Peritumoral endothelial indoleamine 2, 3-dioxygenase expression is an early independent marker of disease relapse in colorectal cancer and is influenced by DNA mismatch repair profile.

Targeting immune checkpoint molecules has become a major new strategy in the treatment of several cancers. Indoleamine 2,3-dioxygenase (IDO)-inhibitors are a potential next-generation immunotherapy, currently investigated in multiple phase I-III trials. IDO is an intracellular immunosuppressive enzyme and its expression/activity has been associated with worse prognosis in several cancers. The aim of this study was to investigate the expression pattern of IDO in colorectal cancer (CRC). In a cohort of 94 CRC patients, primary tumors (PTs) with corresponding tumor-draining lymph nodes (TDLNs, n = 93) and extranodal/distant metastases (n = 27) were retrospectively analyzed by immunohistochemical staining for IDO, CD8 and Foxp3. 45 MSS and 37 MSI-H tumors were selected to compare IDO expression, as these tumors are considered to have different immunogenicity. A highly consistent expression pattern of IDO was observed in the PT, TDLNs and metastases, indicating that immune resistance may be determined very early in the disease course. IDO was expressed both by tumoral cells and host endothelial cells and these expressions were highly correlated (p < 0.001). IDO expression was observed more frequently in the MSI-H subset compared with the MSS subset (43% vs 22% for tumoral expression (p = 0.042) and 38% vs 16% for endothelial expression (p = 0.021)). Endothelial IDO expression was demonstrated to be a negative prognostic marker for recurrence free survival independent of disease stage and DNA mismatch repair (MMR) status (HR 20.67, 95% CI: 3.05-139.94; p = 0.002). These findings indicate that endothelial IDO expression in primary CRC, in addition to the MMR profile, may be helpful in disease stratification.

A Longitudinal Analysis of IDO and PDL1 Expression during Immune- or Targeted Therapy in Advanced Melanoma.

A deepened understanding of the cellular and molecular processes in the tumor microenvironment is necessary for the development of precision immunotherapy (IT). We simultaneously investigated CD3, PDL1, and IDO by immunohistochemistry in paired biopsies from various organs of 43 metastatic melanoma patients treated with IT and targeted therapy (TT). Intraindividual biopsies taken after a period of weeks to months demonstrate discordant results in 30% of the cases. Overlap of IDO and PDL1 increased after therapy. IT only marginally impacted PDL1 expression over time in contrast to TT. Standardized repeated assessments of multiple immune markers in repeated biopsies will generate detailed insights in melanoma's immune evolution and adaption during therapies and might be used to support treatment decisions.

Combination of dabrafenib plus trametinib for BRAF and MEK inhibitor pretreated patients with advanced BRAFV600-mutant melanoma: an open-label, single arm, dual-centre, phase 2 clinical trial.

BACKGROUND: Patients with BRAFV600-mutant melanoma benefit from treatment with the combination of BRAF and MEK inhibitors, but resistance and disease progression develops in most patients. Preclinical studies and case studies have indicated that acquired resistance to BRAF inhibition can be reversible. We aimed to assess the anti-tumour activity of rechallenge with BRAF plus MEK inhibition in a prospective clinical trial. METHODS: In this open-label, single arm, dual-centre, phase 2 academic study in Belgium, patients aged 18 years or older with BRAFV600-mutant melanoma who had previously progressed on BRAF inhibitors (with or without MEK inhibitors) and were off-treatment for at least 12 weeks, were treated with dabrafenib 150 mg orally twice per day plus trametinib 2 mg orally once per day. The primary endpoint was the proportion of patients with investigator-assessed overall response at any time (defined as complete response or partial response according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 confirmed on two occasions, at least 28 days after the first response was recorded). Analyses were done in the intention-to-treat population. The study is ongoing but no longer recruiting patients. This trial is registered with ClinicalTrials.gov, number NCT02296996. FINDINGS: Between April 5, 2014, and Feb 2, 2016, 25 patients were enrolled and initiated treatment in our study. A partial response was documented in eight (32%) of 25 patients (95% CI 15-54; six patients had progressed on previous treatment with dabrafenib plus trametinib and two patients had progressed on previous BRAF inhibitor monotherapy). Stable disease was noted in ten patients (40%; 95% CI 21-61). Rechallenge with dabrafenib plus trametinib was well tolerated. There were no unexpected or grade 4 or 5 treatment-related adverse events. Grade 3 adverse events occurred in two patients (8%; panniculitis [n=1] and pyrexia [n=1]). Serious adverse events which occurred on study were one patient with an Addison crisis triggered by grade 2 pyrexia symptoms that resolved after discontinuation of dabrafenib and trametinib. No patients died as a result of study treatment. INTERPRETATION: Rechallenge with dabrafenib plus trametinib showed anti-tumour activity in patients who had previously progressed on BRAF inhibitors and as such, rechallenge represents a potential new treatment option for these patients. FUNDING: Vlaamse Liga Tegen Kanker, Novartis.

The rationale of indoleamine 2,3-dioxygenase inhibition for cancer therapy.

Indoleamine 2,3-dioxygenase (IDO, also referred to as IDO1) has been demonstrated to be a normal endogenous mechanism of acquired peripheral immune tolerance in vivo. In the field of oncology, IDO expression and/or activity has been observed in several cancer types and has usually been associated with negative prognostic factors and worse outcome measures. This manuscript reviews current available data on the role of IDO in cancer and the current results obtained with IDO inhibition, both in animal models and in phase 1 and 2 clinical trials in humans. Preliminary results with IDO inhibitors, usually combined with other anti-cancer drugs, seem encouraging. Further studies are needed to clarify the conditions in which IDO inhibitors can be of value as an anti-cancer strategy. In addition, further research should address whether the expression of IDO in tissue or blood can be a marker to select patients who can benefit most from IDO inhibition.

A phase II trial of stereotactic body radiotherapy with concurrent anti-PD1 treatment in metastatic melanoma: evaluation of clinical and immunologic response.

BACKGROUND: Antibodies blocking programmed cell death 1 (PD-1) have encouraging responses in patients with metastatic melanoma. Response to anti-PD-1 treatment requires pre-existing CD8+ T cells that are negatively regulated by PD-1-mediated adaptive immune resistance. Unfortunately, less than half of melanoma tumours have these characteristics. Combining anti-PD-1 treatment with other immunomodulating treatments to activate CD8+ T cells is therefore of vital importance to increase response rates and long-term survival benefit in melanoma patients. Both preclinical and retrospective clinical data support the hypothesis that radiotherapy increases the response rates to anti-PD-1 treatment by stimulating the accumulation and activation of CD8+ T cells in the tumour microenvironment. Combining radiotherapy with a PD-1 blocking antibody might therefore increase response rates and even induce long-term survival. The current phase II study will be testing these hypotheses and aims to improve local and distant tumour responses by exploiting the pro-immunogenic effects of radiotherapy in addition to anti-PD-1 treatment. METHODS: The trial will be conducted in patients with metastatic melanoma. Nivolumab or pembrolizumab, both antibodies that target PD-1, will be administrated according to the recommended dosing schedule. Prior to the 2nd cycle, radiotherapy will be delivered in three fractions of 8 Gy to the largest FDG-avid metastatic lesion. The primary endpoint is the proportion of patients with a partial or complete response in non-irradiated metastases according to RECIST v1.1. Secondary endpoints include response rate according to immune related response criteria, metabolic response, local control and survival. To identify peripheral blood biomarkers, peripheral blood mononuclear cells and serum samples will be collected prospectively before, during and after treatment and subjected to flow cytometry and cytokine measurement. DISCUSSION: The current phase II trial aims at exploring the suggested benefits of combining anti-PD-1 treatment and radiotherapy. The translational focus on immunologic markers might be suitable for predicting efficacy and monitoring the effect so to improve patient selection for future clinical applications. ClinicalTrials.gov Identifier NCT02821182.

Quantitative assessment of BRAF V600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors.

BACKGROUND: BRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma. We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors. METHODS: Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treated with dabrafenib and trametinib. RESULTS: 245 plasma samples from 36 patients were analyzed. In 16 patients the first plasma sample was obtained before the first dosing of dabrafenib/trametinib. At baseline, BRAF V600mut ctDNA was detected in 75 % of patients (n = 12/16). BRAF V600mut ctDNA decreased rapidly upon initiation of targeted therapy (p < 0.001) and became undetectable in 60 % of patients (n = 7/12) after 6 weeks of treatment. During treatment, disease progression (PD) was diagnosed in 27 of 36 patients. An increase of the BRAF V600mut ctDNA copy number and fraction, identified PD with a sensitivity of 70 % (n = 19/27) and a specificity of 100 %. An increase in the BRAF V600mut ctDNA fraction was detected prior to clinical PD in 44 % of cases (n = 12/27) and simultaneously with PD in 26 % of patients (n = 7/27). CONCLUSIONS: Quantitative analysis of BRAF V600mut ctDNA in plasma has unique features as a monitoring tool during treatment with BRAF/MEK inhibitors. Its potential as an early predictor of acquired resistance deserves further evaluation.

Comparison of Ex Vivo and In Vivo Dermoscopy in Dermatopathologic Evaluation of Skin Tumors.

IMPORTANCE: Ex vivo dermoscopy (EVD) can be a valuable tool in routine diagnostic dermatopathologic evaluation. OBJECTIVES: To compare in vivo dermoscopy (IVD) and EVD and to provide guidance for routine dermatopathologic evaluations. DESIGN, SETTING, AND PARTICIPANTS: This observational study collected 101 consecutive IVD and EVD images of skin tumors from a private dermatology practice from March 1 to September 30, 2013. Four observers (3 dermatologists and 1 dermatopathologist) blinded to the histopathologic diagnoses independently scored and compared the colors, structures, and vessels of EVD images with those of the corresponding IVD images. Data were analyzed from January 1 to March 31, 2014. MAIN OUTCOMES AND MEASURES: Concordance between the EVD and IVD images and gain or loss of colors, structures, and vessels on EVD relative to IVD images. RESULTS: The final analysis included 404 observations of 101 images. The EVD image was generally similar to the corresponding IVD image but clearly darker, with new areas of blue in 130 of 404 observations (32.2%) and white in 100 of 404 observations (24.8%) and loss of red in 283 of 404 observations (70.0%). Most structures were well preserved. New structureless areas were found in 78 of 404 observations of EVD images (19.3%), and new crystalline structures were detected in 68 of 404 observations of EVD images (16.8%). On EVD images, squames and crusts were lost in 56 of 404 observations (13.9%) and 43 of 404 observations (10.6%), respectively. Blood vessels were lost in 142 of 404 observations of EVD images (35.1%). CONCLUSIONS AND RELEVANCE: The EVD image is an important new tool in dermatopathology and may give direction to targeted tissue processing and examination of skin tumors.

Applications for quantitative measurement of BRAF V600 mutant cell-free tumor DNA in the plasma of patients with metastatic melanoma.

Small fragments of cell-free DNA that are shed by normal and tumor cells can be detected in the plasma of patients with advanced melanoma. Quantitative measurement of BRAF V600 mutant DNA within the cell-free DNA holds promise as a tumor-specific biomarker for diagnosis and therapeutic monitoring in patients with BRAF V600 mutant melanoma. Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations on DNA extracted from 1 ml of plasma is currently under evaluation in a number of ongoing prospective clinical studies. We report five patient cases that indicate the potential applications and utility of quantitative measurements of BRAF V600 mutant cell-free tumor DNA as a diagnostic test and as a therapeutic monitoring tool in stage IV melanoma patients treated with BRAF-targeted therapy or immunotherapy. Finally, we offer novel insights into the dynamics of cell-free tumor DNA in melanoma.

Total-Body Examination vs Lesion-Directed Skin Cancer Screening.

IMPORTANCE: Skin cancer is the most frequent cancer type. It remains unknown if and how screening programs can be organized in a cost-effective manner. OBJECTIVE: To compare the 2 screening strategies of systematic total-body examination (TBE) and lesion-directed screening (LDS), with a focus on the participation rate, detection rate, anxiety, and cost. DESIGN, SETTING, AND PARTICIPANTS: Population-based cross-sectional screenings by a team of 6 dermatologists were organized in 2 sociodemographically similar regions. The TBE was organized in a community of 9325 inhabitants 18 years and older (Wichelen, East Flanders, Belgium) during a 5-day screening (March 14-18, 2014). The LDS was organized in a sociodemographically comparable community (Nevele, East Flanders, Belgium) of 9484 adult inhabitants during a 4-day screening (April 22 and 25-27, 2014). The first population received a personal invitation for a standard TBE. In the second population, individuals were invited for an LDS if they had a lesion meeting 1 or more of the following criteria: ABCD rule (A, asymmetry; B, borders; C, colors; and D, differential structures), ugly duckling sign, new lesion lasting longer than 4 weeks, or red nonhealing lesions. MAIN OUTCOMES AND MEASURES: In total, 1982 individuals were screened, and 47 skin cancers (2.4%) were histologically confirmed, including 9 melanomas (0.5%), 37 basal cell carcinomas (1.9%), and 1 squamous cell carcinoma or Bowen disease (0.1%). RESULTS: The positive predictive value for all suspicious lesions was 56.6% (47 of 83). The participation rate was 17.9% (1668 of 9325) in the TBE group vs 3.3% (314 of 9484) in the LDS group (P < .01). The skin cancer detection rate per 100 participants did not differ significantly between the 2 groups, with rates of 2.3% (39 of 1668) in the TBE group vs 3.2% (8 of 248) in the LDS group (P = .40). The operational effectiveness per 100 invitees was 0.4% (39 of 9325) in the TBE group vs 0.1% (8 of 9484) in the LDS group (P < .01). In addition, LDS was 5.6 times less time consuming than TBE. Participants in the LDS group had significantly higher baseline anxiety levels compared with participants in the TBE group (3.7 vs 3.3 points on a visual analog scale, P < .01). In screenees without a suspicious lesion, anxiety levels significantly dropped after screening. CONCLUSIONS AND RELEVANCE: Total-body examination yielded a higher absolute number of skin cancers. Lesion-directed screening had a similar detection rate of 3.2% (8 of 248) but was 5.6 times less time consuming. When performed by dermatologists, LDS is an acceptable alternative screening method in health care systems with limited budgets or long waiting lists.

Systemic immune changes associated with adjuvant interferon-α2b-therapy in stage III melanoma patients: failure at the effector phase?

Interferon-α (IFN-α) is the only approved adjuvant treatment for high-risk melanoma patients in Europe, but the impact on overall survival is low. Although it is believed that IFN-α exerts its effects through immunomodulation, data on its impact on circulating immune cells are scarce. Flow cytometry was performed on peripheral blood mononuclear cells of eight IFN-α2b-treated stage III melanoma patients and 26 untreated stage III melanoma patients as controls to enumerate myeloid and plasmacytoid dendritic cells (mDC and pDC), monocytic and polymorphonuclear myeloid-derived suppressor cells (mMDSC and pmnMDSC) and cytotoxic and regulatory T-cells (Tregs). The expression of several immunosuppressive markers [indoleamine 2,3-dioxygenase (IDO), programmed-death ligand-1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA4)] was explored. IDO activity in the blood was confirmed by ultra-performance liquid chromatography. Compared with controls, IFN-α2b treatment was associated with increased IDO expression by pDCs (P=0.021) and an increased kynurenine/tryptophan ratio in the serum (P=0.004), compatible with IDO enzyme activity. Furthermore, IFN-α2b-treated patients had a decreased mDC/DC ratio (P=0.002), decreased CD3+ lymphocytes (P=0.034) and increased circulating Treg (P<0.001) and PD-L1+cytotoxic T-cell (P=0.001) frequencies. IDO expression is upregulated in circulating pDCs of high-risk melanoma patients treated with adjuvant IFN-α2b. This is associated with tryptophan consumption in the patients' serum and higher Treg and PD-L1+cytotoxic T-cell frequencies. We hypothesize that in IFN-α2b-treated patients, IDO activity acts as a negative feedback mechanism and might limit the clinical efficacy of IFN-α2b therapy. The underlying mechanism should be explored as this could lead to more efficient immunotherapies.

Clinical significance of plasmacytoid dendritic cells and myeloid-derived suppressor cells in melanoma.

BACKGROUND: Immune markers in the peripheral blood of melanoma patients could provide prognostic information. However, there is currently no consensus on which circulating cell types have more clinical impact. We therefore evaluated myeloid-derived suppressor cells (MDSC), dendritic cells (DC), cytotoxic T-cells and regulatory T-cells (Treg) in a series of blood samples of melanoma patients in different stages of disease. METHODS: Flow cytometry was performed on peripheral blood mononuclear cells of 69 stage I to IV melanoma patients with a median follow-up of 39 months after diagnosis to measure the percentage of monocytic MDSCs (mMDSCs), polymorphonuclear MDSCs (pmnMDSCs), myeloid DCs (mDCs), plasmacytoid DCs (pDCs), cytotoxic T-cells and Tregs. We also assessed the expression of PD-L1 and CTLA-4 in cytotoxic T-cells and Tregs respectively. The impact of cell frequencies on prognosis was tested with multivariate Cox regression modelling. RESULTS: Circulating pDC levels were decreased in patients with advanced (P = 0.001) or active (P = 0.002) disease. Low pDC levels conferred an independent negative impact on overall (P = 0.025) and progression-free survival (P = 0.036). Even before relapse, a decrease in pDC levels was observed (P = 0.002, correlation coefficient 0.898). High levels of circulating MDSCs (>4.13%) have an independent negative prognostic impact on OS (P = 0.012). MDSC levels were associated with decreased CD3+ (P < 0.001) and CD3 + CD8+ (P = 0.017) T-cell levels. Conversely, patients with high MDSC levels had more PD-L1+ T-cells (P = 0.033) and more CTLA-4 expression by Tregs (P = 0.003). pDCs and MDSCs were inversely correlated (P = 0.004). The impact of pDC levels on prognosis and prediction of the presence of systemic disease was stronger than that of MDSC levels. CONCLUSION: We demonstrated that circulating pDC and MDSC levels are inversely correlated but have an independent prognostic value in melanoma patients. These cell types represent a single immunologic system and should be evaluated together. Both are key players in the immunological climate in melanoma patients, as they are correlated with circulating cytotoxic and regulatory T-cells. Circulating pDC and MDSC levels should be considered in future immunoprofiling efforts as they could impact disease management.

Objective responses can be obtained by CTLA-4 inhibition in metastatic melanoma after BRAF inhibitor failure.

The aim of this study was to determine the activity of ipilimumab (ipi)-based therapy after treatment failure with a BRAF inhibitor (BRAFi). Sixty-four patients with unresectable stage III or stage IV BRAF V600-mutant melanoma who were treated sequentially with a BRAFi and ipi-based therapy [ipi as monotherapy or ipi in combination with an autologous mRNA electroporated dendritic cell vaccine (TriMixDC-MEL)] were identified. Thirty-three patients had been treated with a BRAFi before ipi-based therapy (BRAFi-first), and 31 patients had been treated with ipi-based therapy first (ipi-first). In patients treated with a BRAFi first (n=33), the best response on sequential ipi-based therapy was three complete responses and six partial responses (best objective response rate of 27%). In patients treated with ipi-based therapy first (n=31), the best response on ipi-based therapy was 0 complete response and four partial responses (best objective response rate of 13%). The response rate did not differ significantly between the two groups (P=0.14). The median overall survival from the start of ipi-based therapy was 10 months (95% confidence interval: 5.7-14.3) in the BRAFi-first group and 12.3 months (95% confidence interval: 7.4-17.2) in the ipi-first group (P=0.34). We report that objective tumor responses to ipi-based immunotherapy can still be obtained after progression has occurred upon treatment with a BRAFi. A part of this observation might be related to the results obtained with a combination of ipi and TriMixDC-MEL.

A short dermoscopy training increases diagnostic performance in both inexperienced and experienced dermatologists.

Dermoscopy is a clinical tool known to improve the early detection of melanoma and other malignancies of the skin, but only for experienced users. Our aim was to evaluate the effect of short (3-hour) dermoscopy training sessions in both residents and practicing dermatologists. The training improved diagnostic accuracy for both melanocytic and nonmelanocytic neoplasms of the skin and the observed effect was the highest for residents but was also significant for more experienced practicing dermatologists.

Hypomelanoses in children.

Hypomelanosis of the skin is a frequently encountered problem in childhood, being totally innocent or representing the first sign of a multisystem disorder. Medical history, clinical examination, Wood's light investigation, histological analysis of the skin and a multidisciplinary consultation can contribute to a correct and early diagnosis of the different types of hypopigmentations. In the present paper, we present a systematic clinical approach to the differential diagnosis of those skin disorders.

miR-145 overexpression suppresses the migration and invasion of metastatic melanoma cells.

MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression which play important roles in tumorigenesis and cancer metastasis. Since they are often highly deregulated in various types of cancer, miRNAs may be effective treatment targets. miRNA profiling studies of melanoma have led to the identification of several tumor suppressor miRNAs. One of these include miR-145, although functional data proving its specific function are limited. Therefore, in this study, we examined the expression levels of miR-145 in three melanoma cell lines (BLM, FM3P and WM793). Additional gain-of-function experiments revealed that miR-145 exerts an anti-proliferative effect in the primary, non-invasive melanoma cell line, WM793, whereas cell migration and the invasive potential of metastatic melanoma cells was suppressed following transfection with miR-145 mimics. In order to investigate the mechanisms by which miR-145 exerts its invasion suppressor function, we examined the expression level of target genes [fascin homolog 1 (FSCN1), myosin­Va (MYO5A and SOX9] and that of an indirect target (RAB27A) following the overexpression of miR-145. The results showed that SOX9, MYO5A and RAB27A were not involved in the biological effects caused by miR-145 mimics. Surprisingly, we discovered that miR-145 in melanoma, in contrast to many other tumor types, does not necessarily act via the target, FSCN1, since the downregulation of FSCN1 did not inhibit cell proliferation or migration but, on the contrary, increased cell invasion in two out of the three melanoma cell lines examined. Our in vitro data is in accordance with previously reported in vivo data describing the low expression of FSCN1 in malignant melanomas when compared to dysplastic nevi, suggesting that the expression of FSCN1 decreases as the formation and progression stage of melanoma advances. In conclusion, our data provide evidence that miR-145 is an invasion suppressor in metastatic melanoma cells. Despite the fact that it remains unclear which genes or pathways are regulated by miR-145 in melanoma, miR-145 may serve as a useful therapeutic agent in melanoma when re-expressed in situ.

Immune mediated mechanisms of melanocyte destruction: Paving the way for efficient immunotherapeutic strategies against melanoma.

Insights into immune reactions against benign and malignant melanocytes may help the development of more efficient immunotherapeutic treatments for melanoma. The interplay between an active systemic antitumor immunity and a responsive local tumor environment is crucial to achieve effective clinical responses. Increasing evidence confirms this strategy can lead to an adequate and durable immunosurveillance of melanocytes.