients referred to our comprehensive cancer center for evaluation of a melanocytic lesion from January 2010 to January 2011. For each patient, the histopathologic diagnosis from the referring institution was compared with the histopathologic diagnosis from a dermatopathologist at our center. Discordances were classified as major if they resulted in a change in clinical management and minor if they did not. Results.— A total of 1521 cases were included. The concordance rates were 72.2% (52 of 72) for dysplastic nevus, 75.0% (15 of 20) for all other types of nevi, 91.1% (143 of 157) for melanoma in situ, 96.1% (758 of 789) for invasive melanoma, and 99.6% (478 of 480) for metastatic melanoma. Major discordances were found in 20.2% of cases (307 of 1521), and minor discordances were found in 48.8% of cases (742 of 1521). Compared with the guideline-based treatment recommendation based on the referring-institution diagnosis, the guideline-based treatment recommendation based on the cancer center diagnosis was more extensive in 5.9% (89 of 1521) of patients and less extensive in 5.0% (76 of 1521) of patients. Conclusions.— Our findings underscore the importance of secondary histopathologic review of melanocytic lesions by expert dermatopathologists because significant changes in the diagnosis, tumor classification, and/or staging may be identified, thus, resulting in critical changes in recommendations for clinical management.

Reports of cutaneous reactions to COVID-19 vaccination are increasing as the scope of vaccination expands, and more data are available for study. For example, a recent review of 414 registry patients with dermatologic reactions following mRNA COVID-19 vaccination demonstrated that cutaneous eruptions often occurred in individuals without a dermatologic history and typically arose in the vaccinated arm within days.1 The same study also reported a female predominance of 90% in cutaneous reactions to COVID-19 vaccination. Adding to this body of evidence, we, herein, describe an eruption of lichen striatus occurring several days after COVID-19 vaccination in a middle-aged woman. Of note, there is 1 recently reported case of lichen planus that developed 48 hours following the receipt of the Pfizer vaccine in a patient with a history of successfully treated lichen planus.2 Our case, however, to our knowledge, is the first reported instance of lichen striatus following COVID-19 vaccination, with more profound implications on the pathogenesis of this dermatologic condition.

BACKGROUND: In melanoma patients, microscopic tumor in the sentinel lymph node biopsy (SLN) increases the risk of distant metastases but the transition from tumor in the SLN to metastatic disease remains poorly understood. METHODS: Fluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g. tumor) or by fluorescent cell subset markers (e.g. CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune related proteins were collected and normalized to account for system variation and ROI area. RESULTS: Tumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8 years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1 year and highest on the patient with OS > 8 years. CONCLUSION: We demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.

Purpose: Over 60% of patients with melanoma respond to immune checkpoint inhibitor (ICI) therapy, but many subsequently progress on these therapies. Second-line targeted therapy is based on BRAF mutation status, but no available agents are available for NRAS, NF1, CDKN2A, PTEN, and TP53 mutations. Over 70% of melanoma tumors have activation of the MAPK pathway due to BRAF or NRAS mutations, while loss or mutation of CDKN2A occurs in approximately 40% of melanomas, resulting in unregulated MDM2-mediated ubiquitination and degradation of p53. Here, we investigated the therapeutic efficacy of over-riding MDM2-mediated degradation of p53 in melanoma with an MDM2 inhibitor that interrupts MDM2 ubiquitination of p53, treating tumor-bearing mice with the MDM2 inhibitor alone or combined with MAPK-targeted therapy. Experimental Design: To characterize the ability of the MDM2 antagonist, KRT-232, to inhibit tumor growth, we established patient-derived xenografts (PDX) from 15 patients with melanoma. Mice were treated with KRT-232 or a combination with BRAF and/or MEK inhibitors. Tumor growth, gene mutation status, as well as protein and protein–phosphoprotein changes, were analyzed. Results: One-hundred percent of the 15 PDX tumors exhibited significant growth inhibition either in response to KRT-232 alone or in combination with BRAF and/or MEK inhibitors. Only BRAFV600WT tumors responded to KRT-232 treatment alone while BRAFV600E/M PDXs exhibited a synergistic response to the combination of KRT-232 and BRAF/MEK inhibitors. Conclusions: KRT-232 is an effective therapy for the treatment of either BRAFWT or PANWT (BRAFWT, NRASWT) TP53WT melanomas. In combination with BRAF and/or MEK inhibitors, KRT-232 may be an effective treatment strategy for BRAFV600-mutant tumors.

SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently recognized entity with undifferentiated rhabdoid morphology and mutations in the switch/sucrose nonfermenting BRG1-associated factors complex. Patients are typically males in their fifth decade with a history of smoking who present with rapidly progressive intrathoracic disease and follow an aggressive clinical course. Metastatic disease is reported in up to 77% of cases; however, to our knowledge, cutaneous metastasis has not been reported nor has it been reported as the initial manifestation of the disease. Recognizing SMARCA4-DTS from other types of epithelioid tumors that involve the skin is clinically relevant, as targeted therapies for SMARC-deficient tumors are currently being investigated and early clinical trial data show therapeutic benefit.

Diagnosis of invasive fungal disease remains an ongoing challenge for clinicians, while continuously evolving treatment regimens increase patient risk for invasive infection. This case highlights how molecular testing led to the diagnosis of co-infection with two fungal pathogens producing invasive disease in a hematopoietic stem cell transplant recipient with graft-versus-host disease (GVHD). Keywords: Invasive fungal infection, Molecular diagnostics, Ibrutinib, Aspergillus, Cunninghamella

Novel oral anticoagulant (NOAC) medications have revolutionized hematology and cardiology. Recently, NOACs have demonstrated additional promise in dermatology. Specifically, rivaroxaban, a direct factor Xa inhibitor NOAC, has been shown to be successful in the treatment of livedoid vasculopathy. Herein, we describe a patient with systemic lupus erythematosus who presented with painful cutaneous vasculopathy, demonstrated on biopsy with occlusive microvascular fibrin thrombi without evidence of concurrent vasculitis. Interestingly, imaging and laboratory studies did not show evidence of hypercoagulability, arterial disease, or embolic disease. The patient’s vasculopathy and pain progressed despite antiplatelet therapy, often considered first-line in cases of microvascular occlusive disease. However, with rivaroxaban therapy, the patient experienced complete regression of her painful lesions, thereby supporting a further role for NOACs in cutaneous vasculopathy treatment. J Drugs Dermatol. 2020;19(5) doi:10.36849/JDD.2020.4684.

Trichoblastic carcinosarcoma is a rare biphasic adnexal neoplasm. This case report chronicals the eighth occurrence of this tumor published in the English literature and provides a review of the prior publications. Clinically, this tumor presents as an isolated, rapidly growing lesion in elderly patients and is usually cured by complete surgical excision, with no evidence of recurrence or metastasis at follow-up (7/8 cases). Histopathologically, trichoblastic carcinosarcoma is dermal-based, with an epithelial component of basal cells and a mesenchymal component of spindle cells, both of which display malignant features. In addition to a morphologic description of trichoblastic carcinosarcoma, a discussion of the differential diagnoses, including other biphasic neoplasms, is also included. The small number of cases of trichoblastic carcinosarcoma is most likely secondary to under-recognition and underreporting and a larger case volume is needed to more accurately assess the clinical course and treatment strategies.

Checkpoint inhibitors produce durable responses in numerous metastatic cancers, but immune-related adverse events (irAEs) complicate and limit their benefit. IrAEs can affect organ systems idiosyncratically; presentations range from mild and self-limited to fulminant and fatal. The molecular mechanisms underlying irAEs are poorly understood. Here, we report a fatal case of encephalitis arising during anti-programmed cell death receptor 1 therapy in a patient with metastatic melanoma. Histologic analyses revealed robust T cell infiltration and prominent programmed death ligand 1 expression. We identified 209 reported cases in global pharmacovigilance databases (across multiple cancer types) of encephalitis associated with checkpoint inhibitor regimens, with a 19% fatality rate. We performed further analyses from the index case and two additional cases to shed light on this recurrent and fulminant irAE. Spatial and multi-omic analyses pinpointed activated memory CD4+ T cells as highly enriched in the inflamed, affected region. We identified a highly oligoclonal T cell receptor repertoire, which we localized to activated memory cytotoxic (CD45RO+GZMB+Ki67+) CD4 cells. We also identified Epstein-Barr virus-specific T cell receptors and EBV+ lymphocytes in the affected region, which we speculate contributed to neural inflammation in the index case. Collectively, the three cases studied here identify CD4+ and CD8+ T cells as culprits of checkpoint inhibitor-associated immune encephalitis.

Low-grade fibromyxoid sarcoma (LGFMS) is a rare soft tissue sarcoma that usually presents as a deep-seated tumor in young adults; however, they can occur on superficial sites, mostly documented in pediatric age groups. LGFMS presenting on acral sites is not highly emphasized in the general pathology or dermatopathology literature. The case presented is that of a 30-year-old man with a foot mass that was removed 15 years earlier and subsequently recurred as two masses, the first occurring between the third and fourth toes/metatarsal region and the second over the lateral tarsal region. An excisional biopsy showed a relatively circumscribed, bland spindle cell proliferation with hypocellular and hypercellular zones. The cells showed minimal pleomorphism and lacked mitotic activity. Immunohistochemical analysis showed immunoreactivity for MUC4 and break-apart fluorescence in situ hybridization was positive for FUS rearrangement, confirming the diagnosis of LGFMS. There are multiple spindle cell tumors that occur on acral sites which usually generates a list of differential diagnoses; however, LGFMS is not usually discussed in that anatomic location. Awareness of the occurrence of LGFMS on acral sites is important to avoid misdiagnosis of this deceptively benign-appearing tumor.

Although sentinel lymph node (SLN) biopsy is a standard procedure used to identify patients at risk for melanoma recurrence, it fails to risk-stratify certain patients accurately. Because processes in SLNs regulate anti-tumor immune responses, the authors hypothesized that SLN gene expression may be used for risk stratification. The Nanostring nCounter PanCancer Immune Profiling Panel was used to quantify expression of 730 immune-related genes in 60 SLN specimens (31 positive [pSLNs], 29 negative [nSLNs]) from a retrospective melanoma cohort. A multivariate prediction model for recurrence-free survival (RFS) was created by applying stepwise variable selection to Cox regression models. Risk scores calculated on the basis of the model were used to stratify patients into low- and high-risk groups. The predictive power of the model was assessed using the Kaplan–Meier and log-rank tests. During a median follow-up period of 6.3 years, 20 patients (33.3%) experienced recurrence (pSLN, 45.2% [14/31] vs nSLN, 20.7% [6/29]; p = 0.0445). A fitted Cox regression model incorporating 12 genes accurately predicted RFS (C-index, 0.9919). Improved RFS was associated with increased expression of TIGIT (p = 0.0326), an immune checkpoint, and decreased expression of CXCL16 (p = 0.0273), a cytokine important in promoting dendritic and T cell interactions. Independent of SLN status, the model in this study was able to stratify patients into cohorts at high and low risk for recurrence (p

Background Responses to anti-PD-1 antibodies (aPD1) have changed the therapeutic landscape of metastatic melanoma, however predictive biomarkers of resistance are lacking. Beta-catenin pathway activation has been inversely correlated with tumor-infiltrating T lymphocytes in melanoma as well as several other solid tumors.1 However, activating mutations involving this pathway are rare, implying that the modulation of upstream Wnt ligand/Fzd receptor (Wnt/Fzd) signaling could be a critical regulator of anti-tumor immunity. Indeed, expression of certain Wnt ligands has been associated with inferior responses to checkpoint inhibitor immunotherapy in metastatic melanoma patients.2 In addition, we have further found tumor-derived paracrine and autocrine Wnt ligand signaling to drive dendritic cell tolerization and to be associated with escape from aPD1 therapy in transgenic mouse models.3 4 No studies to date have focused on the impact of the various regulators and components of proximal Wnt/Fzd receptor signaling on resistance to aPD1 therapy in melanoma patients. We therefore developed a unique Wnt/Fzd pathway panel using Nanostring technology to examine alterations in Wnt ligands, their receptors, and regulators as a predictor of aPD1 resistance. Methods To test whether this panel could identify aPD1 resistant patients, Nanostring analysis was performed on archival FFPE tissue specimens of 12 responding (R) and 12 nonresponding (NR) metastatic melanoma patients (pts) taken prior to aPD1 monotherapy. Response was assessed radiographically by week 12 RECIST criteria. Results Several components of both canonical and non-canonical Wnt ligand signaling, including regulators of autocrine/paracrine signaling, were upregulated in aPD1 NR pts compared to R pts (figure 1, table 1). GZMB, CD8, and IFNG were among cytotoxic T cell related genes upregulated in Rs. Upregulation of SFRP2 and DKK2 in NR pts, classically negative feedback regulators of Wnt ligands, are a reflection of enhanced Wnt ligand signaling activity. Conclusions This study supports the importance of paracrine and autocrine Wnt ligand signaling in the regulation of effector T cell responses and aPD1 resistance in cancer. In addition to predicting response to aPD1 checkpoint inhibitor immunotherapy, these findings further suggest that this Wnt signaling panel could serve as a predictive marker of immunologic response to Wnt ligand inhibitors, such as the PORCN inhibitors, which are currently under development. We continue to accrue additional pts to further validate these findings. Future studies will include a comparison of pre-treatment and on-treatment biopsies to evaluate these markers as predictors of adaptive aPD1 resistance. Acknowledgements Holly Dressman, PhD of the Duke Center for Genomic and Computational Biology for her assistance with the Nanostring samples; Jenna Goodwin, Carol Ann Wiggs, and Jennifer Nixon of the Duke Clinical Melanoma Research Team for their assistance with the melanoma tissue acquisition protocol; Tadas Rimkus of Nanostring for his assistance with analysis Trial Registration NCT02694965 Ethics Approval This study was approved by Duke University’s Institutional Review Board, protocol number Pro00059349 Consent Not applicable References Luke JJ, B.R., Spranger S, Sweis RF, Lingen MW, Lengyel E, Zha Y, Gajewski TF. Wnt/Beta-catenin pathway activation correlates with immune exclusion across most human cancers. Journal of Clinical Oncology 2016;34(15). Hugo W, et al. Genomic and transcriptomic features of response to anti-pd-1 therapy in metastatic melanoma. Cell 2016;165(1): p. 35–44. DeVito NC, X.C., Zhao F, Evans KS, Theivanthiran B, Lewicki J, Hoey T, Hurwitz H, Strickler JH, Hanks BA. Paracrine wnt-β-catenin signaling inhibition as a strategy to enhance the efficacy of anti-PD-1 antibody (Ab) therapy in a transgenic model of melanoma. Journal of Clinical Oncology, 2017. 35(no. 15_suppl). Zhao F, et al. Paracrine Wnt5a-beta-Catenin signaling triggers a metabolic program that drives dendritic cell tolerization. Immunity 2018;48(1): p. 147–160 e7.

Cutaneous angiosarcoma presents clinically in numerous ways, and can be mistaken for a different clinical entity, particularly when arising at unusual anatomic locations such as the eyelid.A 57-year-old woman presented with a 1-year history of eyelid swelling. Concurrent imaging was also suggestive of an edematous process. Multiple superficial biopsies showed nonspecific dermal inflammation and interstitial edema. A diagnosis of Morbihan disease (chronic and idiopathic lymphedema of the eyelid) was rendered, and the patient was treated with compression and local therapy without clinical improvement. Three years after initial presentation, a diagnostic blepharoplasty was performed revealing a deep dermal vascular proliferation composed of anastomosing vascular channels with an atypical endothelial lining. A diagnosis of cutaneous angiosarcoma was ultimately made.This case illustrates a unique presentation of cutaneous angiosarcoma and the implications of different biopsy techniques in acquiring the correct diagnosis.

Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphomas. Large-cell transformation of MF has been associated with disease progression and overall poor outcome. The expression of CD30, which defines anaplastic large cell lymphoma (ALCL) and lymphomatoid papulosis, might also occur in a subset of patients with MF, with or without large-cell transformation. Brentuximab vedotin is an anti-CD30 monoclonal antibody which has been proven to be a safe and effective therapeutic agent in the treatment of CD30-positive lymphomas, such as Hodgkin lymphoma and ALCL. Recently, brentuximab vedotin has been shown to have a significant clinical activity in treatment-refractory or advanced MF or Sezary syndrome with a wide-range of CD30 expression levels. We report a patient with MF tumor stage with large-cell transformation and low CD30 expression with good response to brentuximab vedotin and unusual extensive xanthomatous changes in the follow-up biopsy.

Primary cutaneous Merkel cell carcinoma (MCC) is an aggressive neuroendocrine malignancy in which lymphovascular invasion (LVI) correlates with more aggressive phenotype. The prognostic significance of LVI detected by D2-40 immunohistochemistry (IHC) in MCC remains controversial. We aimed to determine how LVI detected by D2-40 IHC compares with LVI detected by hematoxylin and eosin (HE) staining in predicting MCC metastasis. Clinical and histopathologic features of MCCs diagnosed and treated in 2002 to 2015 were assembled and included 58 MCC tumors from 58 patients. HE-stained tissue sections and D2-40 IHC studies were reviewed. When LVI was present, the location (peritumoral or intratumoral) and the size of the largest invaded vessel were recorded. LVI findings by HE staining and D2-40 IHC were compared with each other and with histologic features and clinical outcomes. HE staining showed LVI in 37 of 58 cases; D2-40 IHC confirmed LVI in 30 of these cases but failed to confirm LVI in 7. D2-40 IHC also detected 14 cases of LVI not identified on HE staining. Histologically, D2-40-detected LVI was associated with infiltrative growth pattern and nonbrisk lymphoid infiltrate (P = .005 and P = .055, respectively). There was a statistically significant difference between the frequency of detection of peritumoral LVI by HE in comparison to D2-40 IHC (P = .0009). MCCs in which D2-40 IHC-detected both intratumoral and peritumoral LVI were typically larger than MCCs without (mean, 24.5 mm versus 17.3 mm; P = .03) and more frequently metastasized (87% versus 51%; P = .03). D2-40 IHC detection of both intratumoral and peritumoral LVI is associated with metastasis.

Pancreatic carcinomas with acinar differentiation are rare, accounting for 1%-2% of adult pancreatic tumors; they include pancreatic acinar cell carcinoma (PACC), pancreatoblastoma, and carcinomas of mixed differentiation. Patients with PACC have a prognosis better than pancreatic ductal adenocarcinomas but worse than pancreatic neuroendocrine tumors. Reports of overall survival range from 18 to 47 mo. A literature review on PACCs included comprehensive genomic profiling and whole exome sequencing on a series of more than 70 patients as well as other diagnostic studies including immunohistochemistry. Surgical resection of PACC is the preferred treatment for localized and resectable tumors. The efficacy of adjuvant treatment is unclear. Metastatic PACCs are generally not curable and treated with systemic chemotherapy. They are moderately responsive to chemotherapy with different regimens showing various degrees of response in case reports/series. Most of these regimens were developed to treat patients with pancreatic ductal adenocarcinomas or colorectal adenocarcinomas. Review of PACC’s molecular profiling showed a number of gene alterations such as: SMAD4, BRAF, BRCA2, TP53, RB1, MEN1, JAK-1, BRCA-1, BRCA-2, and DNA mismatch repair abnormalities. PACCs had multiple somatic mutations with some targetable with available drugs. Therefore, molecular profiling of PACC should be an option for patients with refractory PACC.

We describe a 44-year-old female with triple-negative breast cancer who developed skin erythaema, sclerosis and contracture of her entire right breast 15 months after completion of post-lumpectomy chemotherapy and radiotherapy, consistent with post-irradiation morphoea (PIM). PIM is a rare complication of breast irradiation that impairs a patient's quality of life. PIM is located usually at the radiation port or in the surrounding tissue. Clinically, PIM is misdiagnosed commonly as lymphoedema and cellulitis in the early inflammatory phase, and recurrent breast cancer, chronic radiodermatitis (CRD), radiation-induced fibrosis (RIF), post-irradiation pseudosclerodermatous panniculitis (PIPP), atypical vascular lesions (AVL) or angiosarcoma (AS) in the late burnout phase. Arriving at the correct diagnosis typically requires a multidisciplinary approach, including a skin biopsy for confirmation. To date, satisfactory treatment of this condition has been challenging. and the clinical outcome after therapy is often unsatisfactory.

The advances in surgical techniques and the introduction of more effective and less toxic immunosuppression regimens have resulted in increased rate of successful transplantation with extended survival of the recipients. Histologic assessment continues to play an essential role in the diagnosis of complications secondary to therapy in this population. In this chapter we discuss the most common cutaneous diseases that may result from hematopoietic stem cell transplantation. Metastatic diseases involving the skin are also discussed.

Pediatric dermatopathology includes a range of diseases, particularly inflammatory and genodermatosis, that pathologists may not be entirely familiar with. Furthermore, children are not biopsied frequently, and this limits the understanding of the histopathologic features in pediatric skin disorders. Some diseases may only be encountered early in life, and the differential diagnosis of certain conditions in children is different from that considered in adults. This chapter covers pediatric dermatopathology in the hospital setting.

Intrinsic resistance of unknown mechanism impedes the clinical utility of inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) in malignancies other than breast cancer. Here we used melanoma patient-derived xenografts (PDXs) to study the mechanisms for CDK4/6i resistance in pre-clinical settings. We observed that melanoma PDXs resistant to CDK4/6i frequently displayed activation of phosphatidyl inositol 3 kinase (PI3K)-AKT pathway, and inhibition of this pathway improved CDK4/6i response in a p21-dependent manner. We showed that a target of p21, CDK2, was necessary for proliferation in CDK4/6i-treated cells. Upon treatment with CDK4/6i, melanoma cells up-regulated cyclin D1, which sequestered p21 and another cyclin-dependent kinase inhibitor, p27, leaving a shortage of p21 and p27 available to bind and inhibit CDK2. Therefore, we tested whether induction of p21 in resistant melanoma cells would render them responsive to CDK4/6i. Because p21 is transcriptionally driven by p53, we coadministrated CDK4/6i with a murine double minute (MDM2) antagonist to stabilize p53, allowing p21 accumulation. This resulted in improved anti-tumor activity in PDXs and in murine melanoma. Furthermore, co-administration of CDK4/6 and MDM2 antagonists with standard of care therapy caused tumor regression. Notably, the molecular features associated with response to CDK4/6 and MDM2 inhibitors in PDXs were recapitulated by an ex vivo organotypic slice culture assay, which could potentially be adopted in clinic for patient stratification. Our findings provide a rationale for co-targeting CDK4/6 and MDM2 in melanoma.

Background Mucoepidermoid pancreatic cancer is a rare entity with only 8 cases reported in the literature. On review of the literature, the authors found that cutaneous metastases in pancreatic cancer are rare and have not been associated with the mucoepidermoid subtype. The authors present the first reported case of cutaneous metastasis in a patient with mucoepidermoid carcinoma of the pancreas. Case presentation A 50-year old white male with a metastatic invasive poorly differentiated mucoepidermoid carcinoma of the pancreas was found to have a slow growing lesion in the skin over his left upper quadrant while undergoing active therapy. The lesion was biopsied and the pathology was consistent with pancreatic origin sharing similar morphologic features when compared with the primary pancreactectomy specimen. Conclusions Mucoepidermoid pancreatic cancer is an exceedingly rare subtype of pancreatic cancer, with very little information regarding its diagnosis, treatment, and patterns of metastases. Here, the authors present the first reported case of cutaneous metastases of mucoepidermoid pancreatic cancer.

Anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma characterized by strong and uniform expression of CD30. Brentuximab vedotin (BV), an anti-CD30 antibody-drug conjugate has been approved by the U.S. FDA for relapsed/refractory systemic ALCL and achieves improved outcomes. We report a 44-year-old African-American man who presented with lymphadenopathy, lip and chest nodules diagnosed as CD30+, ALK-negative ALCL. The patient was treated with BV upon recurrence. While on treatment, the patient developed new-onset nodules on the chest and back. Skin biopsy showed a diffuse dermal infiltrate of medium-to-large atypical lymphocytes with frequent mitosis and scattered eosinophils. Immunohistochemically, the atypical cells displayed the same immunophenotype as previous specimens (CD3+, CD4-/CD8-, CD56-, ALK- and TCR γ-), except for lack of CD30 expression which was attributed to BV treatment effect. The diagnosis was thought to be consistent with ALK-negative ALCL and the patient was continued on BV along with total skin electron beam radiation and the lesions cleared. The patient relapsed 2 months later with extensive disease and expired. In summary, this is the first report in the literature of loss of CD30 expression in ALCL after BV therapy. Awareness of this may prevent a mistaken diagnosis of a CD30-negative secondary T-cell lymphoma.

Colitis is a frequent, clinically-significant immune-related adverse event caused by anti-programmed death-1 (PD-1). The clinical features, timing, and management of colitis with anti-PD-1-based regimens are not well-characterized. Patients with advanced melanoma that received either anti-PD-1 monotherapy (“monotherapy”) or combined with ipilimumab (“combination therapy”) were screened from 8 academic medical centers, to identify those with clinically-relevant colitis (colitis requiring systemic steroids). Of 1261 patients who received anti-PD-1-based therapy, 109 experienced colitis. The incidence was 3.2% (30/937) and 24.4% (79/324) in the monotherapy and combination therapy cohorts, respectively. Patients with colitis from combination therapy had significantly earlier symptom onset (7.2 weeks vs 25.4 weeks, p

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1. Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies. Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice. Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR. See related commentary by Pawelec, p. 5193

BACKGROUND The authors hypothesized that comprehensive genomic profiling of advanced-stage cutaneous squamous cell carcinoma (cSCC) could identify genomic-derived drug targets of therapy for patients with conventional therapy-resistant disease. METHODS Comprehensive genomic profiling of 315 cancer genes was applied to 50 ng of DNA from 122 cSCC cases for the evaluation of all classes of genomic alterations (GAs). Clinically relevant genomic alterations (CRGAs) were defined as those identifying anticancer drugs on the market or in registered clinical trials. RESULTS There were 21 women (17%) and 101 men (83%) with a median age of 64.9 years (range, 21-87 years). Eleven cSCC cases (9%) were histologic AJCC grade 1, 69 (57%) were grade 2, and 42 (34%) were grade 3. The primary cSCC was used for sequencing in 77 cases (63%). Metastatic lesions were sequenced in 37% of cases. There were 1120 total GAs identified (average of 9.2 GAs per tumor), with 100% of cases harboring at least 1 alteration. Of the 122 cSCCs, 107 (88%) harbored at least 1 CRGA (2.5 CRGAs per cSCC) includingNOTCH1 (43%); patched 1 (PTCH1) (11%); BRCA2 (10%); HRAS (8%); ataxia telangiectasia mutated (ATM) (7%); erb-B2 receptor tyrosine kinase 4 (ERBB4) (7%); neurofibromatosis type 1 (NF1) (7%); erb-B2 receptor tyrosine kinase 2 (ERBB2) (6%); phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) (6%); cyclin D1 (CCND1) (6%); epidermal growth factor receptor (EGFR) (5%); and F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase (FBXW7) (5%). CONCLUSIONS In the current study, approximately 88% of patients with cSCC were found to harbor clinically relevant GAs that have the potential to guide the treatment of patients with advanced-stage tumors with targeted therapeutic agents. Cancer 2016;122:249–257. © 2015 American Cancer Society.

3086 Background: Immunotherapies blocking the interaction of CTLA-4 or Programmed Death 1 (PD-1) with their ligands are the standard of care for advanced MEL although many pts fail to benefit from immunotherapy. Herein, we seek to identify epigenetic and genetic signatures associated with lack of response in patients treated with immunotherapy . Methods: We performed whole exome sequencing of 580 cancer-related genes and whole-genome DNA methylation arrays targeting > 850k CpG sites covering promoters, enhancers, and transcription factor sites in 28 MEL samples from patients treated with PD-1 +/- CTLA-4 inhibitors. Findings were correlated with collected clinical history. Results: Findings are summarized in the table . Unsupervised clustering and multi-parametric analysis showed a distinct methylation signature independent of age, sex, stage, site of metastasis, or type of treatment (adj. p

Invasive cutaneous melanoma remains the deadliest among the common forms of skin cancer, and its incidence continues to rise steadily. There is therefore a critical need for continued refinement of the diagnostic algorithm to diagnose melanoma in order to improve our ability to distinguish bona fide melanoma from benign melanocytic proliferations. While the cornerstone of this distinction remains histopathologic assessment, emerging molecular techniques including comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) have provided additional resources to delineate genetic changes distinctive of melanoma and therefore, facilitate its diagnosis and inform its prognosis and treatment. In particular, CGH studies have shown that more than 95 % of melanomas harbor characteristic chromosomal copy number aberrations, whereas benign melanocytic nevi usually lack such changes. The development of a multicolor FISH assay leveraged this fundamental genomic disparity and has improved the sensitivity and specificity of the diagnosis of melanoma—especially among morphologically challenging melanocytic proliferations, providing an additional diagnostically informative test that further refines the appraisal of melanocytic neoplasms in the context of challenging clinical, histologic, and/or immunohistochemical features. This chapter reviews the evolution of our understanding of the genomic changes typical of melanoma and melanocytic nevi beginning from the initial studies applying CGH to melanocytic tumors though the various iterations of the melanoma FISH assay and explores in detail how these assays are practically employed to inform our diagnosis of challenging melanocytic lesions.

Melanoma accounts for most skin cancer-related deaths and has an increasing incidence. Accurate diagnosis and distinction from atypical nevi can be at times difficult using light microscopy alone. Fluorescence in situ hybridization (FISH) and melanoma gene expression score (myPath, Myriad Genetics) have emerged as ancillary tools to further aid in this differential diagnosis. Our aim in this study was to correlate FISH results, gene expression score, consensus histopathologic impression and clinical outcome on a series of 117 challenging melanocytic lesions collected from three separate institutions. The lesions were separated into two groups: 39 histopathologically unequivocal lesions (15 malignant, 24 benign) and 78 challenging lesions interpreted by expert consensus (27 favor malignant, 30 favor benign, and 21 ambiguous). Melanoma-FISH was performed using probes for 6p25, 11q13, 8q24, and 9p21/CEP9 and scored according to established criteria. Analysis by myPath gene expression score was performed and interpreted by the manufacturer as 'benign', 'indeterminate,' or 'malignant'. In the unequivocal group, melanoma-FISH and myPath score showed 97 and 83% agreement with the histopathologic diagnosis, respectively, with 93 and 62% sensitivity, 100 and 95% specificity, and 80% inter-test agreement. In the challenging group, FISH and the myPath score showed 70 and 64% agreement with the histopathologic interpretation, respectively, with 70% inter-test agreement and similar sensitivities and specificities. The inter-test agreement was 73% overall, excluding indeterminate results. Discordant test results occurred in 27/117 cases from both unequivocal and challenging groups. Melanoma-FISH and gene expression score are valuable ancillary tools, though both have limitations and return discordant results in a subset of cases. Follow-up studies with more extensive clinical outcome data are warranted to establish the accuracy of these tests for the classification of melanocytic lesions.

To describe severe lymphedema of the eyelids, known as Morbihan disease, a previously characterized but infrequently reported and poorly understood entity related to rosacea that features solid mid-facial and eyelid lymphedema. Retrospective chart review, histopathologic and immunohistochemical analysis, and pertinent literature consideration. Five cases of Morbihan disease were identified. Histopathologic examination revealed pleomorphic perivascular and perilymphatic inflammation with profound lymphangiectasis and lymph stasis, thus suggesting elements of both rosacea and localized, chronic lymphedema. Multiple therapeutic interventions were performed including systemic anti-inflammatory therapy, surgical debulking, and corticosteroid injection. Extreme eyelid edema associated with characteristic skin changes and histopathologic findings represents an entity known as Morbihan disease which is rare and difficult to treat. While multiple modalities have been employed with variable results, future therapeutic considerations may include the use of targeted biologic agents.

Introduction: The purpose was to investigate how inhibition of MDM2 and CDK4/6 would affect growth of melanoma tumors with deleted CDKN2A in vivo. Methods: B16F0 and WM115 cells were cultured with increasing concentrations of MDM2 inhibitors nutlin-3a, RG7112, or RG7388. Viability was monitored by CellTiter-Blue assay. Induction of p21, MDM2, and BAX expression was monitored by qRT-PCR. C57BL/6J mice (n=40) were injected subcutaneously into both flanks with 50,000 B16F0 cells, a CDKN2A-deficient mouse melanoma cell line. Tumors grew for one week, then mice were randomized into four treatment groups: vehicle (100 µL 0.5% methylcellulose) (n=10), CDK4/6 inhibitor palbociclib 100 mg/kg (n=10), RG7388 150 mg/kg (n=10), and combination palbociclib 100 mg/kg + RG7388 150 mg/kg (n=10). Drugs were given daily via oral gavage. Tumor volumes were measured every 2-3 days, and mice with tumors >15 mm in diameter were euthanized. After 12 days of therapy, tumors were excised and weighed. At the time of euthanasia, mice were bled to evaluate AST and ALT levels. Four tumors per group were randomly selected for H&E staining to evaluate necrosis and IHC for CD3 (T cell marker), Ki67 (proliferation marker), and cleaved caspase 3 (apoptosis marker). The percent of necrosis and number of cells that were positive on IHC were evaluated by a pathologist blinded to the groupings. A mixed-effects model was used to compare tumor growth rate and tumor weights. Kruskal-Wallis and post-hoc Wilcoxon were used for other analyses. Results: In vitro experiments showed RG7388 was the most potent inhibitor of B16F0 and WM115 viability and the strongest inducer of p21 and MDM2. In vivo experiments revealed B16F0 tumor growth rate was significantly reduced in mice treated with palbociclib (p Citation Format: Lauren Slesur, Anna Vilgelm, Rami Al-Rohil, Ann Richmond. Investigating the effect of CDK4/6 and MDM2 inhibition on melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B166.

Immune checkpoint inhibitors (ICIs) have made a profound impact on the treatment of a variety of cancers. However, as with any systemic treatment, toxicities are inevitable. With most classes of cancer therapies, toxicities are relatively predictable based on clinical trial safety data and therefore can be handled with prophylactic or supportive care measures. However, ICIs are unique in their ability to cause rare but severe auto-immune toxicities. The molecular underpinnings of these toxicities, as well as unique features of the patient, tumor, or affected tissue, have not been extensively explored. We recently reported a small case series of two patients with myocarditis resulting in death arising following combination ICI therapy (Johnson et al, N Engl J Med, 2016). High lymphocytic infiltration, coupled with PD-L1 expression was present in the affected myocardium and skeletal muscle. Common T cell clones were identified between the affected tissue and tumor, and abnormal expression of muscle-specific transcripts was identified in the associated tumor, suggesting release of peripheral tolerance to tumor-expressed self-antigens.To expand upon our reported study, we collected healthy and afflicted tissue from a series of cancer patients with immune-related colitis, myocarditis (MC), and encephalopathy following ICI treatment. We hypothesize that molecular analysis of these tissues will identify causal factors in the etiology of these toxicities, and how to better predict, prevent, and treat them. Thus, we performed molecular characterization of the immune infiltrate and diseased tissue microenvironment. A total of 20 affected (colon, cardiac, brain) and non-diseased control specimens were examined by spatial digital profiling (nanoString). This process generates a spatial heatmap of digital counts of 20 selected immunology and cellular markers and proteins across each specimen. Using this technology, the landscape of inflammation in ICI-affected organs can be resolved for insights into the mechanism whereby ICI-mediated auto-immunity occurs. Targeted RNAseq for selected immuno-oncology mRNA targets was also performed. In initial RNA sequencing analyses of MC cases, affected myocardium, skeletal muscle, and patient-matched tumors all demonstrated expression of immune activation markers (e.g. interferon-gamma and granzyme B), expression of PD-L1, and muscle-specific genes. In the expanded population, including colitis, digital spatial profiling analyses and targeted NGS (RNAseq) are underway. Although data analyses are incomplete at the time of this abstract, this work will be the largest and most comprehensive analysis of the molecular underpinnings of ICI-mediated auto-immune toxicity reported to date. These data should offer clarity in the mechanisms and features of these adverse events, how to prevent or predict them with precision medicine, and how to treat them when they do occur. Citation Format: Justin M. Balko, Daniel Y. Wang, Yu Wang, Rami Al-Rohil, Margaret Compton, Jeffery A. Sosman, Igor Puzanov, Bret Mobley, Robert D. Hoffman, Yaomin Xu, Javid J. Moslehi, Chanjuan Shi, Douglas B. Johnson. Advanced molecular characterization of severe autoimmune toxicities associated with checkpoint inhibitor therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 588. doi:10.1158/1538-7445.AM2017-588

3076 Background: Immune checkpoint inhibitors (ICIs; anti-CTLA-4 and anti-PD-1) have shown clinical success in many cancers, but may cause rare irSAE. The molecular features of irSAE have not been extensively explored. Therefore, we characterized the immune composition of tissue affected by ICI-mediated inflammation with a focus on colitis and neurologic toxicity. Methods: We performed retrospective T-cell receptor (TCRβ) sequencing, RNA-sequencing (HTG EdgeSeq; > 2500 immune-related genes), and digital spatial profiling (NanoString) for 20 protein markers in 10 regions across tissues representing ICI-induced colitis, autoimmune inflammation (e.g. Crohn’s) and normal colon. Matched tumors were also included in a subset. We also analyzed the encephalitic and healthy brain of a unique presentation of anti-PD-1-induced encephalitis. Results: Patient-matched melanoma and colitis biopsies (n = 3) demonstrated shared T cell clones in all samples ranging from several shared clones to several hundred (0.4%, 2.7%, and 3% of rearrangements, respectively), including high-frequency clones. Shared TCRβ sequences were also identified among and between colon-irSAE and Crohn’s specimens. Gene expression patterns of inflammation in colon-irSAE resembled that of Crohn’s disease in principle components and clustering analysis, highlighting likeness between these diseases/SAEs. NanoString digital spatial profiling of regions of inflammation across samples showed higher CD68 and PD-L1 positivity in colon-irSAE specimens versus normal colon or Crohn’s specimens and reduced beta-catenin levels in both Crohn’s and colon-irSAE specimens relative to normal controls. Finally, we detected a high degree of TCR clonality in the encephalitic brain, including a single sequence present in ~20% of > 12,000 T cells, suggesting a distinct antigen-specific response. Conclusions: We report the molecular characteristics of irSAE in colon and brain specimens from patients receiving ICIs. Highly clonal TCRβ sequences were frequently detected, particularly in a unique case of encephalitis-irSAE. Furthermore, we identify molecular distinctions and similarities between autoimmune and colon-irSAEs at the gene expression and proteomic levels.

Background. Intrahepatic cholangiocarcinoma (ICC) is a subtypeofprimarylivercancerthatisrarelycurablebysurgeryand is rapidly increasing in incidence. Relapsed ICC has a poor prognosis, and current systemic nontargeted therapies are commonlyextrapolatedfromthose used in othergastrointestinalmalignancies.Wehypothesizedthat genomic profiling of clinicalICCsampleswouldidentifygenomicalterationsthatare linked to targeted therapies and that could facilitate a personalized approach totherapy. Methods. DNA sequencing of hybridization-capturedlibraries was performed for 3,320 exons of 182 cancer-related genes and 36 introns of 14 genes frequently rearranged in cancer. Sample DNA was isolated from 40 mm of 28 formalin-fixed paraffin-embedded ICC specimens and sequenced to high coverage. Results.Themostcommonlyobservedalterationswerewithin ARID1A (36%), IDH1/2 (36%), and TP53 (36%) as well as amplification of MCL1 (21%).Twenty cases (71%) harbored at least one potentially actionable alteration, including FGFR2 (14%), KRAS (11%), PTEN (11%), CDKN2A (7%), CDK6 (7%), ERBB3 (7%), MET (7%), NRAS (7%), BRCA1 (4%), BRCA2 (4%), NF1(4%),PIK3CA(4%),PTCH1(4%),andTSC1(4%).Four(14%) of the ICC cases featured novel gene fusions involving the tyrosine kinases FGFR2 and NTRK1 (FGFR2-KIAA1598, FGFR2BICC1, FGFR2-TACC3, and RABGAP1L-NTRK1). Conclusion.Two thirds of patients in this study harbored genomic alterations that are associated with targeted therapies and that have the potential to personalize therapy selection for to individual patients. The Oncologist 2014; 19:235–242 Implications for Practice:The recenttranslation of next-generation DNA sequencingtechnology fromthe researchlaboratory to clinical practice has enabled oncologists to personalize therapy decisions for each patient by targeting the genomic alterations driving the disease. For tumors such as primary cholangiocarcinoma of the liver, this new ability to determine all of the major genomic alterations (base substitutions, short insertions and deletions, copy number changes, homozygous deletions, and gene fusions)onverysmallformalin-fixedparaffinembeddedclinicalsamplesholdsgreatpromisethatlesstoxictargetedtherapiesmay be available for patients currently being treated with conventional “one size fits all” approaches.

9522Background: Although cutaneous cBCC and cSCC are the most frequent primary human cancers, primary treatment is nearly always curative, and metastatic disease (mcBCC and mcSCC) rarely develops. ...

Background Expression of integrin α3β1 is associated with tumor progression, metastasis, and poor prognosis in several cancers, including breast cancer. Moreover, preclinical studies have revealed important pro-tumorigenic and pro-metastatic functions for this integrin, including tumor growth, survival, invasion, and paracrine induction of angiogenesis. Our previously published work in a preclinical breast cancer model showed that integrin α3β1 promotes expression of cyclooxygenase-2 (COX2/PTGS2), a known driver of breast cancer progression. However, the clinical significance of this regulation was unknown. The objective of the current study was to assess the clinical relevance of the relationship between integrin α3β1 and COX2 by testing for their correlated expression among various forms of human breast cancer. Methods Immunohistochemistry was performed to assess co-expression of α3 and COX2 in specimens of human invasive ductal carcinoma (IDC), either on a commercial tissue microarray (n = 59 samples) or obtained from Albany Medical Center archives (n = 68 samples). Immunostaining intensity for the integrin α3 subunit or COX2 was scored, and Spearman’s rank correlation coefficient analysis was performed to assess their co-expression across and within different tumor subtypes or clinicopathologic criteria. Results Although expression of integrin α3 or COX2 varied among clinical IDC samples, a statistically significant, positive correlation was detected between α3 and COX2 in both tissue microarrays (rs = 0.49, p