Markus Hecht

Radiosensitization by BRAF inhibitor therapy—mechanism and frequency of toxicity in melanoma patients

BACKGROUND Recent evidence suggests that ionizing radiation may be associated with unexpected side-effects in melanoma patients treated with concomitant BRAF inhibitors. A large multicenter analysis was carried out to generate reliable safety data and elucidate the mechanism. METHODS A total of 161 melanoma patients from 11 European skin cancer centers were evaluated for acute and late toxicity, of whom 70 consecutive patients received 86 series of radiotherapy with concomitant BRAF inhibitor therapy. To further characterize and quantify a possible radiosensitization by BRAF inhibitors, blood samples of 35 melanoma patients were used for individual radiosensitivity testing by fluorescence in situ hybridization of chromosomal breaks after ex vivo irradiation. RESULTS With radiotherapy and concomitant BRAF inhibitor therapy the rate of acute radiodermatitis ≥2° was 36% and follicular cystic proliferation was seen in 13% of all radiotherapies. Non-skin toxicities included hearing disorders (4%) and dysphagia (2%). Following whole-brain radiotherapy, rates of radiodermatitis ≥2° were 44% and 8% (P < 0.001) for patients with and without BRAF inhibitor therapy, respectively. Concomitant treatment with vemurafenib induced acute radiodermatitis ≥2° more frequently than treatment with dabrafenib (40% versus 26%, P = 0.07). In line with these findings, analysis of chromosomal breaks ex vivo indicated significantly increased radiosensitivity for patients under vemurafenib (P = 0.004) and for patients switched from vemurafenib to dabrafenib (P = 0.002), but not for patients on dabrafenib only. No toxicities were reported after stereotactic treatment. CONCLUSION Radiotherapy with concomitant BRAF inhibitor therapy is feasible with an acceptable increase in toxicity. Vemurafenib is a more potent radiosensitizer than dabrafenib.BACKGROUND Recent evidence suggests that ionizing radiation may be associated with unexpected side-effects in melanoma patients treated with concomitant BRAF inhibitors. A large multicenter analysis was carried out to generate reliable safety data and elucidate the mechanism. METHODS A total of 161 melanoma patients from 11 European skin cancer centers were evaluated for acute and late toxicity, of whom 70 consecutive patients received 86 series of radiotherapy with concomitant BRAF inhibitor therapy. To further characterize and quantify a possible radiosensitization by BRAF inhibitors, blood samples of 35 melanoma patients were used for individual radiosensitivity testing by fluorescence in situ hybridization of chromosomal breaks after ex vivo irradiation. RESULTS With radiotherapy and concomitant BRAF inhibitor therapy the rate of acute radiodermatitis ≥2° was 36% and follicular cystic proliferation was seen in 13% of all radiotherapies. Non-skin toxicities included hearing disorders (4%) and dysphagia (2%). Following whole-brain radiotherapy, rates of radiodermatitis ≥2° were 44% and 8% (P < 0.001) for patients with and without BRAF inhibitor therapy, respectively. Concomitant treatment with vemurafenib induced acute radiodermatitis ≥2° more frequently than treatment with dabrafenib (40% versus 26%, P = 0.07). In line with these findings, analysis of chromosomal breaks ex vivo indicated significantly increased radiosensitivity for patients under vemurafenib (P = 0.004) and for patients switched from vemurafenib to dabrafenib (P = 0.002), but not for patients on dabrafenib only. No toxicities were reported after stereotactic treatment. CONCLUSION Radiotherapy with concomitant BRAF inhibitor therapy is feasible with an acceptable increase in toxicity. Vemurafenib is a more potent radiosensitizer than dabrafenib.

Cytotoxic effect of efavirenz is selective against cancer cells and associated with the cannabinoid system.

Background:Recently, a regression of precancerous lesions in HIV-1-infected patients after initiation of HAART was reported. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) as efavirenz (EFV) might be mediators of this effect, as they are known to have a cytotoxic effect on tumour cells. A potential mechanism involved in this effect may be the activation of the cannabinoid receptor to mediate tumour toxicity. Methods:Several tumour-derived and fibroblast cell lines were studied. Cytotoxicity of EFV was evaluated by Annexin-Pi staining. The expression of the cannabinoid receptors CB1, CB2 and GPR55 was analysed by western blot, quantitative reverse transcriptase (qRT-PCR) and fluorescence activated cell sorting. The influence of the cannabinoid agonists and antagonists on the effects of EFV was investigated. Furthermore, the effect of EFV on the phosphorylation state of the growth factors Erk, Akt and the tumour suppressor protein p53 was tested. Results:EFV revealed a selective cytotoxic effect on several tumour cell lines, whereas primary fibroblasts were not affected. The cytotoxic effect was associated with the expression of CB1. The combination of EFV with cannabinoid agonists showed an increase in toxicity. The phosphorylation state of Erk and Akt was not affected by EFV, whereas p53 showed an increased phosphorylation. Conclusion:EFV has a selective cytotoxic effect on several tumour cells. Furthermore, EFV led to an activating phosphorylation of the tumour suppressor protein p53 going in line with earlier reports that EFV may be antitumourigenic and a potential cytostatic drug. The observed synergistic effect with cannabinoid agonists implicates an involvement of the cannabinoid system.

Increased skin and mucosal toxicity in the combination of vemurafenib with radiation therapy

BackgroundPalliative radiotherapy is often required for patients with metastatic malignant melanoma in the case of bone or brain metastases. Since BRAF inhibitor therapy is highly efficient in V600-mutated melanomas, there is hesitation to stop it during radiotherapy. Consequently, radiotherapy under simultaneous vemurafenib treatment is frequently needed.Case reportWe report the case of a patient receiving palliative radiotherapy of spinal bone metastases before and during vemurafenib therapy. The skin reactions were quantitatively scored using computer-assisted digital image evaluation.ResultsRadiotherapy without vemurafenib was tolerated very well, whereas radiotherapy under simultaneous vemurafenib treatment resulted in accentuated skin reactions. Furthermore, the patient developed dysphagia and had to be hospitalized for parenteral nutrition. In the quantitative analysis, there was a twofold increase in pigmentation and erythema of the irradiated skin area of the thoracic spine when vemurafenib was combined with radiotherapy compared with radiotherapy treatment alone. This is the first reported case of a patient showing no complications during radiotherapy without vemurafenib but remarkable skin and mucosal toxicity under concurrent vemurafenib therapy. Thus, a genetically conditioned individually elevated radiosensitivity can definitely be excluded. Compared with other reported cases, radiosensitization was not limited to the skin, but also affected the esophageal mucosa.ConclusionVemurafenib is a strong radiosensitizer. Patients receiving radiotherapy under simultaneous vemurafenib treatment should be monitored very closely.ZusammenfassungHintergrundBei Patienten mit metastasiertem Melanom ist die palliative Bestrahlung von Knochen- oder Hirnmetastasen häufig erforderlich. Da eine Therapie mit BRAF-Inhibitoren bei Patienten mit V600-mutierten Melanomen hoch effektiv ist, sollte man sie während einer Strahlentherapie nicht unterbrechen. Daher ist eine Strahlentherapie unter laufender Behandlung mit Vemurafenib häufig erforderlich.MethodenHier wird der Fall eines Patienten dargestellt, der palliative Bestrahlungen von Wirbelkörpermetastasen vor und während einer Behandlung mit Vemurafenib erhalten hat. Bei diesem Fallbericht wurde die Stärke der Hautreaktionen mit computerassistierter digitaler Bildanalyse quantitativ ausgewertet.ErgebnisDie Strahlentherapie ohne Vemurafenib wurde gut vertragen, wogegen es bei der Bestrahlung unter simultaner Vemurafenib-Therapie zu verstärkten Hautreaktionen kam. Darüber hinaus entwickelte der Patient eine Mukositis mit ausgeprägten Schluckbeschwerden, so dass er zur parenteralen Ernährung stationär aufgenommen werden musste. Verglichen zur alleinigen Bestrahlung waren Pigmentierung und Rötung in der quantitativen Auswertung bei der Kombination von Bestrahlung mit Vemurafenib auf das Doppelte erhöht. Dies ist der erste berichtete Fall eines Patienten, bei dem ohne Vemurafenib eine Strahlentherapie komplikationslos durchführbar war und in Kombination mit Vemurafenib Haut- und Schleimhauttoxizität auftraten. Daher kann eine genetisch bedingte individuell erhöhte Strahlenempfindlichkeit definitiv ausgeschlossen werden. Verglichen mit anderen berichteten Fällenbetraf die erhöhte Strahlenempfindlichkeit nicht nur die Haut, sondern auch die Schleimhaut des Ösophagus.SchlussfolgerungVemurafenib wirkt stark strahlensensibilisierend. Patienten, die unter simultaner Behandlung mit Vemurafenib bestrahlt werden, sollten engmaschig überwacht werden.

Chemoradiation Increases PD-L1 Expression in Certain Melanoma and Glioblastoma Cells

Immunotherapy approaches currently make their way into the clinics to improve the outcome of standard radiochemotherapy (RCT). The programed cell death receptor ligand 1 (PD-L1) is one possible target that, upon blockade, allows T cell-dependent antitumor immune responses to be executed. To date, it is unclear which RCT protocol and which fractionation scheme leads to increased PD-L1 expression and thereby renders blockade of this immune suppressive pathway reasonable. We therefore investigated the impact of radiotherapy (RT), chemotherapy (CT), and RCT on PD-L1 surface expression on tumor cells of tumor entities with differing somatic mutation prevalence. Murine melanoma (B16-F10), glioblastoma (GL261-luc2), and colorectal (CT26) tumor cells were treated with dacarbazine, temozolomide, and a combination of irinotecan, oxaliplatin, and fluorouracil, respectively. Additionally, they were irradiated with a single dose [10 Gray (Gy)] or hypo-fractionated (2 × 5 Gy), respectively, norm-fractionated (5 × 2 Gy) radiation protocols were used. PD-L1 surface and intracellular interferon (IFN)-gamma expression was measured by flow cytometry, and IL-6 release was determined by ELISA. Furthermore, tumor cell death was monitored by AnnexinV-FITC/7-AAD staining. For first in vivo analyses, the B16-F10 mouse melanoma model was chosen. In B16-F10 and GL261-luc2 cells, particularly norm-fractionated and hypo-fractionated radiation led to a significant increase of surface PD-L1, which could not be observed in CT26 cells. Furthermore, PD-L1 expression is more pronounced on vital tumor cells and goes along with increased levels of IFN-gamma in the tumor cells. In melanoma cells CT was the main trigger for IL-6 release, while in glioblastoma cells it was norm-fractionated RT. In vivo, fractionated RT only in combination with dacarbazine induced PD-L1 expression on melanoma cells. Our results suggest a tumor cell-mediated upregulation of PD-L1 expression following in particular chemoradiation that is not only dependent on the somatic mutation prevalence of the tumor entity.

Efavirenz Has the Highest Anti-Proliferative Effect of Non-Nucleoside Reverse Transcriptase Inhibitors against Pancreatic Cancer Cells

Background Cancer prevention and therapy in HIV-1-infected patients will play an important role in future. The non-nucleoside reverse transcriptase inhibitors (NNRTI) Efavirenz and Nevirapine are cytotoxic against cancer cells in vitro. As other NNRTIs have not been studied so far, all clinically used NNRTIs were tested and the in vitro toxic concentrations were compared to drug levels in patients to predict possible anti-cancer effects in vivo. Methods Cytotoxicity was studied by Annexin-V-APC/7AAD staining and flow cytometry in the pancreatic cancer cell lines BxPC-3 and Panc-1 and confirmed by colony formation assays. The 50% effective cytotoxic concentrations (EC50) were calculated and compared to the blood levels in our patients and published data. Results The in vitro EC50 of the different drugs in the BxPC-3 pancreatic cancer cells were: Efavirenz 31.5μmol/l (= 9944ng/ml), Nevirapine 239μmol/l (= 63786ng/ml), Etravirine 89.0μmol/l (= 38740ng/ml), Lersivirine 543μmol/l (= 168523ng/ml), Delavirdine 171μmol/l (= 78072ng/ml), Rilpivirine 24.4μmol/l (= 8941ng/ml). As Efavirenz and Rilpivirine had the highest cytotoxic potential and Nevirapine is frequently used in HIV-1 positive patients, the results of these three drugs were further studied in Panc-1 pancreatic cancer cells and confirmed with colony formation assays. 205 patient blood levels of Efavirenz, 127 of Rilpivirine and 31 of Nevirapine were analyzed. The mean blood level of Efavirenz was 3587ng/ml (range 162–15363ng/ml), of Rilpivirine 144ng/ml (range 0-572ng/ml) and of Nevirapine 4955ng/ml (range 1856–8697ng/ml). Blood levels from our patients and from published data had comparable Efavirenz levels to the in vitro toxic EC50 in about 1 to 5% of all patients. Conclusion All studied NNRTIs were toxic against cancer cells. A low percentage of patients taking Efavirenz reached in vitro cytotoxic blood levels. It can be speculated that in HIV-1 positive patients having high Efavirenz blood levels pancreatic cancer incidence might be reduced. Efavirenz might be a new option in the treatment of cancer.

Clinical outcome of concomitant vs interrupted BRAF inhibitor therapy during radiotherapy in melanoma patients

This corrects the article DOI: 10.1038/bjc.2017.85

Spatial distribution of FoxP3+ and CD8+ tumour infiltrating T cells reflects their functional activity

Background Regulatory and cytotoxic T cells are key players in the hosts anticancer immune response. We studied the spatial distribution of FoxP+ and CD8+ cells to identify potential interactions. Methods In 202 patients 103 pre-radiochemotherapy biopsies and 153 post-radiochemotherapy tumour specimens of advanced rectal cancer were available and an immunohistochemical double staining of FoxP3+ and CD8+ tumour-infiltrating lymphocytes was performed to investigate cell density and cell-to-cell distances. Results FoxP3+ cells decreased after radiochemotherapy by a factor of 3 while CD8+ cells remained nearly unchanged. High epithelial (p=0.033) and stromal (p=0.009) FoxP3+ cell density was associated with an improved overall survival. Cell-to-cell distances of randomly distributed cells were simulated and compared to observed cell-to-cell distances. Observed distances shorter than the simulated, random distances were hypothesized to represent FoxP3+ cells actively interacting with CD8+ cells. Epithelial short distances were associated with a favourable prognosis while the opposite was true for the stromal compartment. Conclusion The analysis of cell-to-cell distances may offer a tool to predict outcome, maybe by identifying functionally active, interacting infiltrating inflammatory cells in different tumour compartments.

Ex Vivo Apoptosis in CD8+ Lymphocytes Predicts Rectal Cancer Patient Outcome.

Background. Apoptotic rates in peripheral blood lymphocytes can predict radiation induced normal tissue toxicity. We studied whether apoptosis in lymphocytes has a prognostic value for therapy outcome. Methods. Lymphocytes of 87 rectal cancer patients were ex vivo irradiated with 2 Gy, 8 Gy, or a combination of 2 Gy ionizing radiation and Oxaliplatin. Cells were stained with Annexin V and 7-Aminoactinomycin D and apoptotic and necrotic rates were analyzed by multicolor flow cytometry. Results. After treatment, apoptotic and necrotic rates in CD8+ cells are consistently higher than in CD4+ cells, with lower corresponding necrotic rates. Apoptotic and necrotic rates of CD4+ cells and CD8+ cells correlated well within the 2 Gy, 8 Gy, and 2 Gy and Oxaliplatin arrangements (p ≤ 0.009). High apoptotic CD8+ rates after 2 Gy, 8 Gy, and 2 Gy + Oxaliplatin treatment were prognostically favorable for metastasis-free survival (p = 0.009, p = 0.038, and p = 0.009) and disease-free survival (p = 0.013, p = 0.098, and p = 0.013). Conclusions. Ex vivo CD8+ apoptotic rates are able to predict the patient outcome in regard to metastasis-free or disease-free survival. Patients with higher CD8+ apoptotic rates in the peripheral blood have a more favorable prognosis. In addition to the prediction of late-toxicity by utilization of CD4+ apoptotic rates, the therapy outcome can be predicted by CD8+ apoptotic rates.

Durvalumab verbessert die Prognose beim lokal fortgeschrittenen nicht-kleinzelligen Bronchialkarzinom nach definitiver Radiochemotherapie

Hintergrund und Ziel Bei 60–80% der Patienten kommt es nach definitiver Radiochemotherapie (RCT) von lokal fortgeschrittenen nicht-kleinzelligen Bronchialkarzinomen (NSCLC) im weiteren Verlauf zum Progress. Eine Immuntherapie durch Inhibitoren des PD-1/PD-L1-Signalwegs ist in der Behandlung des metastasierten Bronchialkarzinoms bereits etabliert. In der hier kommentierten Studie wird die Wirksamkeit einer Erhaltungstherapie mit dem PD-L1-Antagonisten Durvalumab nach definitiver RCT beim lokal fortgeschrittenen NSCLC im Stadium III untersucht.

Cytotoxic effect of Efavirenz in BxPC‑3 pancreatic cancer cells is based on oxidative stress and is synergistic with ionizing radiation

The non-nucleoside reverse transcriptase inhibitor (NNRTI) Efavirenz is frequently used in human immunodeficiency virus treatment, but also efficient against cancer in mouse models. Its radiosensitizing effect makes it a promising drug for combination with radiotherapy. The efficacy of Efavirenz combined with irradiation was assessed with immunostaining of DNA-damage markers and colony formation assays in BxPC-3 pancreatic cancer cells. Gene expression and protein phosphorylation of the Efavirenz-sensitive BxPC-3 cells was compared to the resistant primary fibroblasts SBL-5. Oxidative stress, mitochondrial damage and cell death were studied with live-cell microscopy and flow cytometry. Combined Efavirenz and radiation significantly increased the number of γH2AX and phospho-ataxia telangiectasia mutated foci. Efavirenz and ionizing radiation had a synergistic effect using the clonogenic survival assay. Efavirenz selectively induced cell death in the BxPC-3 cells. The differing gene expression of cell cycle and apoptotic regulator genes in both cell cultures after Efavirenz treatment match with this selective effect against cancer cells. In the phosphoprotein array, an early phosphorylation of extracellular signal-related kinase 1/2 and p38 mitogen-activated protein kinase was notably detected in the cancer cells. The phosphorylation of AKT decreased in the cancer cells whereas it increased in the fibroblasts. Oxidative stress and mitochondrial membrane depolarization appeared in the cancer cells immediately after Efavirenz treatment, but not in the fibroblasts. Efavirenz has an anti-cancer effect against pancreatic cancer mainly by the induction of oxidative stress. The antitumor potential of Efavirenz and radiotherapy are additive.