Circulating Tumor DNA in Patients with Desmoid Fibromatosis during Active Surveillance.

BACKGROUND: Sporadic desmoid fibromatosis (DF) is a rare locally aggressive tumor characterized by mutation in exon 3 of CTNNB1 (T41A, S45F, and S45P). Standard of care is active surveillance (AS), but 30% require treatment. DF clinical course is unpredictable and identification of prognostic markers is needed to tailor strategy. In this prospective study, we investigated the consistency between mutation detected in tumor biopsies with that detected in plasma by digital droplet PCR (ddPCR) and the association between circulating tumor DNA (ctDNA) abundancy with clinical outcome. PATIENTS AND METHODS: A total of 56 patients and 10 healthy donors were included. CTNNB1 mutation status of DF biopsies was determined by Sanger and in case of WT CTNNB1 with NGS. In matched plasma samples at enrollment and during AS at specific timepoints, we evaluated cfDNA quantity and ctDNA. RESULTS: ctDNA levels were measured in 46 patients with CTNNB1 mutation. Detection rate for T41A, S45F and S45P was 68%, 42% and 100%, respectively. S45P variant has been detected in all patients with S45P mutation. Longitudinal assessment of ctDNA during AS in nine patients (four with regression and five with progression as first event according to RECIST) showed a concordance between the event and ctDNA level change in six out of nine patients tested (4/5 with progression and 2/4 with regression). CONCLUSIONS: Results of ctDNA analysis support its potential clinical implementation as diagnostic tool in specific clinical scenarios where biopsy can be challenging. A prospective clinical trial needs to be performed to evaluate the potential role of ctDNA as predictive biomarker.

Extracellular vesicles miR-574-5p and miR-181a-5p as prognostic markers in NSCLC patients treated with nivolumab.

Immune checkpoint inhibitors (ICIs) have revolutionized the management of advanced non-small cell lung cancer (NSCLC), although patient survival is still unsatisfactory. Accurate predictive markers capable of personalizing the treatment of patients with NSCLC are still lacking. Circulating extracellular vesicles involved in cell-to-cell communications through miRNAs (EV-miRs) transfer are promising markers. Plasma from 245 patients with advanced NSCLC who received nivolumab as second-line therapy was collected and analyzed. EV-miRnome was profiled on 174/245 patients by microarray platform, and selected EV-miRs were validated by qPCR. A prognostic model combining EV-miR and clinical variables was built using stepwise Cox regression analysis and tested on an independent patient cohort (71/245). EV-PD-L1 gene copy number was assessed by digital PCR. For 54 patients with disease control, EV-miR changes at best response versus baseline were investigated by microarray and validated by qPCR. EV-miRNome profiling at baseline identified two EV-miRs (miR-181a-5p and miR-574-5p) that, combined with performance status, are capable of discriminating patients unlikely from those that are likely to benefit from immunotherapy (median overall survival of 4 months or higher than 9 months, respectively). EV-PD-L1 digital evaluation reported higher baseline copy number in patients at increased risk of mortality, without improving the prognostic score. Best response EV-miRNome profiling selected six deregulated EV-miRs (miR19a-3p, miR-20a-5p, miR-142-3p, miR-1260a, miR-1260b, and miR-5100) in responding patients. Their longitudinal monitoring highlighted a significant downmodulation already in the first treatment cycles, which lasted more than 6 months. Our results demonstrate that EV-miRs are promising prognostic markers for NSCLC patients treated with nivolumab.

Spatial distribution of tumour immune infiltrate predicts outcomes of patients with high-risk soft tissue sarcomas after neoadjuvant chemotherapy.

BACKGROUND: Anthracycline-based neoadjuvant chemotherapy (NAC) may modify tumour immune infiltrate. This study characterized immune infiltrate spatial distribution after NAC in primary high-risk soft tissue sarcomas (STS) and investigate association with prognosis. METHODS: The ISG-STS 1001 trial randomized STS patients to anthracycline plus ifosfamide (AI) or a histology-tailored (HT) NAC. Four areas of tumour specimens were sampled: the area showing the highest lymphocyte infiltrate (HI) at H&E; the area with lack of post-treatment changes (highest grade, HG); the area with post-treatment changes (lowest grade, LG); and the tumour edge (TE). CD3, CD8, PD-1, CD20, FOXP3, and CD163 were analyzed at immunohistochemistry and digital pathology. A machine learning method was used to generate sarcoma immune index scores (SIS) that predict patient disease-free and overall survival (DFS and OS). FINDINGS: Tumour infiltrating lymphocytes and PD-1+ cells together with CD163+ cells were more represented in STS histologies with complex compared to simple karyotype, while CD20+ B-cells were detected in both these histology groups. PD-1+ cells exerted a negative prognostic value irrespectively of their spatial distribution. Enrichment in CD20+ B-cells at HI and TE areas was associated with better patient outcomes. We generated a prognostic SIS for each tumour area, having the HI-SIS the best performance. Such prognostic value was driven by treatment with AI. INTERPRETATION: The different spatial distribution of immune populations and their different association with prognosis support NAC as a modifier of tumour immune infiltrate in STS. FUNDING: Pharmamar; Italian Ministry of Health [RF-2019-12370923; GR-2016-02362609]; 5 × 1000 Funds-2016, Italian Ministry of Health; AIRC Grant [ID#28546].

Modulation of faecal miRNAs highlights the preventive effects of a Mediterranean low-inflammatory dietary intervention.

BACKGROUND: Dietary interventions have been proposed as therapeutic approaches for several diseases, including cancer. A low-inflammatory Mediterranean dietary intervention, conducted as a pilot study in subjects with Familial Adenomatous Polyposis (FAP), reduced markers of local and systemic inflammation. We aim to determine whether this diet may modulate faecal microRNA (miRNA) and gene expression in the gut. METHODS: Changes in the faecal miRNome were evaluated by small RNA sequencing at baseline (T0), after the three-month intervention (T1), and after an additional three months (T2). Changes in the transcriptome of healthy rectal mucosa and adenomas were evaluated by RNA sequencing at T0 and T2. The identification of validated miRNA-gene interactions and functional analysis of miRNA targets were performed using in silico approaches. RESULTS: Twenty-seven subjects were included in this study. It was observed that the diet modulated 29 faecal miRNAs (p < 0.01; |log2 Fold Change|>1), and this modulation persisted for three months after the intervention. Levels of miR-3612-3p and miR-941 correlated with the adherence to the diet, miR-3670 and miR-4252-5p with faecal calprotectin, and miR-3670 and miR-6867 with serum calprotectin. Seventy genes were differentially expressed between adenoma and normal tissue, and most were different before the dietary intervention but reached similar levels after the diet. Functional enrichment analysis identified the proinflammatory ERK1/2, cell cycle regulation, and nutrient response pathways as commonly regulated by the modulated miRNAs and genes. CONCLUSIONS: Faecal miRNAs modulated by the dietary intervention target genes that participate in inflammation. Changes in levels of miRNAs and genes with oncogenic and tumour suppressor functions further support the potential cancer-preventive effect of the low-inflammatory Mediterranean diet. CLINICAL TRIAL NUMBER REGISTRATION: NCT04552405, Registered in ClinicalTrials.gov.

Immunological characterization of a long-lasting response in a patient with metastatic triple-negative breast cancer treated with PD-1 and LAG-3 blockade.

In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.

Comprehensive genomic profiling on metastatic Melanoma: results from a network screening from 7 Italian Cancer Centres.

BACKGROUND: The current therapeutic algorithm for Advanced Stage Melanoma comprises of alternating lines of Targeted and Immuno-therapy, mostly via Immune-Checkpoint blockade. While Comprehensive Genomic Profiling of solid tumours has been approved as a companion diagnostic, still no approved predictive biomarkers are available for Melanoma aside from BRAF mutations and the controversial Tumor Mutational Burden. This study presents the results of a Multi-Centre Observational Clinical Trial of Comprehensive Genomic Profiling on Target and Immuno-therapy treated advanced Melanoma. METHODS: 82 samples, collected from 7 Italian Cancer Centres of FFPE-archived Metastatic Melanoma and matched blood were sequenced via a custom-made 184-gene amplicon-based NGS panel. Sequencing and bioinformatics analysis was performed at a central hub. Primary analysis was carried out via the Ion Reporter framework. Secondary analysis and Machine Learning modelling comprising of uni and multivariate, COX/Lasso combination, and Random Forest, was implemented via custom R/Python scripting. RESULTS: The genomics landscape of the ACC-mela cohort is comparable at the somatic level for Single Nucleotide Variants and INDELs aside a few gene targets. All the clinically relevant targets such as BRAF and NRAS have a comparable distribution thus suggesting the value of larger scale sequencing in melanoma. No comparability is reached at the CNV level due to biotechnological biases and cohort numerosity. Tumour Mutational Burden is slightly higher in median for Complete Responders but fails to achieve statistical significance in Kaplan-Meier survival analysis via several thresholding strategies. Mutations on PDGFRB, NOTCH3 and RET were shown to have a positive effect on Immune-checkpoint treatment Overall and Disease-Free Survival, while variants in NOTCH4 were found to be detrimental for both endpoints. CONCLUSIONS: The results presented in this study show the value and the challenge of a genomics-driven network trial. The data can be also a valuable resource as a validation cohort for Immunotherapy and Target therapy genomic biomarker research.

Multistep tumor genetic evolution and changes in immunogenicity trigger immune-mediated disease eradication in stage IV melanoma: lessons from a single case.

Durable remissions are observed in 10%-20% of treated patients with advanced metastatic melanoma but the factors associated with long-term complete clinical responses are largely unknown. Here, we report the molecular characteristics of tumor evolution during disease progression along a 9-year clinical course in a patient with advanced disseminated melanoma who received different treatments, including trametinib, ipilimumab, radiation, vemurafenib, surgical tumor debulking and a second ipilimumab course, ultimately achieving complete long-term disease remission.Longitudinal analyses of therapies-resistant metastatic tumors revealed the effects of different treatments on tumor's microenvironment and immunogenicity, ultimately creating a milieu favorable to immunotherapy response. Monitoring of the temporal dynamics of T cells by analysis of the T cell receptor (TCR) repertoire in the tumor and peripheral blood during disease evolution indicated that T-cell clones with common TCR rearrangements, present at low levels at baseline, were maintained and expanded after immunotherapy, and that TCR diversity increased. Analysis of genetic, molecular, and cellular components of the tumor depicted a multistep process in which treatment with kinase inhibitors strongly conditioned the immune microenvironment creating an inflamed milieu converting cold into hot tumors, while ipilimumab impacted and increased the TCR repertoire, a requirement for tumor rejection.Since the optimal sequencing of treatment with antibodies targeting immune checkpoints and kinase inhibitors for advanced melanoma is still clinically debated, this case indicates that immunotherapy success is possible even after progression on targeted therapy.

Pathologic complete response after neoadjuvant chemotherapy/(re)chemoradiation for pelvic relapse of rectal cancer undergoing complex pelvic surgery: more frequent than expected?

PURPOSE: To estimate the rate of pathologic complete response (pCR) after neoadjuvant chemotherapy/(re)chemoradiation and its impact on survival in locally recurrent rectal cancer (LRRC) and to identify predictors of pCR or differences between neoadjuvant treatments. METHODS: Among 394 LRRC patients treated at the National Cancer Institute of Milan (Italy), 74 (27.8%) were treated with neoadjuvant chemotherapy with or without (re)chemoradiation before surgery. The pCR rate was estimated, and its impact on 5-year survival was evaluated with the Kaplan-Meier survival method. Univariate analysis was performed to find pre-treatment predictors of pCR. RESULTS: After surgery, in 12 (16.2%) patients, a pCR was observed. All patients who reached pCR had R0 margins after surgery; among the 62 non-pCR patients, R0 margins were obtained in 29 (46.8%) cases only (p = 0.0004). pCR patients showed a significantly higher 5-year overall survival compared to non-pCR cases (33.3% vs. 21.0%, p = 0.045) and a trend toward better 5-year re-local recurrence-free survival. On univariate analysis, no predictor of pCR was found in the present study based on pre-treatment features. CONCLUSION: Since pCR is significantly associated to R0 resection and 5-year overall survival, pCR could be a target for LRRC cure. However, pCR is currently unpredictable based on pre-treatment features.

Development of a Molecular Blood-Based Immune Signature Classifier as Biomarker for Risks Assessment in Lung Cancer Screening.

BACKGROUND: Low-dose CT (LDCT) screening trials have shown that lung cancer early detection saves lives. However, a better stratification of the screening population is still needed. In this respect, we generated and prospectively validated a plasma miRNA signature classifier (MSC) able to categorize screening participants according to lung cancer risk. Here, we aimed to deeply characterize the peripheral immune profile and develop a diagnostic immune signature classifier to further implement blood testing in lung cancer screening. METHODS: Peripheral blood mononuclear cell (PBMC) samples collected from 20 patients with LDCT-detected lung cancer and 20 matched cancer-free screening volunteers were analyzed by flow cytometry using multiplex panels characterizing both lymphoid and myeloid immune subsets. Data were validated in PBMC from 40 patients with lung cancer and 40 matched controls and in a lung cancer specificity set including 27 subjects with suspicious lung nodules. A qPCR-based gene expression signature was generated resembling selected immune subsets. RESULTS: Monocytic myeloid-derived suppressor cell (MDSC), polymorphonuclear MDSC, intermediate monocytes and CD8+PD-1+ T cells distinguished patients with lung cancer from controls with AUCs values of 0.94/0.72/0.88 in the training, validation, and lung cancer specificity set, respectively. AUCs raised up to 1.00/0.84/0.92 in subgroup analysis considering only MSC-negative subjects. A 14-immune genes expression signature distinguished patients from controls with AUC values of 0.76 in the validation set and 0.83 in MSC-negative subjects. CONCLUSIONS: An immune-based classifier can enhance the accuracy of blood testing, thus supporting the contribution of systemic immunity to lung carcinogenesis. IMPACT: Implementing LDCT screening trials with minimally invasive blood tests could help reduce unnecessary procedures and optimize cost-effectiveness.

Efficacy of mRNA anti-SARS-CoV-2 vaccination and dynamics of humoral immune response in patients with solid tumors: results from the institutional registry of an Italian tertiary cancer center.

Background: Systemic immunosuppression characterizing cancer patients represents a concern regarding the efficacy of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, and real-world evidence is needed to define the efficacy and the dynamics of humoral immune response to mRNA-based anti-SARS-CoV-2 vaccines. Methods: We conducted an observational study that included patients with solid tumors who were candidates for mRNA anti-SARS-CoV-2 vaccination at the Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. The primary objective was to monitor the immunologic response to the mRNA anti-SARS-CoV-2 vaccination in terms of anti-spike antibody levels. All the patients received two doses of the mRNA-1273 vaccine or the BNT162b2 vaccine. Healthcare workers served as a control group of healthy subjects. Results: Among the 243 patients included in the present analysis, 208 (85.60%) and 238 (97.94%) resulted seroconverted after the first and the second dose of vaccine, respectively. Only five patients (2.06%) had a negative titer after the second dose. No significant differences in the rate of seroconversion after two vaccine doses were observed in patients as compared with the control group of healthy subjects. Age and anticancer treatment class had an independent impact on the antibody titer after the second dose of vaccination. In a subgroup of 171 patients with available data about the third timepoint, patients receiving immunotherapy with immune checkpoint inhibitors seem to have a higher peak of antibodies soon after the second dose (3 weeks after), but a more pronounced decrease at a late timepoint (3 months after). Conclusions: The systemic immunosuppression characterizing cancer patients did not seem to dramatically affect the humoral response to anti-SARS-CoV-2 mRNA vaccines in our population of patients with solid tumors. Further investigation is needed to dissect the interplay between immunotherapy and longitudinal dynamics of humoral response to mRNA vaccines, as well as to analyze the cellular response to mRNA vaccines in cancer patients.

Genetic Layout of Melanoma Lesions Is Associated with BRAF/MEK-Targeted Therapy Resistance and Transcriptional Profiles.

The genetic landscape of melanoma resistance to targeted therapy with small molecules inhibiting BRAF and MEK kinases is still largely undefined. In this study, we portrayed in detail the somatic alterations of resistant melanoma and explored the associated biological processes and their integration with transcriptional profiles. By targeted next-generation sequencing and whole-exome sequencing analyses, a list of 101 genes showing imbalance in metastatic tumors from patients with a complete/durable response or disease progression during therapy with vemurafenib or with dabrafenib and trametinib was defined. Classification of altered genes in functional categories indicated that the mutational pattern of both resistant tumors and melanoma cell lines was enriched in gene families involved in oncogenic signaling pathways and in DNA repair. Integration of genomic and transcriptomic features showed that the enrichment of mutations in gene sets associated with anabolic processes, chromatin alterations, and IFN-α response determined a significant positive modulation of the same gene signatures at the transcriptional level. In particular, MTORC1 signaling was enriched in tumors from poorly responsive patients and in resistant tumors excised from treated patients. Results indicate that genetic patterns are associated with melanoma resistance to targeted therapy and disclose the underlying key molecular pathways to define drug combinations for improved personalized therapies.

Low baseline neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios predict increased overall survival in locally recurrent rectal cancer despite R1 margins.

BACKGROUND: Prognostic features in locally recurrent rectal cancer (LRRC), beyond R0 surgery, are unknown. AIMS: Aim of the present study was to evaluate the prognostic role of peripheral immune estimators, such as neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR), on survival outcomes in LRRC patients. METHODS: 184 LRRC patients treated at the National Cancer Institute of Milan (Italy) were included. Optimal cut-off values for NLR and PLR were determined. Kaplan-Meier curves and multivariate Cox analyses were used to assess the 5-yr overall survival (OS) according to NLR and PLR, also considering margins status. RESULTS: NLR >3.9 (hazard ratio [HR] 3.96, P = 0.049), PLR >275 (HR 5.39, P = 0.002) and size on imaging (HR 1.36, P = 0.044) were associated to worse OS. R+ patients with NLR >3.9 showed a significantly lower 5-yr OS compared to NLR ≤3.9 (13.5% vs. 36.7%, P < 0.0001). Also PLR >275 was related with a lower 5-yr OS compared to PLR ≤275 in R+ patients (6.4% vs. 36.8%, P = 0.0003). Conversely, NLR and PLR were irrelevant in case of R0 surgery. CONCLUSION: NLR and PLR predict 5-yr OS in LRRC, also identifying a subset of R+ patients with a similar expected survival compared to R0 cases.

3D tumor explant as a novel platform to investigate therapeutic pathways and predictive biomarkers in cancer patients.

Immunotherapy with immune checkpoint inhibitors can induce durable clinical responses in different human malignancies but the number of responding patients remains globally modest. The limited therapeutic efficacy of ICI depends on multiple factors, among which the immune suppressive features of the tumor microenvironment play a key role. For this reason, experimental models that enable dissection of the immune-hostile tumor milieu components are required to unravel how to overcome resistance and obtain full-fledged anti-tumor immunity. Recent evidence supports the usefulness of 3D ex vivo systems in retaining features of tumor microenvironment to elucidate molecular and immunologic mechanisms of response and resistance to immune checkpoint blockade. In this perspective article we discuss the recent advances in patient-derived 3D tumor models and their potential in support of treatment decision making in clinical setting. We will also share our experience with dynamic bioreactor tumor explant culture of samples from melanoma and sarcoma patients as a reliable and promising platform to unravel immune responses to immune checkpoint inhibitors.

Fasting-Mimicking Diet Is Safe and Reshapes Metabolism and Antitumor Immunity in Patients with Cancer.

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. SIGNIFICANCE: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy.This article is highlighted in the In This Issue feature, p. 1.

Extracellular vesicles in anti-tumor immunity.

To what extent extracellular vesicles (EVs) can impact anti-tumor immune responses has only started to get unraveled. Their nanometer dimensions, their growing number of subtypes together with the difficulties in defining their origin hamper their investigation. The existence of tumor cell lines facilitated advance in cancer EV understanding, while capturing information about phenotypes and functions of immune cell EVs in this context is more complex. The advent of immunotherapy with immune checkpoint inhibitors has further deepened the need to dissect the impact of EVs during immune activation and response, not least to contribute unraveling and preventing the generation of resistance occurring in the majority of patients. Here we discuss the factors that influence anddrive the immune response in cancer patients in the context of cancer therapeutics and the roles or possible functions that EVs can have in this scenario. With immune cell-derived EVs as leitmotiv, we will journey from EV discovery and subtypes through physiological and pathological functions, from similarities with tumor EVs to measures to revert detrimental consequences on immune responses to cancer.

Assessing the safety, tolerability and efficacy of PLGA-based immunomodulatory nanoparticles in patients with advanced NY-ESO-1-positive cancers: a first-in-human phase I open-label dose-escalation study protocol.

INTRODUCTION: The undiminished need for more effective cancer treatments stimulates the development of novel cancer immunotherapy candidates. The archetypical cancer immunotherapy would induce robust, targeted and long-lasting immune responses while simultaneously circumventing immunosuppression in the tumour microenvironment. For this purpose, we developed a novel immunomodulatory nanomedicine: PRECIOUS-01. As a PLGA-based nanocarrier, PRECIOUS-01 encapsulates a tumour antigen (NY-ESO-1) and an invariant natural killer T cell activator to target and augment specific antitumour immune responses in patients with NY-ESO-1-expressing advanced cancers. METHODS AND ANALYSIS: This open-label, first-in-human, phase I dose-escalation trial investigates the safety, tolerability and immune-modulatory activity of increasing doses of PRECIOUS-01 administered intravenously in subjects with advanced NY-ESO-1-expressing solid tumours. A total of 15 subjects will receive three intravenous infusions of PRECIOUS-01 at a 3-weekly interval in three dose-finding cohorts. The trial follows a 3+3 design for the dose-escalation steps to establish a maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D). Depending on the toxicity, the two highest dosing cohorts will be extended to delineate the immune-related parameters as a readout for pharmacodynamics. Subjects will be monitored for safety and the occurrence of dose-limiting toxicities. If the MTD is not reached in the planned dose-escalation cohorts, the RP2D will be based on the observed safety and immune-modulatory activity as a pharmacodynamic parameter supporting the RP2D. The preliminary efficacy will be evaluated as an exploratory endpoint using the best overall response rate, according to Response Evaluation Criteria in Solid Tumors V.1.1. ETHICS AND DISSEMINATION: The Dutch competent authority (CCMO) reviewed the trial application and the medical research ethics committee (CMO Arnhem-Nijmegen) approved the trial under registration number NL72876.000.20. The results will be disseminated via (inter)national conferences and submitted for publication to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04751786.

Genetic Variants and Somatic Alterations Associated with MITF-E318K Germline Mutation in Melanoma Patients.

The MITF-E318K variant has been implicated in genetic predisposition to cutaneous melanoma. We addressed the occurrence of MITF-E318K and its association with germline status of CDKN2A and MC1R genes in a hospital-based series of 248 melanoma patients including cohorts of multiple, familial, pediatric, sporadic and melanoma associated with other tumors. Seven MITF-E318K carriers were identified, spanning every group except the pediatric patients. Three carriers showed mutated CDKN2A, five displayed MC1R variants, while the sporadic carrier revealed no variants. Germline/tumor whole exome sequencing for this carrier revealed germline variants of unknown significance in ATM and FANCI genes and, in four BRAF-V600E metastases, somatic loss of the MITF wild-type allele, amplification of MITF-E318K and deletion of a 9p21.3 chromosomal region including CDKN2A and MTAP. In silico analysis of tumors from MITF-E318K melanoma carriers in the TCGA Pan-Cancer-Atlas dataset confirmed the association with BRAF mutation and 9p21.3 deletion revealing a common genetic pattern. MTAP was the gene deleted at homozygous level in the highest number of patients. These results support the utility of both germline and tumor genome analysis to define tumor groups providing enhanced information for clinical strategies and highlight the importance of melanoma prevention programs for MITF-E318K patients.

Nanobiotechnology and Immunotherapy: Two Powerful and Cooperative Allies against Cancer.

A number of novel cancer therapies have recently emerged that have rapidly moved from the bench to the clinic. Onco-immunotherapies, such as immune checkpoint blockade inhibitors and adoptive cell therapies, have revolutionized the field, since they provide a way to induce strong anti-tumor immune responses, which are able to fight cancer effectively. However, despite showing great efficacy in hematological and some solid tumors, unresponsiveness, development of therapy resistance and the development of serious adverse effects, limit their capacity to impact the vast majority of tumors. Nanoparticle-based delivery systems are versatile vehicles for a wide variety of molecular cargoes and provide an innovative strategy to improve conventional onco-immunotherapies. They can be finely tuned to release their contents in the tumor microenvironment, or to deliver combinations of adjuvants and antigens in the case of nanovaccines. In this review, we summarize the recent advancements in the field of nanobiotechnology, to remodel the tumor microenvironment and to enhance immunotherapies.

Liquid Biopsy and Radiological Response Predict Outcomes Following Discontinuation of Targeted Therapy in Patients with BRAF Mutated Melanoma.

BACKGROUND: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity after sustained response are unknown. MATERIALS AND METHODS: This retrospective case series analysis conducted at a single Cancer Center in Italy included patients with BRAF mutated metastatic melanoma treated with a BRAF inhibitor as a single agent or in combination with a MEK inhibitor between June 1, 2011 and January 1, 2020 and interrupted treatment due to cumulative toxicity after achieving complete response (CR) or long-lasting partial response (PR; i.e. >12 months). RESULTS: We included 24 patients with a median treatment duration of 59.4 months (95% confidence interval [CI], 55.4-63.4; range, 12-88). CR and PR were achieved in 71% and 29% of patients, respectively. At a median follow-up after treatment discontinuation of 37.8 months (95% CI, 33.7-41.9), the 12-month progression-free survival after discontinuation (dPFS) rate was 70.8% (95% CI 54.8-91.6) and 24-month dPFS rate was 58.3% (95% CI, 41.6-81.8). Baseline patient and tumor characteristics as well as treatment duration and best response did not significantly impact on dPFS. Patients with CR and negative circulating tumor DNA (ctDNA) at time of discontinuation had a significantly improved dPFS compared with patients with either radiological residual disease or ctDNA positivity (p = .007). No patient in CR with undetectable ctDNA experienced progression. CONCLUSION: The risk of progression is high even in patients with sustained sensitivity to BRAF/MEK inhibitors. Integration of liquid biopsy in clinical trials investigating the optimal management of patients with sustained sensitivity to BRAF/MEK inhibitors is warranted. IMPLICATIONS FOR PRACTICE: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity are unknown. This study analyzed patients with sustained responses (median treatment duration 59.4 months). Twelve- and 24-month progression-free survival following discontinuation were 70.8% and 58.3%, respectively. Complete response and negative circulating tumor DNA at time of discontinuation are promising prognostic biomarkers in this setting.