Untreated Melanoma Patients Research Articles

The Use of ctDNA for BRAF Mutation Testing in Routine Clinical Practice in Patients with Advanced Melanoma.

Simple SummaryAssessment of BRAF mutation status is mandatory in advanced, previously untreated melanoma patients since it is present in 40–50% of cases and allows treatment with specific inhibitors. The testing is usually performed on the primary tumor or metastatic lesion; however, in some cases, liquid biopsy and analysis of circulating tumor DNA in the blood can be used. The aim of our study was to evaluate the clinical utility of plasma circulating tumor DNA analysis for BRAF mutation. We identified 46 patients (21 female, 25 male) who underwent such a procedure. A BRAF mutation was found in 45.7% of liquid biopsies and 44.8% of tissue samples. In 18 patients, therapy with BRAF/MEK inhibitors was initiated on the basis of the result of liquid biopsy. Our study confirms the clinical utility of BRAF mutation detection in liquid biopsy.Assessment of BRAF mutation status is mandatory in advanced, treatment-naïve melanoma patients. Liquid biopsy can be an alternative in cases with inadequate or unavailable tumor tissue. The aim of our study was to evaluate the clinical utility of plasma circulating tumor DNA analysis for BRAF mutation testing and to assess outcomes of therapy with BRAF/MEK inhibitors initiated based on the liquid biopsy results. This was a retrospective single-center analysis of 46 patients (21 female, 25 male) with advanced melanoma who underwent circulating tumor DNA (ctDNA) BRAF mutation testing. A BRAF mutation was found in 45.7% (21/46) of liquid biopsies and 44.8% (13/29) of tissue samples. In patients with both ctDNA and tissue samples (n = 29), the concordance between the results of both tests was 82.8%. A BRAF mutation was detected in 7/17 (41.2%) patients with only ctDNA analysis. In 18 patients, therapy with BRAF/MEK inhibitors was initiated on the basis of the result of liquid biopsy. The objective response rate was 77.8 %, and the median PFS was 6.0 months. Our study confirms the clinical utility of BRAF mutation detection in plasma ctDNA. This study provides initial real-world data showing that treatment with BRAF/MEK inhibitors could be commenced based on liquid biopsy results.

Comparative efficacy of dabrafenib+trametinib versus treatment options for metastatic melanoma in first-line settings.

Aim: The objective was to systematically review the literature and assess the relative efficacy of agents approved in first-line settings via network meta-analysis. Materials & methods: A literature review was conducted via searching different medical databases. The eligibility criteria included Phase II or III randomized controlled trialsthat had enrolled treatment-naive adult patients with advanced/metastatic melanoma. Results: The network meta-analysis results suggested that dabrafenib+trametinib significantly prolongs the survival outcomes compared with the monotherapies and had comparable efficacy profile compared with encorafenib+binimetinib and cobimetinib+vemurafenib. In comparison with immunotherapies, the results varied for progression-free survivaland overall survival.Conclusion: Long-term survival data of dabrafenib+trametinib establishes the combination as one of the preferred treatment optionsfor previously untreated melanoma patients.

Autoantibodies as predictors for survival and immune-related adverse events in checkpoint inhibition therapy of metastasized melanoma.

10011 Background: Increasing evidence suggests that the B cell response in cancer patients is an important component of anti-tumour immunity. Autoantibodies targeting tumour and self-antigens may serve as biomarkers of anti-tumour and auto-immunity. As they can be measured in patient' sera, they have great potential as clinical routine biomarkers. The objective of this study was to explore if autoantibodies are associated with survival and immune related adverse events (irAE) in patients with metastatic melanoma under checkpoint inhitibor (CPI) therapy. Methods: Pre-treatment serum samples from 333 metastatic melanoma patients receiving CPI therapy at 5 European centers were retrospectively used to identify autoantibody signatures for survival and irAE. We designed a cancer immunotherapy antigen array comprising 832 autoimmune and tumour antigens as well as immune and cancer pathway proteins. Statistical tests were separately performed for patients treated with anti-CTLA4 (alone or in combination) and with anti-PD1 antibody monotherapy. Cox-regression analysis and univariate statistical tests were applied for biomarker discovery. Progression free and overall survival was measured from treatment initiation to tumor progression or death date. irAE were recorded including onset date and grade (CTC-AE). Results: For each therapy group we identified a set of autoantibody reactivities in untreated melanoma patients that were associated with the development of irAEs and/or survival. The identified autoantibodies target a diverse set of antigens comprising neoantigens (p53), cancer testis antigens (MAGEB4), paraneoplastic antigens (gephyrin), autoantigens (ribosomal proteins, Nor-90) and FGFR1. Autoantigens that correlated with irAE and survival were e.g. anti-MAGEB4 and anti-FGFR1. Elevated anti-MAGEB4 pre-treatment levels were associated with longer overall survival (p = 0.002, HR = 0.77) and the development of irAEs (p = 0.002, HR = 1.27) in ipilimumab +/- nivolumab treated patients. Higher pre-treatment anti-FGFR1 antibodies were associated with shorter survival (p = 0.008, HR = 1.27) and a lower a lower frequency of irAEs (p = 0.04, HR = 0.69) in these patients. Conclusions: We identified autoantibody targets suggesting a diverse B cell response to antigens expressed in tumours and those associated with autoimmunity. Depending on the specific antigen, the immune response towards those antigens may be associated with anti-tumour or pro-tumour responses.

Therapeutic melanoma vaccine with cancer stem cell phenotype represses exhaustion and maintains antigen-specific T cell stemness by up-regulating BCL6

ABSTRACT We developed a therapeutic, gene-modified, allogeneic melanoma vaccine (AGI-101H), which, upon genetic modification, acquired melanoma stem cell-like phenotype. Since its initial clinical trial in 1997, the vaccine has resulted in the long-term survival of a substantial fraction of immunized patients (up to 20 years). Here, we investigated the potential molecular mechanisms underlying the long-lasting effect of AGI-101H using transcriptome profiling of patients’ peripheral T lymphocytes. Magnetically-separated T lymphocytes from AGI-101H-immunized long-term survivors, untreated melanoma patients, and healthy controls were subjected to transcriptome profiling using the microarray analyses. Data were analyzed with a multitude of bioinformatics tools (WebGestalt, DAVID, GSEA) and the results were validated with RT-qPCR. We found substantial differences in the transcriptomes of healthy controls and melanoma patients (both untreated and AGI-101H-vaccinated). AGI-101H immunization induced similar profiles of peripheral T cells as tumor residing in untreated patients. This suggests that whole stem cells immunization mobilizes analogous peripheral T cells to the natural adaptive anti-melanoma response. Moreover, AGI-101H treatment activated the TNF-α and TGF-β signaling pathways and dampened IL2-STAT5 signaling in T cells, which finally resulted in the significant up-regulation of a BCL6 transcriptional repressor, a known amplifier of the proliferative capacity of central memory T cells and mediator of a progenitor fate in antigen-specific T cells. In the present study, high levels of BCL6 transcripts negatively correlated with the expression of several exhaustion markers (CTLA4, KLRG1, PTGER2, IKZF2, TIGIT). Therefore, Bcl6 seems to promote a progenitor fate for cancer-experienced T cells from AGI-101H-vaccinated patients by repressing the exhaustion markers.

Abstract IA05: Selective pressure by activated T cells identifies de novo and adaptive resistance mechanisms in melanoma

Abstract Melanoma has been the tumor type in which immune checkpoint antibodies have been directed for the longest time in clinical trials and clinical practice. Compared to many other advanced solid tumors, a significant minority of patients have manifestations of metastatic disease that are amenable to minimally invasive biopsies to fuel translational research. With CTLA-4 blocking antibodies, the response rate was only 10% and insights into predictive biomarkers only began to emerge once large numbers of patients had been treated and with long-term follow-up. However, with PD-1/PD-L1 blocking antibodies response rates as high as 40% have been observed in previously untreated melanoma patients, making it far more tractable to glean insights into differences between responders and nonresponders in smaller number of patients. Emerging data from analysis of pretreatment tumor biopsies strongly support the greater, but still not high, likelihood of patients responding to PD-1/PD-L1 antibody-based therapy if there are robust numbers of CD8+ T cells infiltrating into a tumor prior to the start of therapy. Response prediction appears to be improved by assessment of T-cell infiltration into tumors early in the course of therapy. Modest improvement in response prediction appears to be achieved by accounting for the presence of regulatory T cells, with those tumors having the highest effector CD8+/Treg ratio being the most likely to respond. Additional predictive accuracy is contributed by a marker of T-cell activation, such as granzyme B, which would only be scored as positive if there are T cells present in the first place. While many additional modulators of T-cell entry and function have been implicated as mechanistically related to immune checkpoint antibody efficacy/resistance, none have been shown to have add predictive accuracy for response. A parallel body of evidence has developed indicating that the presence of predicted mutated neoantigens can predict response to immune checkpoint antibodies in melanoma. It is notable that melanoma has the highest number of mutations per megabase of any common solid tumor, which is thought to associate with the high response rate (40%) compared to other cancers in which PD-1/PD-L1 antibodies have demonstrated efficacy leading to regulatory approvals (15-20% response rates). The current state-of-the-art approach for predicting the presence of mutated antigens is algorithm-based, matching altered peptide sequences with a patient's HLA type to rank potential high-affinity binders. A very small number of such antigens have been validated by the presence of specific T-cell clones that recognize those antigens in patients. Nonetheless, response prediction appears robust and roughly compared to that achieved with phenotypic markers of tumor recognition described above, particularly when predicted mutated neoantigens are clonal. The most recent area of active investigation pertains to the phenotype of tumors with de novo resistance to immune checkpoint antibodies and mechanisms of acquire resistance. Early work suggests that the same phenotype of resistance associated with MAP kinase pathway targeted therapy (tumor cells that lose expression of melanocyte markers in favor of neural crest markers) predicts nonresponse to PD-1 antibodies. Very few patients with progression following response have been interrogated to date, but genetic loss of components of the MHC complex and inactivating mutations in the interferon signaling pathway in tumor cells appear to account for a significant proportion of cases. Citation Format: Keith T. Flaherty. Selective pressure by activated T cells identifies de novo and adaptive resistance mechanisms in melanoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr IA05.

Untreated stage IV melanoma patients exhibit abnormal monocyte phenotypes and decreased functional capacity.

Monocytes may contribute to tumor progression in part by mediating tumor-induced immunosuppression. Alterations to the monocyte populations and functions in untreated patients with late-stage melanoma are not fully understood. To characterize these alterations, we compared the frequency, phenotype, and functional capacity of peripheral blood monocytes and other myeloid cells in untreated, newly diagnosed stage IV melanoma patients (n = 18) with those in healthy volunteers. Stage IV untreated melanoma patients exhibited a sizeable decrease in the percentage of monocytes (P < 0.0001) that included a drop in the percentage of the CD14(+)CD16(-) classical monocyte pool (P = 0.006). Although there was not a significant difference in the CD14(+)HLA-DR(low/-) monocyte population between the patients with melanoma and the healthy volunteers, the HLA-DR levels were considerably lower in the patients' CD14(+)CD16(+) intermediate (P < 0.0001) and CD14(low)CD16(+) nonclassical monocyte populations (P = 0.001). Decreased surface expression of CD86 (P = 0.0006) and TNFRII (P = 0.0001) and increased expression of tissue factor and PD-L1 (P = 0.003) were identified on monocytes from patients with melanoma. Furthermore, these monocytes had decreased ability to upregulate CD80 expression and cytokine production following stimulation with agonist of Toll-like receptor 3 (TLR3). Peripheral blood dendritic cell subsets were decreased in untreated stage IV melanoma patients. Our study demonstrates that untreated late-stage melanoma patients exhibit monocytopenia in addition to phenotypic and functional deficiencies that may negatively affect their immune function. These findings open new avenues into examining the role of monocyte populations in melanoma development.

Phase II trial of RO4929097 Notch gamma-secretase inhibitor in metastatic melanoma: SWOG S0933.

8525 Background: The Notch pathway regulates expression of genes for cell cycle, tissue-specific differentiation, and vasculogenesis. Notch target genes affect melanomagenesis, and Notch levels can influence stemlike versus differentiated tumor cells. Gamma-secretase, which activates intracellular Notch, can be inhibited to kill melanoma cells. We designed this trial to test RO4929097 in pts with melanoma and its effects on T lymphocytes and tumor gene expression. Methods: To assess 6-month progression-free survival (PFS) and 1-year overall survival (OS) in advanced, untreated melanoma patients (pts), a 2-stage accrual design was used. Correlative studies: markers of Notch pathway activation in archived or fresh tumor and T cell functional assays pre-treatment (Rx) and at week 3. Rx dose was 20 mg orally on 3 consecutive days, weekly. Results: 33 pts were Rx’d in stage 1 (median age 61 [range 32-85]; 70% male; 42% elevated LDH; 30% unknown primary; 24% bone mets; 36% liver; 55% lung; 55% lymph node, skin or soft tissue). The clinical outcomes did not meet criteria for stage 2 accrual. One pt had a confirmed PR of 7 months’ (mo) duration. The median PFS was 1.4 mo, [95% confidence interval, c.i. 1.3-2.7], the 6-mo PFS was 11% [95% c.i 3%-33%], and the 1-year OS was 45% [95% c.i. 23%-90%]. Treatment was well-tolerated with no grade (gr) 4-5 tox. The most common gr 2 drug tox were fatigue and nausea in 4 patients (12%) each, and only 4 of 7 gr 3 tox were considered drug-related (1 increased ALT, 1 QTc prolongation, 1 bradycardia, 1 lymphopenia). Pre- and week 3 on-Rx peripheral T cell samples assayed for IL-2 (23 pts) and IFN-γ (22 pts) secretion to Staphylococcal enterotoxin A showed no significant change, in contrast to in vitro gamma-secretase inhibitors which blocked T cell activation. Pre- and on-Rx tumor biopsies in one pt showed no decrease in the Notch target Hey1. Conclusions: RO4929097 at this dose and schedule has limited activity in molecularly-unselected pts with melanoma. Lack of effect on T cell function and tumor Hey1 expression suggests that sustained target inhibition might not have been achieved. Supported in part by PHS Cooperative Agreements, NCI, DHHS CA32102 and CA38926. ClinicalTrials.gov identifier: NCT01120275.

Screening tools for interferon-related cognitive decline in melanoma patients.

8539 Background: Interferon α-2b (IFN) is the only approved adjuvant therapy for high-risk melanoma. IFN-related cognitive deficiencies and mood alterations have been reported. Antidepressants are able to remedy mood, but therapies targeting the cognitive effects of IFN are lacking. Development of therapies requires validation of quantitative measures of cognitive dysfunction in this population. Methods: Melanoma patients who received 1 month high-dose IFN (n = 9), were compared to IFN eligible patients (> stage IIA) who declined therapy (n = 10). Five cognitive function tests were administered: mini-mental status exam, Hopkins Verbal Learning Test-Revised (HVLT), controlled oral word association test (COWAT), Ruff 2 and 7 and Trail Making Tests A and B. The Profile of Mood States-Short Form (POMS) was used to assess mood changes. Test scores were compared as the change from baseline to after IFN or 1-3 months of observation. Welch's t test was used to determine significance. Results: Significant differences were seen in the COWAT, HVLT and POMS. COWAT results showed a significant decrease in word generation (p = 0.013) in IFN patients. HVLT results showed a significant decrease in immediate recall (p = 0.017), but no change in delayed recall or recognition. Significant self-reported mood changes included increased fatigue (p = 0.034), decreased vigor (p = 0.046) and increased confusion (p = 0.034). 8 of 9 IFN patients scored 1 standard deviation (SD) below the observation patient mean on at least one cognitive function test. 5 patients scored 1 SD below on either the HVLT or COWAT. 3 patients scored 1 SD below on the HVLT while 4 scored 1 SD below on the COWAT. Conclusions: In this pilot study, COWAT and HVLT results demonstrate neurocognitive differences between IFN-treated and untreated melanoma patients. It may be possible to use these tests to screen for cognitive dysfunction in IFN patients. Based on these preliminary results, the combination of COWAT and the HVLT immediate recall item is most sensitive. COWAT alone may be more practical than the combination, as it is freely available, easy to administer, and has a sensitivity of 80% vs. the combination. These results must be validated in a larger population and correlated with functional impairment. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merck Schering-Plough Merck, Schering-Plough

In vivo activation of melanoma-specific CD8+ T cells by endogenous tumor antigen and peptide vaccines. A comparison to virus-specific T cells

Many new types of vaccines against infectious or malignant diseases are currently being proposed. Careful characterization of the induced immune response is required in assessing their efficiency. While in most studies human tumor antigen-specific T cells are analyzed after in vitro re-stimulation, we investigated these T cells directly ex vivo using fluorescent tetramers. In peripheral blood lymphocytes from untreated melanoma patients with advanced disease, a fraction of tumor antigen (Melan-A/MART-1)-specific T cells were non-naive, thus revealing tumor-driven immune activation. After immunotherapy with synthetic peptides plus adjuvant, we detected tumor antigen-specific T cells that proliferated and differentiated to memory cells in vivo in some melanoma patients. However, these cells did not present the features of effector cells as found in cytomegalovirus specific T cells analyzed in parallel. Thus, peptide plus adjuvant vaccines can lead to activation and expansion of antigen specific CD8(+) T cells in PBL. Differentiation to protective CD8(+) effector cells may, however, require additional vaccine components that stimulate T cells more efficiently, a major challenge for the development of future immunotherapy.

NONRANDOM DISTRIBUTION OF MUTAGEN-INDUCED CHROMOSOME BREAKS IN LYMPHOCYTES OF PATIENTS WITH DIFFERENT MALIGNANCIES

Data regarding specific chromosomal alterations in most solid neoplasms are scarce because the complex changes observed in tumor biopsies are often a challenge to interpret. The present investigation using chromosomal banding, was designed to analyze exact regions of spontaneous and mutagen-induced lymphocytic chromosomal breaks and investigate if they are unique for different cancers. Tissue from three groups of individuals were included in the study, viz., normal individuals, untreated head and neck cancer patients, and untreated melanoma patients. For every individual three samples were analyzed for spontaneous, bleomycin (radiomimetic)-induced and 4-nitroquinoline-N-oxide(4NQO) (ultraviolet rays mimetic)-induced chromosome damage. The results revealed that in melanoma patients, chromosomes 1, 6, and 9 showed a significantly higher number of breaks than other chromosomes. A clustering of breaks was observed at loci 1p32, 1q32, 6p21, 6q21 and 9q11. Among the head and neck cancer patients, a significantly larger number of breaks was found in chromosomes 3 and 7 with clustering of breaks mainly in regions 3p21, 3q21, 7q22 and 7q32. Thus, it was found that regardless of the mutagen used, specific chromosomes are more susceptible to breakage than others. Our results indicate that chromosomal fragility is specific for particular cancers and that challenging the cells with mutagens reveals it at a more pronounced rate. Mutagen-induced chromosome breaks appear to be nonrandom affecting different chromosomes in different cancers. Clustering of breaks that occur in specific regions of these chromosomes might provide definite clues for molecular analysis and further in-depth studies of cancer predisposed individuals.

Heterogeneity of the T-cell and macrophage infiltrate in cutaneous and subcutaneous metastases of melanoma patients

To examine the intra-individual heterogeneity of the local inflammatory infiltrate in metastases of untreated melanoma patients we analysed 42 skin and subcutaneous metastases from 11 patients. Metastases were excised on the same date (10 patients) or consecutively (six patients), and processed in a two-step immunoperoxidase technique using monoclonal antibodies directed against T-cells (CD3, CD25) and two macrophage subpopulations (RM 3/1, 27E10). The number of positively stained T-cells and macrophages were counted for each lesion in representative high power fields. The mean values were calculated. We observed limited heterogeneity among T-cells (CD3+) and activated T-cells (CD25+) in simultaneously excised metastases. Contrary to this, markedly heterogeneous CD3+ infiltrates were found in metastases taken on different dates, even if there was a time difference of only 1 day between excisions, suggesting spontaneous variations. However, activated T-cells in follow-up biopsies showed only limited heterogeneity. In spite of inter-individual variations, metastases taken simultaneously, as well as consecutively, showed little intra-individual heterogeneity with respect to macrophages. Our findings should be taken into account for comparative studies of the infiltrate in metastatic melanoma during immunotherapy.