Max Schreuer

Characterization of the in vivo immune network of IDO, tryptophan metabolism, PD-L1, and CTLA-4 in circulating immune cells in melanoma

In melanoma, both the induction of immunosuppression by tumor cells and the inflammatory antitumor response can induce an upregulation of counter-regulatory mechanisms such as indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and CTLA-4+ regulatory T-cells (Tregs) in the tumor microenvironment. Even though these immunosuppressive mediators are targets for immunotherapy, research investigating their expression in the peripheral blood is lacking. We therefore, performed flow cytometry on PBMCs of stage I–IV melanoma patients. IDO expression was detected in plasmacytoid dendritic cells (pDC) and monocytic myeloid-derived suppressor cells (mMDSC), and increased in advanced disease stage (p = 0.027). Tryptophan breakdown confirmed the functional activity of IDO and was linked with increased PD-L1+ cytotoxic T-cells (p = 0.009), relative lymphopenia (p = 0.036), and a higher mDC/pDC ratio (p = 0.002). High levels of circulating PD-L1+ cytotoxic T-cells were associated with increased CTLA-4 expression by Tregs (p = 0.005) and MDSC levels (p = 0.033). This illustrates that counter-regulatory immune mechanisms in melanoma should be considered as one interrelated signaling network. Moreover, both increased PD-L1+ T-cells and CTLA-4 expression in Tregs conferred a negative prognosis, indicating their in vivo relevance. Remarkably, circulating CTLA-4, IDO, and pDC levels were altered according to prior invasion of the sentinel lymph node and IDO expression in the sentinel was associated with more IDO+ PBMCs. We conclude that the expression of IDO, PD-L1, and CTLA-4 in the peripheral blood of melanoma patients is strongly interconnected, associated with advanced disease and negative outcome, independent of disease stage. Combination treatments targeting several of these markers are therefore likely to exert a synergistic response.

Peritumoral indoleamine 2,3‐dioxygenase expression in melanoma: an early marker of resistance to immune control?

Indoleamine 2,3‐dioxygenase (IDO) is an emerging immunomodulating factor in cancer. IDO expression in tumour‐negative sentinel lymph nodes (SLNs) of patients with melanoma has a negative prognostic value.

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.

Granulomatous nephritis and dermatitis in a patient with BRAF V600E mutant metastatic melanoma treated with dabrafenib and trametinib.

A 61-year-old man was diagnosed with stage IIIB BRAF V600E mutant melanoma in October 2012. He was treated with a combination therapy of dabrafenib and trametinib. He remained in complete remission for 18 months and the treatment was well tolerated after dose reduction because of pyrexia. In March 2013, he developed bilateral pitting edema of the legs with an erythematous, slightly infiltrated rash on his back and upper arms. His face was edematous, with a heliotrope rash-like aspect. Eye examination showed bilateral blepharitis. Additional blood test showed inflammation and acute kidney injury Rifle category failure. A skin and kidney biopsy indicated a granulomatous inflammation. A complete workup for other causes of granulomatous inflammation was negative. Treatment with dabrafenib and trametinib was stopped and corticosteroids were initiated, with a rapid beneficial effect on both the kidney function and skin rash. When corticosteroids were halted after 1 month, a rapid decline in the kidney function was observed. After reintroduction of corticosteroids, kidney function normalized and steroids could be tapered gradually over 6 months. To our knowledge, interstitial nephritis has not been described in patients on BRAF-targeted nor MEK-targeted therapy for melanoma, although it has been described in a melanoma patient treated with the immune checkpoint inhibitor, ipilimumab. Currently, the patient has no sign of local or distal recurrence of melanoma, notwithstanding that treatment with dabrafenib and trametinib has been stopped for 10 months and no other antimelanoma therapy was initiated.

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

Interferon-&agr; (IFN-&agr;) 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-&agr; 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-&agr;2b-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-&agr;2b 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-&agr;2b-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-&agr;2b. 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-&agr;2b-treated patients, IDO activity acts as a negative feedback mechanism and might limit the clinical efficacy of IFN-&agr;2b therapy. The underlying mechanism should be explored as this could lead to more efficient immunotherapies.

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.

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.

Illustrative cases for monitoring by quantitative analysis of BRAF/NRAS ctDNA mutations in liquid biopsies of metastatic melanoma patients who gained clinical benefits from anti-PD1 antibody therapy

Anti-programmed death 1 (PD-1) monoclonal antibodies improve the survival of metastatic melanoma patients. Predictive or monitoring biomarkers for response to this therapy could improve the clinical management of these patients. To date, no established biomarkers are available for monitoring the response to immunotherapy. Tumor- specific mutations in circulating tumor DNA (ctDNA) such as BRAF and NRAS mutations for melanoma patients have been proposed for monitoring of immunotherapy response. We present seven illustrative cases for the use of ctDNA BRAF and NRAS mutations’ monitoring in plasma. The cases described exemplify four distinct clinical benefit patterns: rapid and durable complete response (CR), early progression, followed by CR, CR followed by early progression after interrupting treatment and long-term disease stabilization. These representative cases suggest that comprehensive BRAF/NRAS ctDNA monitoring during anti-PD1 therapy is informative and can be of added value for the monitoring of melanoma patients gaining clinical benefit on anti-PD1 treatment. An important advantage of our approach is that using the cartridge system on the Idylla platform for mutation analysis, the results become available the same day 2 h after plasma collection. Therefore, in the future, the ctDNA level can be an element in the clinical management of the patients.

Abstract B003: A randomized controlled phase II clinical trial on mRNA electroporated autologous dendritic cells for stage III/IV melanoma patients who are disease-free following the local treatment of macrometastases

Autologous monocyte-derived synthetic mRNA electroporated dendritic cells (TriMixDC-MEL) are immunogenic and can induce anti-tumor activity in pretreated advanced melanoma patients when administered by the intradermal (id) and intravenous (iv) route. We investigated the safety and activity of TriMixDC-MEL in stage III/IV melanoma patients who are disease free following the local treatment of macrometastases. We performed a randomized, controlled, non-comparative phase II clinical trial where TriMixDC-MEL was administered iv (20.106 DCs) and id (4.106 DCs) at 2 separate sites of the body every 2 weeks for a total of 4 administrations and a fifth administration after 16 weeks in patients randomized to the treatment arm. Patients on the control arm did not receive treatment but were allowed to “cross-over” and be treated with TriMixDC-MEL at the time of non-salvageable recurrence. Tumor evaluations on both arms were performed by total-body 18-FDG-PET/CT every 12 weeks. The primary endpoint was the percentage of patients who were alive and free from melanoma macrometastases at 1 year following randomization. Patients were allowed to undergo local salvage treatments for loco-regional melanoma recurrences during the study (week 0-52). If local salvage treatment during the study resulted in a disease-free status that was confirmed at any of the planned assessment, but not earlier than 16 weeks after the salvage treatment, these patients were considered not to have reached the primary endpoint of the study. Between November 2012 and November 2014, 41 eligible patients were randomized between the TriMixDC-MEL treatment arm (n = 21) and control-arm (n = 20). Baseline characteristics (22M/19F; median age 57.5y (range 24-81); AJCC stage III/IV-M1a/-M1b/-M1c: 33/1/6/1 patients, ulcerated primary melanoma: 12 patients) were well balanced between both groups. After a median follow-up of 28 months (range 15 to 40 months) 21 patients experienced a non-salvageable melanoma recurrence (7 on the DC- and 14 on the control-arm). The rate of patients who were disease-free at 1 year (evaluable population = 41 patients) was higher in the TriMixDC-MEL treated group (68% [95%CI 46-86] vs. 35% [14-55]). The time-to-non-salvageable melanoma recurrence was significantly lower in the TriMixDC-MEL treated group (log-rank p = .021). Seven patients in the TriMixDC-MEL group and 3 patients in the control arm were offered local salvage therapy at first recurrence (surgery: 9 patients; RT: 1pt). TriMixDC-MEL was well tolerated (there were no grade ≥3 AE related to TriMixDC-MEL). AE were limited to: local skin injection reactions (grade 1-2; 17/21 patients), flu-like symptoms (grade 1-2; 4/21 patients), and post-infusion chills (grade 1-2; 4 [19%] patients). The results of this non-comparative randomized controlled phase II clinical trial of TriMixDC-MEL id/iv versus observation support the further evaluation of TriMixDC-MEL as a well-tolerated adjuvant therapy for melanoma patients following the resection of macrometastases. Citation Format: Bart Neyns, Yanina Jansen, Jurgen Corthals, Sofie Wilgenhof, Max Schreuer, Carlo Heirman, Kris Thielemans. A randomized controlled phase II clinical trial on mRNA electroporated autologous dendritic cells for stage III/IV melanoma patients who are disease-free following the local treatment of macrometastases [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B003.