Five-year analysis of neoadjuvant dabrafenib and trametinib for stage III melanoma.

BACKGROUND: Neoadjuvant dabrafenib plus trametinib has a high pathological response rate and impressive short-term survival in patients with resectable stage III melanoma. We report five-year outcomes from the phase II NeoCombi trial. METHODS: NeoCombi (NCT01972347) was a single-arm, open-label, single-centre, phase II trial. Eligible patients were adults (aged ≥18) with histologically-confirmed, resectable, RECIST-measurable AJCC 7th ed. clinical stage IIIB-C BRAF V600E/K-mutant melanoma and Eastern Co-operative Oncology Group performance status ≤1. Patients received 52 weeks of treatment with 150 mg dabrafenib (orally twice per day) plus 2 mg trametinib (orally once per day), with complete resection of the pre-therapy tumour bed at Week 12. RESULTS: Between August 20, 2014, and April 19, 2017, 35 patients were enrolled. At data cut-off (August 17, 2021), the median follow-up was 60 months (95% CI 56-72). Overall, 21 of 35 (60%) patients recurred, including twelve (57%) with first recurrence in locoregional sites (followed by later distant recurrence in six) and nine (43%) with first recurrence in distant sites, including three in the brain. Most recurrences occurred within two years, with no recurrences beyond three years. At five years, recurrence-free survival was 40% (95% CI 27-60), distant metastasis-free survival was 57% [95% CI 42-76%], and overall survival was 80% (95% CI 67-94). Five-year survival outcomes were stratified by pathological response: recurrence-free survival was 53% with pCR versus 28% with non-pCR (p=0.087), distant metastasis-free survival was 59% versus 55% (p=0.647), and overall survival was 88% versus 71% (p=0.205), respectively. CONCLUSIONS: Neoadjuvant dabrafenib plus trametinib has high pathological response rates in clinical stage III melanoma, but low rates of recurrence-free survival, similar to those achieved with adjuvant targeted therapy alone. Patients with a pCR to dabrafenib plus trametinib still had a high risk of recurrence, unlike that seen with immunotherapy where recurrences are rare.

Stroma-infiltrating T cell spatiotypes define immunotherapy outcomes in adolescent and young adult patients with melanoma.

The biological underpinnings of therapeutic resistance to immune checkpoint inhibitors (ICI) in adolescent and young adult (AYA) melanoma patients are incompletely understood. Here, we characterize the immunogenomic profile and spatial architecture of the tumor microenvironment (TME) in AYA (aged ≤ 30 years) and older adult (aged 31-84 years) patients with melanoma, to determine the AYA-specific features associated with ICI treatment outcomes. We identify two ICI-resistant spatiotypes in AYA patients with melanoma showing stroma-infiltrating lymphocytes (SILs) that are distinct from the adult TME. The SILhigh subtype was enriched in regulatory T cells in the peritumoral space and showed upregulated expression of immune checkpoint molecules, while the SILlow subtype showed a lack of immune activation. We establish a young immunosuppressive melanoma score that can predict ICI responsiveness in AYA patients and propose personalized therapeutic strategies for the ICI-resistant subgroups. These findings highlight the distinct immunogenomic profile of AYA patients, and individualized TME features in ICI-resistant AYA melanoma that require patient-specific treatment strategies.

CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

Inter-rater concordance of basal cell carcinoma subtypes: influences on reporting format and opportunities for further classification modifications.

Diagnosis of basal cell carcinoma (BCC) higher risk subtypes influences management strategies because of their propensity to recur locally. Subtyping is prone to inter-observer variability, and subtyping definitions are inconsistently applied. This study sought to compare the interobserver reproducibility of individual BCC subtypes using the 4th edition World Health Organization (WHO) Classification of Skin Tumours (CoST) definitions, with classification into lower and higher risk histological subtype groups. Ninety-one BCC cases were rated by seven pathologists, noting the presence of BCC subtype(s), and providing a higher or lower risk subtype grouping per case. Raters were provided with definitions as per the 4th edition WHO CoST for 10 listed BCC subtypes. Surgical specimen type was noted. Subgroup analysis was performed to exclude cases when the tumour deep front was not well visualised, or there was tangential sectioning (n = 6). Light's kappa was used to assess inter-rater reliability. From the total group (n = 91), five BCC subtypes showed a sufficient number of ratings for computing a κ statistic. From these five subtypes, superficial subtype showed substantial inter-rater agreement (κ = 0.64), and the other four subtypes showed moderate inter-rater agreement [nodular (κ = 0.45), sclerosing/morphoeic (κ = 0.45), infiltrating (κ = 0.49) and micronodular (κ = 0.57)]. Two-tiered rating into either higher or lower risk subtype showed substantial inter-rater agreement (κ = 0.72). Our results suggest a need to more precisely define BCC subtypes. We suggest reporting BCC subtype using a two-tiered risk grouping, followed by specific subtypes present. Further studies examining the inter-rater reliability of less common BCC subtypes are required.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

In vivo reflectance confocal microscopy can detect the invasive component of lentigo maligna melanoma: Prospective analysis and case-control study.

BACKGROUND: Lentigo maligna (LM), a form of melanoma in situ, has no risk of causing metastasis unless dermal invasive melanoma (LMM) supervenes. Furthermore, the detection of invasion impacts prognosis and management. OBJECTIVE: To assess the accuracy of RCM for the detection of invasion component on LM/LMM lesions. METHODS: In the initial case-control study, the performance of one expert in detecting LMM at the time of initial RCM assessment of LM/LMM lesions was recorded prospectively (n = 229). The cases were assessed on RCM-histopathology correlation sessions and a panel with nine RCM features was proposed to identify LMM, which was subsequently tested in a subset of initial cohort (n = 93) in the matched case-control study by two blinded observers. Univariable and multivariable logistic regression models were performed to evaluate RCM features predictive of LMM. Reproducibility of assessment of the nine RCM features was also evaluated. RESULTS: A total of 229 LM/LMM cases evaluated by histopathology were assessed blindly and prospectively by an expert confocalist. On histopathology, 210 were LM and 19 were LMM cases. Correct identification of an invasive component was achieved for 17 of 19 LMM cases (89%) and the absence of a dermal component was correctly diagnosed in 190 of 210 LM cases (90%). In the matched case-control (LMM n = 35, LM n = 58), epidermal and junctional disarray, large size of melanocytes and nests of melanocytes were independent predictors of LMM on multivariate analysis. The interobserver analysis demonstrated that these three features had a fair reproducibility between the two investigators (K = 0.4). The multivariable model including those three features showed a high predictive performance AUC = 74% (CI 95% 64-85%), with sensitivity of 63% (95% CI 52-78%) and specificity of 79% (CI 95% 74-88%), and likelihood ratio of 18 (p-value 0.0026). CONCLUSION: Three RCM features were predictive for identifying invasive melanoma in the background of LM.

Clinical Features Associated with Outcomes and Biomarker Analysis of Dabrafenib plus Trametinib Treatment in Patients with BRAF-Mutant Melanoma Brain Metastases.

PURPOSE: This study aimed to identify baseline clinical features associated with the outcomes of patients enrolled in the COMBI-MB phase II study of dabrafenib and trametinib treatment in patients with V600 BRAF-mutant metastatic melanoma with melanoma brain metastases (MBM). Exploratory biomarker analysis was also conducted as part of the synergistic COMBI-BRV trial (BRV116521), to identify molecular and immunologic changes associated with dabrafenib in MBMs and extracranial metastases (ECM). PATIENTS AND METHODS: Post hoc analysis was performed for baseline features of patients (n = 125) enrolled in COMBI-MB. Analyses were performed to identify baseline clinical features associated with intracranial response rate (ICRR), progression-free survival (PFS), and overall survival (OS).Exploratory biomarker analysis was performed on biospecimen collected in the COMBI-BRV trial in which patients with BRAF-mutant, resectable MBM were treated with dabrafenib for 10 to 14 days prior to craniotomy. Accessible ECM were resected or biopsied at the time of craniotomy. Biospecimens underwent molecular and immunologic profiling for comparative analyses. RESULTS: In COMBI-MB baseline treatment with corticosteroids was independently associated with lower ICRR [39% vs. 63%; OR, 0.323; 95 % confidence interval (CI), 0.105-0.996; P = 0.049] and shorter PFS (HR, 1.93; 95% CI, 1.06-3.51; P = 0.031). Additional significant associations identified in the multivariate analysis were improved PFS in patients with a BRAFV600E genotype (HR, 0.565; 95% CI, 0.321-0.996; P = 0.048) and improved OS in patients with Eastern Cooperative Oncology Group 0 (HR, 0.44; 95% CI, 0.25-0.78; P = 0.005). CONCLUSIONS: Corticosteroid treatment was associated with reduced ICRR and PFS in COMBI-MB, similar to results with immunotherapy for MBMs. Baseline corticosteroid treatment is a key factor to consider in MBM patient management and clinical trial design/interpretation.

Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes.

Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes. SIGNIFICANCE: This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma. This article is highlighted in the In This Issue feature, p. 2711.

Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome.

While the tumor immune microenvironment (TIME) of metastatic melanoma has been well characterized, the primary melanoma TIME is comparatively poorly understood. Additionally, although the association of tumor-infiltrating lymphocytes with primary melanoma patient outcome has been known for decades, it is not considered in the current AJCC melanoma staging system. Detailed immune phenotyping of advanced melanoma has revealed multiple immune biomarkers, including the presence of CD8+ T-cells, for predicting response to immunotherapies. However, in primary melanomas, immune biomarkers are lacking and CD8+ T-cells have yet to be extensively characterized. As recent studies combining immune features and clinicopathologic characteristics have created more accurate predictive models, this study sought to characterize the TIME of primary melanomas and identify predictors of patient outcome. We first phenotyped CD8+ T cells in fresh stage II primary melanomas using flow cytometry (n = 6), identifying a CD39+ tumor-resident CD8+ T-cell subset enriched for PD-1 expression. We then performed Opal multiplex immunohistochemistry and quantitative pathology-based immune profiling of CD8+ T-cell subsets, along with B cells, NK cells, Langerhans cells and Class I MHC expression in stage II primary melanoma specimens from patients with long-term follow-up (n = 66), comparing patients based on their recurrence status at 5 years after primary diagnosis. A CD39+CD103+PD-1- CD8+ T-cell population (P2) comprised a significantly higher proportion of intratumoral and stromal CD8+ T-cells in patients with recurrence-free survival (RFS) ≥5 years vs those with RFS <5 years (p = 0.013). Similarly, intratumoral B cells (p = 0.044) and a significantly higher B cell density at the tumor/stromal interface were associated with RFS. Both P2 and B cells localized in significantly closer proximity to melanoma cells in patients who remained recurrence-free (P2 p = 0.0139, B cell p = 0.0049). Our results highlight how characterizing the TIME in primary melanomas may provide new insights into how the complex interplay of the immune system and tumor can modify the disease outcomes. Furthermore, in the context of current clinical trials of adjuvant anti-PD-1 therapies in high-risk stage II primary melanoma, assessment of B cells and P2 could identify patients at risk of recurrence and aid in long-term treatment decisions at the point of primary melanoma diagnosis.

Comprehensive Clinical, Histopathologic, and Molecular Analysis and Long-term Follow-up of Patients With Nodal Blue Nevi.

Blue nevi are benign, melanocytic neoplasms that show a range of clinical and morphologic patterns and include common/dendritic, cellular, and atypical cellular subtypes. Like other nevi, they most commonly occur in skin but can occasionally involve lymph nodes where they may be misinterpreted as representing metastatic melanoma. Moreover, whether benign blue nevi can metastasize to lymph nodes and their natural history and prognostic significance has been the subject of great controversy. To date, few cases of nodal blue nevi have been reported in the literature, and those reports have had limited clinical follow-up and supporting molecular data. This study sought to determine the clinical, pathologic, and molecular features of blue nevi involving lymph nodes, clarify their clinical significance, provide evidence for understanding their pathogenesis, and highlight potential pitfalls in the interpretation of lymph nodes with an ultimate aim of improving patient care. Thirteen cases of blue nevi involving lymph nodes were identified in the archives of Royal Prince Alfred Hospital, Sydney, Australia (1984-2018). A detailed assessment of the clinical and pathologic features of each case was performed, including an evaluation of all available immunohistochemical stains. Extended clinical follow-up was available for 9 patients. Droplet digital polymerase chain reaction for GNAQ Q209L, Q209P and GNA11 Q209L mutations was performed on 7 cases of blue nevi within lymph nodes together with matching cutaneous (presumed primary) blue nevi in 2 cases. All cases showed typical histologic features of blue nevi. BAP1 was retained in all cases (n=7). There were no recurrence or metastasis of blue nevus in any case on long-term clinical follow-up (n=9, median follow-up, 12 y). The majority of cases (n=5 of 7 evaluated) had GNAQ and GNA11 driver mutations. The 2 patients with a matched primary cutaneous blue nevus and regionally associated nodal blue nevus had the same GNAQ Q209L mutation in both sites in each patient. We conclude that blue nevi can involve lymph nodes and are associated with benign clinical behavior, and probably represent so-called "benign" metastasis. Awareness of these lesions is important when evaluating lymph nodes to avoid misdiagnosis as metastatic melanoma.

Pathologist initiated reflex BRAF mutation testing in metastatic melanoma: experience at a specialist melanoma treatment centre.

Testing for BRAF mutations in metastatic melanoma is pivotal to identifying patients suitable for targeted therapy and influences treatment decisions regarding single agent versus combination immunotherapy. Knowledge of BRAF V600E immunohistochemistry (IHC) results can streamline decisions during initial oncology consultations, prior to DNA-based test results. In the absence of formal guidelines that require pathologist initiated ('reflex') BRAF mutation testing, our institution developed a local protocol to perform BRAF V600E IHC on specimens from all stage III/IV melanoma patients when the status is otherwise unknown. This study was designed to evaluate the application of this protocol in a tertiary referral pathology department. A total of 408 stage III/IV melanoma patients had tissue specimens accessioned between 1 January and 31 March in three consecutive years (from 2019 to 2021), reported by 32 individual pathologists. The BRAF mutation status was established by pathologists in 87% (352/408) of cases. When a prior BRAF mutation status was previously known, as confirmed in linked electronic records (202/408), this status had been communicated by the clinician on the pathology request form in 1% of cases (3/202). Pathologists performed BRAF V600E IHC in 153 cases (74% of cases where the status was unknown, 153/206) and testing was duplicated in 5% of cases (20/408). Reflex BRAF IHC testing was omitted in 26% of cases (53/206), often on specimens with small volume disease (cytology specimens or sentinel node biopsies) despite adequate tissue for testing. Incorporating BRAF IHC testing within routine diagnostic protocols of stage III/IV melanoma was both feasible and successful in most cases. Communication of a patient's BRAF mutation status via the pathology request form will likely improve implementation of pathologist initiated BRAF mutation testing and may result in a reduction of duplicate tests. To improve pathologist reflex testing rates, we advocate for the use of an algorithmic approach to pathologist initiated BRAF mutation testing utilising both IHC and DNA-based methodologies for stage III/IV melanoma patients.

Dermoscopic features and screening strategies for the detection of small-diameter melanomas.

BACKGROUND: Around 70% of cutaneous malignant melanomas (MMs) develop de novo, and small-diameter or 'tiny' lesions are expected to represent the earliest manifestation of most MMs. AIM: To describe the clinical, histopathological and dermoscopic features of tiny MMs, and to investigate the impact of imaging tools, including total body photography (TBP) and sequential digital dermoscopy imaging (SDDI) in their detection. METHODS: Consecutive MMs diagnosed over 2 years in a referral centre were retrospectively included. Tiny MMs were defined as MMs with a diameter of ≤ 5 mm on dermoscopy. Dermoscopic features and the performance of four imaging methods were evaluated. RESULTS: Of the 312 MMs included, 86 (27.6%) measured ≤ 5 mm, and 44.2% of these were invasive. Tiny MMs were more frequently excised for being new and/or changing compared with nontiny MMs (77.9% vs. 50.9%; P < 0.001). Half of the tiny MMs would have been missed by the dermoscopic seven-point checklist (48.2%) or the three-point checklist (49.4%), while Menzies' method and the revised pattern analysis correctly identified respectively 65.9% and 63.5% of the tiny MMs. The most frequent positive features for tiny MMs were asymmetry in structure or colour (77.6%), brown dots (65.9%), irregular dots and globules (76.5%) and atypical pigment network (44.7%). Dermoscopic features predictive of invasion in tiny MMs were atypical vascular pattern (OR = 26.5, 95% CI 1.5-475.5, P < 0.01), shiny white lines (OR = 12.4, 95% CI 0.7-237.8, P = 0.04) and grey/blue structures (OR = 3.7, 95% CI 1.3-10.5, P = 0.01). CONCLUSION: Tiny MMs are frequently invasive and represent a clinical, dermoscopic and histopathological challenge. Dermoscopy alone has suboptimal diagnostic accuracy. Early diagnosis relies on the detection of new or changing lesions aided by TBP and SDDI.

Complex melanoma of the scalp: Diagnosis and management aided by confocal microscopy.

Melanomas of lentigo maligna subtype are a steadily growing problem and frequently represent a clinical challenge. A case is reported of a complex melanoma of the scalp illustrating the critical role of confocal microscopy for optimal diagnosis and management.

Multiomic profiling of checkpoint inhibitor-treated melanoma: Identifying predictors of response and resistance, and markers of biological discordance.

We concurrently examine the whole genome, transcriptome, methylome, and immune cell infiltrates in baseline tumors from 77 patients with advanced cutaneous melanoma treated with anti-PD-1 with or without anti-CTLA-4. We show that high tumor mutation burden (TMB), neoantigen load, expression of IFNγ-related genes, programmed death ligand expression, low PSMB8 methylation (therefore high expression), and T cells in the tumor microenvironment are associated with response to immunotherapy. No specific mutation correlates with therapy response. A multivariable model combining the TMB and IFNγ-related gene expression robustly predicts response (89% sensitivity, 53% specificity, area under the curve [AUC], 0.84); tumors with high TMB and a high IFNγ signature show the best response to immunotherapy. This model validates in an independent cohort (80% sensitivity, 59% specificity, AUC, 0.79). Except for a JAK3 loss-of-function mutation, for patients who did not respond as predicted there is no obvious biological mechanism that clearly explained their outlier status, consistent with intratumor and intertumor heterogeneity in response to immunotherapy.

BRAF mutation testing for patients diagnosed with stage III or stage IV melanoma: practical guidance for the Australian setting.

Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now among the possible treatment options for patients with BRAF mutation-positive stage III or stage IV melanoma. This makes prompt BRAF mutation testing an important step in the management of patients diagnosed with stage III or IV melanoma; one that can help better ensure that the optimal choice of systemic treatment is initiated with minimal delay. This article offers guidance about when and how BRAF mutation testing should be conducted when patients are diagnosed with melanoma in Australia. Notably, it recommends that pathologists reflexively order BRAF mutation testing whenever a patient is found to have American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage III or IV melanoma (i.e., any metastatic spread beyond the primary tumour) and that patient's BRAF mutation status is hitherto unknown, even if BRAF mutation testing has not been specifically requested by the treating clinician (in Australia, Medicare-subsidised BRAFV600 mutation testing does not need to be requested by the treating clinician). When performed in centres with appropriate expertise and experience, immunohistochemistry (IHC) using the anti-BRAF V600E monoclonal antibody (VE1) can be a highly sensitive and specific means of detecting BRAFV600E mutations, and may be used as a rapid and relatively inexpensive initial screening test. However, VE1 immunostaining can be technically challenging and difficult to interpret, particularly in heavily pigmented tumours; melanomas with weak, moderate or focal BRAFV600E immunostaining should be regarded as equivocal. It must also be remembered that other activating BRAFV600 mutations (including BRAFV600K), which account for ∼10-20% of BRAFV600 mutations, are not detected with currently available IHC antibodies. For these reasons, if available and practicable, we recommend that DNA-based BRAF mutation testing always be performed, regardless of whether IHC-based testing is also conducted. Advice about tissue/specimen selection for BRAF mutation testing of patients diagnosed with stage III or IV melanoma is also offered in this article; and potential pitfalls when interpreting BRAF mutation tests are highlighted.

Clinical and Molecular Heterogeneity in Patients with Innate Resistance to Anti-PD-1 +/- Anti-CTLA-4 Immunotherapy in Metastatic Melanoma Reveals Distinct Therapeutic Targets.

While immune checkpoint inhibitors targeting the CTLA-4 and PD-1 receptors have significantly improved outcomes of many patients with metastatic melanoma, there remains a group of patients who demonstrate no benefit. In this study, we sought to characterise patients who do not respond to anti-PD-1-based therapies based on their clinical, genetic and immune profiles. Forty patients with metastatic melanoma who did not respond to anti-PD-1 +/- anti-CTLA-4 treatment were identified. Targeted RNA sequencing (n = 37) was performed on pretreatment formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Patients clustered into two groups based on the expression profiles of 26 differentially expressed genes: an immune gene rich group (n = 17) expressing genes associated with immune and T cell signalling, and a second group (n = 20) expressing genes associated with metabolism, signal transduction and neuronal signalling. Multiplex immunohistochemistry validated significantly higher densities of tumour-infiltrating lymphocytes (TILs) and macrophages in the immune gene-rich group. This TIL-high subset of patients also demonstrated higher expression of alternative immune-regulatory drug targets compared to the TIL-low group. Patients were also subdivided into rapid progressors and other progressors (cut-off 2 mo progression-free survival), with significantly lower TILs (p = 0.04) and CD68+ macrophages (p = 0.0091) in the rapid progressors. Furthermore, a trend towards a higher tumour burden was observed in rapid progressors (p = 0.06). These data highlight the need for a personalised and multilayer (clinical and molecular) approach for identifying the most appropriate treatments for anti-PD-1 resistant patients and provides insight into how individual treatment strategies can be achieved.

Transplant-associated penile Kaposi sarcoma managed with single agent paclitaxel chemotherapy: a case report.

BACKGROUND: Kaposi's sarcoma is an uncommon complication in renal transplant patients, and typically presents with cutaneous lesions on the lower extremities. Penile involvement has been reported only rarely. Management of cutaneous-limited disease is primarily reduction of immunosuppression and conversion to an mTOR-inhibitor, whereas the treatment of disseminated disease in transplant patients is more variable. CASE PRESENTATION: A 75-year-old male, originally from Somalia, received a deceased-donor kidney transplant for diabetic and hypertensive nephropathy. Seven months post-transplant he presented with lower limb lesions, oedema and bilateral deep vein thromboses. He then developed a fast-growing painful lesion on his penile shaft. A biopsy of this lesion confirmed KS, and a PET scan demonstrated disseminated disease in the lower extremities, penis and thoracic lymph nodes. His tacrolimus was converted to sirolimus, and his other immunosuppression was reduced. He was treated with single agent paclitaxel chemotherapy in view of his rapidly progressing, widespread disease. The penile lesion completely resolved, and the lower extremity lesions regressed significantly. His kidney allograft function remained stable throughout treatment. CONCLUSION: This case illustrates a rare presentation of an uncommon post-transplant complication and highlights the need for a high index of suspicion of KS in transplant patients presenting with atypical cutaneous lesions. It serves to demonstrate that the use of single agent paclitaxel chemotherapy, switch to an mTORi and reduction in immunosuppression where possible produces excellent short-term outcomes, adding to the body of evidence for this management strategy in disseminated Kaposi's sarcoma.

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.See related Spotlight on p. 600.

Programmed death ligand-1 (PD-L1) as a predictive marker for immunotherapy in solid tumours: a guide to immunohistochemistry implementation and interpretation.

Immunotherapy with checkpoint inhibitors is well established as an effective treatment for non-small cell lung cancer and melanoma. The list of approved indications for treatment with PD-1/PD-L1 checkpoint inhibitors is growing rapidly as clinical trials continue to show their efficacy in patients with a wide range of solid tumours. Clinical trials have used a variety of PD-L1 immunohistochemical assays to evaluate PD-L1 expression on tumour cells, immune cells or both as a potential biomarker to predict response to immunotherapy. Requests to pathologists for PD-L1 testing to guide choice of therapy are rapidly becoming commonplace. Thus, pathologists need to be aware of the different PD-L1 assays, methods of evaluation in different tumour types and the impact of the results on therapeutic decisions. This review discusses the key practical issues relating to the implementation of PD-L1 testing for solid tumours in a pathology laboratory, including evidence for PD-L1 testing, different assay types, the potential interchangeability of PD-L1 antibody clones and staining platforms, scoring criteria for PD-L1, validation, quality assurance, and pitfalls in PD-L1 assessment. This review also explores PD-L1 IHC in solid tumours including non-small cell lung carcinoma, head and neck carcinoma, triple negative breast carcinoma, melanoma, renal cell carcinoma, urothelial carcinoma, gastric and gastroesophageal carcinoma, colorectal carcinoma, hepatocellular carcinoma, and endometrial carcinoma. The review aims to provide pathologists with a practical guide to the implementation and interpretation of PD-L1 testing by immunohistochemistry.