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Since their first approval 25 years ago, monoclonal antibodies (mAbs) have become important targeted cancer therapeutics. However, dermatologic toxicities associated with non-immune checkpoint inhibitor (non-ICI) mAbs may complicate the course of cancer treatment. Data on the incidence and types of these reactions are limited.A comprehensive review was conducted on dermatologic toxicities associated with different classes of non-ICI mAbs approved for treatment of solid tumors and hematologic malignancies. The review included prospective Phase 1, 2, and 3 clinical trials; retrospective literature reviews; systematic reviews/meta-analyses; and case series/reports.Dermatologic toxicities were associated with several types of non-ICI mAbs. Inflammatory reactions were the most common dermatologic toxicities, manifesting as maculopapular, urticarial, papulopustular/acneiform, and lichenoid/interface cutaneous adverse events (cAEs) with non-ICI mAbs. Immunobullous reactions were rare and a subset of non-ICI mAbs were associated with the development of vitiligo cAEs.Dermatologic toxicities of non-ICI mAbs are diverse and mostly limited to inflammatory reactions. Awareness of the spectrum of the histopathologic patterns of cAE from non-ICI mAbs therapy is critical in the era of oncodermatology and oncodermatopathology.

The immunohistochemical (IHC) marker PReferentially expressed Antigen in MElanoma (PRAME) has shown promise in the diagnosis of melanocytic lesions. A few studies have investigated PRAME IHC expression in acral melanomas, but PRAME expression in subungual melanomas is largely unknown. We evaluated the utility of PRAME IHC expression in distinguishing subungual melanomas (SUM) and non-subungual acral melanomas (AM) from acral nevi (AN).Twenty-two SUM, 20 AM, and 14 AN were identified. IHC studies were performed using an anti-PRAME antibody. The percentage of lesional cells with PRAME expression was recorded and categorized as follows: 0%, 0; 1%-25%, 1+; 26%-50%, 2+; 51%-75%, 3+; and75%, 4+. Patient demographics and other relevant clinicopathologic parameters were recorded.Diffuse (4+) PRAME IHC expression was identified in 55% (12/22) SUM and 70% (14/20) AM, respectively. Any PRAME expression (1+ to 4+) was identified in 73% (16/22) SUMs and 95% (19/20) AM, respectively. One of 14 (7%) AN exhibited PRAME expression; interestingly, the pattern of expression was diffuse.In our study, PRAME IHC expression was useful in identifying AM, including SUM. However, there are exceptions of PRAME-negative melanomas and PRAME-positive nevi.

Trichorhinophalangeal syndrome type 1 (TRPS1) immunohistochemistry has been gaining popularity in recent years in the field of surgical pathology for its utility as a highly sensitive and specific marker for breast carcinomas, including those with triple-negative phenotype. More recent data suggest TRPS1 may also prove its utility in the diagnosis of mesenchymal tumors arising in the breast parenchyma, including malignant phyllodes tumors and primary chondrosarcomas and osteosarcomas of the breast. However, little is known about TRPS1 expression in nontumor cells, such as stromal fibroblasts/myofibroblasts of dermal granulation tissues and scars. Here, we describe our unique experience with TRPS1-positive cells, morphologically consistent with reactive fibroblasts/myofibroblasts, seen in dermal granulation tissues and scars from breast skin specimens of a 51-year-old woman with a history of bilateral invasive ductal carcinomas of the breast, status after bilateral total mastectomy and chemoradiation, who presented with nonhealing wounds on the chests. To the best of our knowledge, this is the first reported case of strong TRPS1 expression in dermal granulation tissue/scar. As the usage of TRPS1 immunohistochemistry in routine clinical practice, including in the field of dermatopathology, will likely increase over time, awareness of this potential diagnostic pitfall is important to avoid overinterpretation of the findings.

Merkel cell carcinoma (MCC) is an aggressive, highly metastatic, cutaneous neuroendocrine malignancy with poor prognosis. Here, we describe a MCC excision specimen with a rare case of tumor-associated amyloid deposition in the absence of residual tumor cells. A 72-year-old man presented with a lesion of 5-6 months' duration on his left elbow, clinically thought to be a ganglion cyst. The biopsy specimen revealed a Stage IIA MCC with classic histomorphologic and immunophenotypic findings, with tumor extending to the tissue edges. The patient underwent wide local excision with negative margins and a negative sentinel lymph node biopsy. Although the patient did not receive any presurgical chemotherapy, immunotherapy, or targeted therapy, the re-excision specimen showed only amphophilic, feathery deposits that were salmon-pink with Congo red stain and further confirmed as amyloid by electron microscopy; there were no residual carcinoma cells. Amyloid deposition in MCC has been described in rare case reports. Our case was extraordinary in that there was only amyloid deposition and an associated granulomatous reaction, without identifiable MCC cells. This case demonstrates that amyloid deposition may be evidence of a prior MCC at the site of a prior procedure and may warrant careful evaluation for residual MCC.

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Supplemental Figure 1. Representative images of nuclear grade. Histopathologic grade was defined according to the nuclear morphology and cytoplasmic evidence of sebaceous differentiation comprising the predominance (>50%) of the tumor cells. (A) High grade tumors exhibited enlarged irregular nuclei with high nuclear:cytoplasmic ratio and only focal evidence of sebaceous differentiation (intracytoplasmic vacuoles indenting the nucleus; H&E, 200x). (B) Intermediate grade tumors exhibited similar enlarged nuclei but with lower nuclear:cytoplasmic ratio and more obvious evidence of sebaceous differentiation (H&E, 200x). (C) Well-differentiated tumors showed only slightly enlarged nuclei and frank evidence of sebaceous differentiation (H&E, 200x).

This table contains multivariable Cox Analyses on survival models in validation set

Supplemental Figure 1. Correlation between dermal mitotic rate and patient survival. Kaplan-Meier estimate of (A) overall survival and (B) disease-specific survival based on dermal mitotic rate (mitotic figures/mm2). Supplemental Figure 2. Derivation of proposed T-category for Primary Vulvar Melanoma. (A, B, C) We first assigned patients with PVM to a T-category according to the AJCC for PCM (7th Edition). (A) Shows the AJCC schema and the number of patients assigned to each category. (B) Kaplan Meier DSS curves for patients stratified by AJCC T-categories demonstrate robust prognostic differences for patients with PVM and pT1 and to an extent pT2 disease from those with pT3 and pT4 disease, but less robust stratification among those patients with PVMs {less than or equal to}2.00 mm; pT1 versus pT2 (isolated in C) or >2.0 mm; pT3 and pT4. (D, E, F) Because both tumor thickness and mitotic rate were independent prognostic factors for DSS and because mitotic rate showed a natural prognostic separation at 2 mitotic figures/mm2 (as shown in Figure 1D), we first asked whether a T-category according to the conventional AJCC cut-offs ({less than or equal to}1.0, 1.01-2.0, 2.01-4.0 and >4.0 mm) subsequently modified by mitotic rate (a: 2.0 mm (pT3 and pT4) had a short DSS independent of mitotic rate. More importantly, among patients with a tumor thickness {less than or equal to}2.0 mm (pT1 or pT2), only those with mitotic rate {greater than or equal to}2/mm2 had a short DSS, which was essentially superimposable with patients with pT3 and pT4 PVM in this schema. (F) Kaplan-Meier survival curve for patients with pT1 and pT2 PVMs show striking differences in survival according to mitotic rate (

Supplemental Table 1. Summary of the clinical and histopathologic characteristics of Merkel cell carcinoma patients. Supplemental Table 2. Summary of antibodies used in this study. Supplemental Table 3. Correlation between tumor associated immune infiltrate and co-localized expression of CD31 and B7-H3 in Primary MCCs.

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Supplementary Data from Integrated Molecular and Clinical Analysis of AKT Activation in Metastatic Melanoma

Kaplan-Meier estimate of (A) overall survival and (B) disease-specific survival based on dermal mitotic rate (mitotic figures/mm2).

Clinical outcomes by combined markers of CD74 and MIF in tumor cells in stage III melanoma patients in discovery (A) and validation (B) datasets. Kaplan-Meier analyses are shown for OS and RFS for their expression by percentage of staining.

Purpose:In patients with cutaneous melanoma, metastasis in a nonsentinel lymph node (non-SLN) is a strong independent adverse prognostic factor. However, patients with a tumor-involved SLN no longer routinely undergo completion lymph node dissection (CLND). We hypothesized that SLN tumor burden may predict non-SLN tumor burden.Experimental Design:We compared tumor burden parameters between SLN and non-SLN in patients with cutaneous melanoma who underwent SLN biopsy with a positive SLN during 2003 to 2008 at The University of Texas MD Anderson Cancer Center.Results:We identified 336 eligible patients with a positive SLN. Of these, 308 (92%) underwent CLND, and 35 (10%) had non-SLN metastasis. The median follow-up time was 6.0 years. For patients with maximum diameter of tumor in the SLN ≤2.0 mm, >2.0–5.0 mm, and >5.0 mm, non-SLN metastasis was detected in 5 of 200 patients (3%), 10 of 63 patients (16%), and 20 of 57 patients (35%), and the mean maximum diameters of the non-SLN tumor deposits were 0.09, 1.56, and 2.71 mm, respectively (P < 0.0001). The percentage of patients with both subcapsular and intraparenchymal non-SLN tumor was higher for patients with SLN tumor in both locations than for patients with SLN tumor in only one location (P < 0.0001). Extranodal extension in a non-SLN was more common in patients with extranodal extension in an SLN (P = 0.003).Conclusions:In patients with cutaneous melanoma who undergo CLND, SLN tumor burden predicts non-SLN tumor burden. SLN tumor burden parameters provide accurate prognostic stratification independent of non-SLN status and should be considered for incorporation into future staging systems and integrated risk models.

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Biomarkers that guide therapy selection are gaining unprecedented importance as targeted therapy options increase in scope and complexity. In conjunction with high-throughput molecular techniques, therapy-guiding biomarker assays based upon immunohistochemistry (IHC) have a critical role in cancer care in that they inform about the expression status of a protein target. Here, we describe the validation procedures for four clinical IHC biomarker assays—PTEN, RB, MLH1, and MSH2—for use as integral biomarkers in the nationwide NCI-Molecular Analysis for Therapy Choice (NCI-MATCH) EAY131 clinical trial. Validation procedures were developed through an iterative process based on collective experience and adaptation of broad guidelines from the FDA. The steps included primary antibody selection; assay optimization; development of assay interpretation criteria incorporating biological considerations; and expected staining patterns, including indeterminate results, orthogonal validation, and tissue validation. Following assay lockdown, patient samples and cell lines were used for analytic and clinical validation. The assays were then approved as laboratory-developed tests and used for clinical trial decisions for treatment selection. Calculations of sensitivity and specificity were undertaken using various definitions of gold-standard references, and external validation was required for the PTEN IHC assay. In conclusion, validation of IHC biomarker assays critical for guiding therapy in clinical trials is feasible using comprehensive preanalytic, analytic, and postanalytic steps. Implementation of standardized guidelines provides a useful framework for validating IHC biomarker assays that allow for reproducibility across institutions for routine clinical use. Clin Cancer Res; 24(3); 521–31. ©2017 AACR.

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Purpose: Primary vulvar melanoma (PVM) is the second most common vulvar malignancy. Despite their distinct anatomic site and unique molecular–genetic alterations, PVMs are staged according to the American Joint Committee on Cancer (AJCC) guidelines for primary cutaneous melanomas (PCM). However, whether parameters derived for PCM also apply to PVM remain a critical yet largely unexplored clinical question. The objective of this study was to determine the parameters predictive of survival in PVM.Experimental Design: We retrospectively reviewed 100 patients with PVM and determined associations between clinical and histopathologic parameters and disease-specific survival (DSS) and overall survival (OS).Results: Univariate Cox regression analysis demonstrated older age (>56 years), greater tumor thickness, higher dermal mitotic rate, ulceration, lymphovascular invasion, perineural invasion, microscopic satellitosis, and absence of precursor nevus associated with decreased OS. Furthermore, age, midline, and/or multifocal involvement, greater tumor thickness, higher dermal mitotic rate, ulceration, lack of regression, lymphovascular invasion, perineural invasion, and microscopic satellitosis associated with decreased DSS. Multivariate analysis demonstrated tumor thickness, dermal mitotic rate, lymphovascular invasion, microscopic satellitosis, and absence of precursor nevus independently predicted shorter OS. Only tumor thickness and increased dermal mitotic rate (≥2/mm2) independently predicted reduced DSS. In comparison with the AJCC T-category, a novel, bivariate T-category based only on tumor thickness and dermal mitotic rate robustly predicted OS and DSS in our patient cohort.Conclusions: In the largest single institutional study of PVM, we demonstrate a combination of tumor thickness and mitotic rate comprise a simple but robust T-category to direct staging and prognosis. Clin Cancer Res; 23(8); 2093–104. ©2016 AACR.

Supplementary Table 2. Mean per sample coverage information.

Supplemental Figure 1. Dynamic range of co-localized expression of CD31 and B7-H3 in primary Merkel cell carcinoma. Each CD31+ and B7-H3+ cell centroid was tabulated as a series of (X, Y) coordinates (cell centroids). The frequency of their respective overlap for each of the 52 primary MCCs was calculated within a given distance as indicated: blue boxes show G-function colocalization index applying a 1 pixel (1.57 �m) minimum distance of overlapped expression as our cutoff, and green boxes show G-function colocalization index applying a 2 pixel (3.14 �m) minimum distance of overlapped expression as our cutoff.

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Purpose:Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy whose pathogenesis and prognosis are related to the integrity of the host immune system. Despite promising clinical responses to immune-checkpoint blockade, response and resistance remain unpredictable, underscoring a critical need to delineate novel prognostic biomarkers and/or therapeutic targets for this disease.Experimental Design: Expression of immune-regulatory markers (PD-L2, B7-H3, B7-H4, IDO-1, ICOS, TIM3, LAG3, VISTA, and OX-40) was assessed using singlet chromogenic IHC in 10 primary MCCs. Multiplex immunofluorescence quantified CD31 and B7-H3 expression in 52 primary and 25 metastatic MCCs. B7-H3 and CD31 expressions were tabulated as a series of independent (X,Y) cell centroids. A spatial G-function, calculated based on the distribution of distances of B7-H3+ (X,Y) cell centroids around the CD31+ (X,Y) cell centroids, was used to estimate a colocalization index equivalent to the percentage of CD31-positive cell centroids that overlap with a B7-H3–positive cell centroid.Results:Primary and metastatic MCCs exhibit a dynamic range of colocalized CD31 and B7-H3 expression. Increasing colocalized expression of B7-H3 with CD31 significantly associated with increased tumor size (P = 0.0060), greater depth of invasion (P = 0.0110), presence of lymphovascular invasion (P = 0.0453), and invasion beyond skin (P = 0.0428) in primary MCC. Consistent with these findings, increasing colocalized expression of B7-H3 and CD31 correlated with increasing vascular density in primary MCC, but not metastatic MCC.Conclusions:Our results demonstrate that colocalized expression of B7-H3/CD31 is a poor prognostic indicator and suggest therapies targeting B7-H3 may represent an effective approach to augmenting immune-activating therapies for MCC.

Purpose: This phase I clinical trial was conducted to determine the safety, efficacy, and molecular effects of sorafenib with temsirolimus in patients with advanced melanoma.Patients and Methods: Patients with stage IV or unresectable or recurrent stage III melanoma and Eastern Cooperative Oncology Group performance status of 0 to 1 were eligible. Sorafenib was given orally once or twice daily and temsirolimus was given i.v. weekly, both starting on day 1, with a 4-week cycle. Responses were assessed every 2 cycles per Response Evaluation Criteria in Solid Tumors criteria. Consenting patients with accessible tumors underwent optional tumor biopsies before treatment and after the second infusion of temsirolimus. Tumor biopsies were analyzed for activating mutations in BRAF and NRAS, and for expression of P-extracellular signal-regulated kinase (P-ERK) and P-S6 proteins.Results: A total of 25 patients were accrued to the study. The maximum tolerated doses were sorafenib 400 mg every morning and 200 mg every evening and temsirolimus 25 mg i.v. weekly. Dose-limiting toxicities included thrombocytopenia, hand-foot syndrome, serum transaminase elevation, and hypertriglyceridemia. There were no complete or partial responses with the combination; 10 patients achieved stabilization of disease as their best response. The median progression-free survival was 2.1 months. Matching pretreatment and day 15 tumor biopsies showed marked inhibition of P-S6 with treatment in 3 of 4 evaluable patients, but minimal inhibition of P-ERK.Conclusions: Combination therapy with sorafenib and temsirolimus resulted in significant toxicity at higher dose levels, failed to achieve any clinical responses in genetically unselected patient population, and did not inhibit P-ERK. Clin Cancer Res; 18(4); 1120–8. ©2012 AACR.

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Purpose: Merkel cell carcinoma (MCC) is an aggressive cancer with frequent metastasis and death with few effective therapies. Because programmed death ligand-1 (PD-L1) is frequently expressed in MCC, immune checkpoint blockade has been leveraged as treatment for metastatic disease. There is therefore a critical need to understand the relationships between MCPyV status, immune profiles, and patient outcomes.Experimental Design: IHC for CD3, CD8, PD-1, PD-L1, and MCPyV T-antigen (to determine MCPyV status) was performed on 62 primary MCCs with annotated clinical outcomes. Automated image analysis quantified immune cell density (positive cells/mm2) at discrete geographic locations (tumor periphery, center, and hotspot). T-cell receptor sequencing (TCRseq) was performed in a subset of MCCs.Results: No histopathologic variable associated with overall survival (OS) or disease-specific survival (DSS), whereas higher CD3+ (P = 0.004) and CD8+ (P = 0.037) T-cell density at the tumor periphery associated with improved OS. Higher CD8+ T-cell density at the tumor periphery associated with improved DSS (P = 0.049). Stratifying MCCs according to MCPyV status, higher CD3+ (P = 0.026) and CD8+ (P = 0.015) T-cell density at the tumor periphery associated with improved OS for MCPyV+ but not MCPyV− MCC. TCRseq revealed clonal overlap among MCPyV+ samples, suggesting an antigen-specific response against a unifying antigen.Conclusions: These findings establish the tumor-associated immune infiltrate at the tumor periphery as a robust prognostic indicator in MCC and provide a mechanistic rationale to further examine whether the immune infiltrate at the tumor periphery is relevant as a biomarker for response in ongoing and future checkpoint inhibitor trials in MCC. Clin Cancer Res; 22(22); 5553–63. ©2016 AACR.

Supplemental Table 3. Summary of TP53 and RB1 mutational status, immunohistochemical detection of TP53 and RB and histopathologic features in primary/locally recurrent ocular adnexal (OA) sebaceous carcinomas.

Table S1A. Relationship between RFS and MSS, and size of tumor deposit in the SLN using the cut-off values of < 0.1 mm, 0.1-1.0 mm, and > 1.0 mm (univariate analysis); Table S1B. Relationship between SLN and non-SLN tumor burden using the cut-off values of < 0.1 mm, 0.1-1.0 mm, and > 1.0 mm (univariate analysis)

Supplemental Figure 2. Correlation between B7-H3/CD31 G-function co-localization index in MCPyV-positive MCCs. Plots showing the correlation B7-H3/CD31 G-function co-localization index for each primary MCPyV-positive MCC plotted against tumor size (A), depth of invasion (B), invasion beyond skin (C) and metastasis beyond SLN (D). G-function calculated according to a 1 pixel (1.57 �m) radius.

Purpose: Activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway has been implicated in melanoma based primarily on the prevalence of mutations in PTEN and NRAS. To improve our understanding of the regulation and clinical significance of the PI3K-AKT pathway in melanoma, we quantitatively measured the levels of phosphorylated AKT, its substrate GSK3α/β, and its negative regulator PTEN in clinical metastases. Results were compared with mutational status, clinical outcomes, and sites of metastasis.Experimental Design: DNA and protein were isolated from dissected frozen melanoma metastases (n = 96). Activating mutations of BRAF, NRAS, AKT, PIK3CA, and KIT were detected by mass spectroscopy genotyping. Phosphorylated AKT (Ser473 and Thr308), P-GSK3α/β, and PTEN protein expression were measured by reverse-phase protein array. A panel of human melanoma cells lines (n = 58) was analyzed for comparison.Results: BRAF-mutant tumors had higher levels of P-AKT-Ser473 (P = 0.01), P-AKT-Thr308 (P = 0.002), and P-GSK3α/β (P = 0.08) than NRAS-mutant tumors. Analysis of individual tumors showed that almost all tumors with elevated P-AKT had low PTEN levels; NRAS-mutant tumors had normal PTEN and lower P-AKT. Similar results were observed in melanoma cell lines. Stage III melanoma patients did not differ in overall survival based on activation status of the PI3K-AKT pathway. Brain metastases had significantly higher P-AKT and lower PTEN than lung or liver metastases.Conclusions: Quantitative interrogation of the PI3K-AKT pathway in melanoma reveals unexpected significant differences in AKT activation by NRAS mutation and PTEN loss, and hyperactivation of AKT in brain metastases. These findings have implications for the rational development of targeted therapy for this disease. (Clin Cancer Res 2009;15(24):7538–46)

(A) Kaplan-Meier plot for age and disease specific survival applying a cut off of 71y. (B) Kaplan-Meier plot for sex and disease specific survival. (C) Kaplan-Meier plot for MCPyV status and disease specific survival. (D) Kaplan-Meier plot for perineural invasion and disease specific survival.

PDF file - 416KB, Supplementary methods, figures and tables. Fig. S1. Gene expression profiling of patient-matched brain and extracranial metastases. Fig. S2. PTEN gene expression in BM_03 (homozygous copy loss) and EM_03 (one copy loss). Gene expression was measured by microarray. Fig. S3. Quantitative RT-PCR measurement of SGK3, SGSM2, and ELOVL2 expression in five pairs of brain metastases (BM) and extracranial metastases (EM). Fig. S4. Log2 expression levels of RB_pS807_S811 in seven pairs of matched metastases. Fig. S5 Examples of PTEN-absent tumors. PTEN IHC of BM_03 and EM_03 were photographed at 00X magnification. Fig. S6 GSK3/_pS21/S9 IHC staining in 26 pairs of matched brain metastases (BM) and extracranial metastases (EM). Fig. S7. Results of RB_pS807_S811 IHC in 25 pairs of matched brain metastases (BM) and extracranial metastases (EM). Fig. S8. Kaplan-Meier survival analysis of nude mice with BRAF-mutant melanoma brain metastases treated with BKM120 and/or LGX818. Fig. S9. AKT_pS473 level in subcutaneously (sc) and intracranially (ic) implanted melanomas. Table S1. List of clinical samples analyzed. Table S2. Clinical features and genetic aberrations of matched samples analyzed by high-throughput techniques. Table S3. Hotspot mutations analyzed by Sequenom. Table S4. Antibodies used in RPPA analysis. Table S5. Genes with copy number differences (P

Supplementary Table 1. Summary of mutations identified in the entire cohort of primary, locally recurrent and metastatic tumors.

Supplemental Figure 3. Correlation between B7-H3/CD31 G-function co-localization index in MCPyV-negative MCCs. Plots showing the correlation B7-H3/CD31 G-function co-localization index for each primary MCPyV-negative MCC plotted against the frequency of associated malignancies (A) and lymphovascular invasion (B). G-function calculated according to a 1 pixel (1.57 �m) radius.