Critical role of reactive oxygen species (ROS) for synergistic enhancement of apoptosis by vemurafenib and the potassium channel inhibitor TRAM-34 in melanoma cells

Inhibition of MAP kinase pathways by selective BRAF inhibitors, such as vemurafenib and dabrafenib, have evolved as key therapies of BRAF-mutated melanoma. However, tumor relapse and therapy resistance have remained as major problems, which may be addressed by combination with other pathway inhibitors. Here we identified the potassium channel inhibitor TRAM-34 as highly effective in combination with vemurafenib. Thus apoptosis was significantly enhanced and cell viability was decreased. The combination vemurafenib/TRAM-34 was also effective in vemurafenib-resistant cells, suggesting that acquired resistance may be overcome. Vemurafenib decreased ERK phosphorylation, suppressed antiapoptotic Mcl-1 and enhanced proapoptotic Puma and Bim. The combination resulted in enhancement of proapoptotic pathways as caspase-3 and loss of mitochondrial membrane potential. Indicating a special mechanism of vemurafenib-induced apoptosis, we found strong enhancement of intracellular ROS levels already at 1 h of treatment. The critical role of ROS was demonstrated by the antioxidant vitamin E (α-tocopherol), which decreased intracellular ROS as well as apoptosis. Also caspase activation and loss of mitochondrial membrane potential were suppressed, proving ROS as an upstream effect. Thus ROS represents an initial and independent apoptosis pathway in melanoma cells that is of particular importance for vemurafenib and its combination with TRAM-34

Open-label, multicenter, single-arm phase II DeCOG-study of ipilimumab in pretreated patients with different subtypes of metastatic melanoma

Background: Ipilimumab is an approved immunotherapy that has shown an overall survival benefit in patients with cutaneous metastatic melanoma in two phase III trials. As results of registrational trials might not answer all questions regarding safety and efficacy of ipilimumab in patients with advanced melanoma seen in daily clinical practice, the Dermatologic Cooperative Oncology Group conducted a phase II study to assess the efficacy and safety of ipilimumab in patients with different subtypes of metastatic melanoma. Patients and methods: We undertook a multicenter phase II study in melanoma patients irrespective of location of the primary melanoma. Here we present data on patients with pretreated metastatic cutaneous, mucosal and occult melanoma who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals. Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria. Adverse events (AEs),including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0. Primary endpoint was the OS rate at 12 months. Results: 103 pretreated patients received at least one dose of ipilimumab, including 83 cutaneous, seven mucosal and 13 occult melanomas. 1-year OS rates for cutaneous, mucosal and occult melanoma were 38 %,14 % and 27 %,respectively. Median OS was 6.8 months (95 % CI 5.3-9.9) for cutaneous, 9.6 months (95 % CI 1.6-11.1) for mucosal, and 9.9 months (lower 95 % CI 2.3, upper 95 % CI non-existent) for occult melanoma. Overall response rates for cutaneous, mucosal and occult melanoma were 16 %,17 % and 11 %,respectively. Eleven patients had partial response (16 %) and ten patients experienced stable disease (14 %),none achieved a complete response. Treatment-related AEs were observed in 71 patients (69 %),including 20 grade 3-4 events (19 %). No new and unexpected safety findings were noted. Conclusions: Ipilimumab is a treatment option for pretreated patients with advanced cutaneous melanoma seen in daily routine. Toxicity was manageable when treated as per protocol-specific guidelines

Long-term therapy of interferon-alpha induced pulmonary arterial hypertension with different PDE-5 inhibitors: a case report

BACKGROUND: Interferon alpha2 is widely used in hepatitis and high-risk melanoma. Interferon-induced pulmonary arterial hypertension as a side effect is rare. CASE PRESENTATION: We describe a melanoma patient who developed severe pulmonary arterial hypertension 30 months after initiation of adjuvant interferon alpha2b therapy. Discontinuation of interferon did not improve pulmonary arterial hypertension. This patient could be treated successfully with phosphodiesterase-5 inhibitor therapy. CONCLUSION: This is only the 5th case of interferon-induced pulmonary arterial hypertension and the first documented case where pulmonary arterial hypertension was not reversible after termination of interferon alpha2 therapy. If interferon alpha2 treated patients develop respiratory symptoms, pulmonary arterial hypertension should be considered in the differential diagnosis. For these patients phosphodiesterase-5 inhibitors, e.g. sildenafil or vardenafil, could be an effective therapeutic approach

Optimized Peptides and Peptide Pools for Stimulation of Antigen Specific T-Cells in Cytomegalovirus

HINTERGRUND: Noch immer stellt die Primärinfektion bzw. die Reaktivierung des humanen Cytomegalievirus (HCMV) bei Immunkompromitierten ein großes klinisches Problem dar. Die T-Zell-vermittelte Immunität spielt eine zentrale Rolle bei der Kontrolle der Infektion. Die Untersuchung der T-Zell-Antwort ist daher von größter Wichtigkeit. Peptide zur Stimulation von T-Zellen können hierbei hilfreich sein. Wie Peptide und Peptidpools in Abhängigkeit von ihrem Einsatzziel optimal konfiguriert sein sollten, steht bisher nicht abschließend fest. In dieser Arbeit sollte daher geklärt werden, wie die Peptide und Peptidpools konfiguriert sein müssen, um möglichst optimal spezifische T-Zell- Antworten zu erfassen. Stellvertretend wurden dazu Peptide der Proteine pp65 und IE1 aus HCMV gewählt. Es sollte geklärt werden, ob unterschiedliche Anforderungen an die eingesetzten Peptide gestellt werde, wenn CD4 und CD8 T-Zellen simultan durchgeführt werden und welchen Einfluss das Epitop flankierende Aminosäuren auf die Stimulationseffizienz des Peptides haben. Es sollte zudem geklärt werden, ob eine pp65-Gesamtmischung aus mit dem Verfahren der Spotsynthese hergestellten 9-AS-Peptiden realisierbar ist, die in ihrer Fähigkeit CD8 T-Zellen zu stimulieren mit einer Mischung aus 15-AS-Peptiden vergleichbar ist. METHODEN: Dazu wurden 15-AS-Peptide und 9- bzw. 10-AS- Peptide eingesetzt zur Stimulation von PBMC von insgesamt 11 Spendern mit bekannten Reaktivitäten gegenüber Epitopen aus den Virusproteinen pp65 und IE1. Teilweise wurden die Peptide mittels Festphasen-Peptid-Synthese eigenständig synthetisiert. Die resultierenden T-Zell-Reaktionen wurden durch Färbung von intrazellulärem Interferon-gamma durchflusszytometrisch sichtbar gemacht. ERGEBNISSE: Kurze Peptide stimulieren CD8 T-Zellen effektiver als korrespondierende längere 15-AS-Peptide. Eine pp65-Gesamtmischung schien sinnvoll, die aus kurzen überlappenden 9-AS-Peptiden bestand. Diese Mischung erzielte vergleichbare Ergebnisse wie die konventionelle aus 15-AS-Peptiden bestehende. Wir verlängerten definierte kurze Peptide um einzelne AS sowohl C- als auch N-terminal. Die Addition von AS hatte sehr unterschiedliche Auswirkungen auf die Stimulationseffektivität in Abhängigkeit von der angehängten AS, ob diese C- oder N-terminal angehängt wurde und dem Individuum mit seiner eigenen Ausstattung an HLA-Molekülen, Peptidasen sowie anderen noch nicht bekannten Faktoren. Zum Screening von CD4 T-Zell-Antworten sind Pools mit 15-AS-Peptide und 12 Überlappungen mindestens so effizient wie die entsprechenden rekombinanten Proteine. Falls kürzere Peptide verwendet werden, könnten einige CD4-Antworten verloren gehen. Es wurde gezeigt, dass auch 9-AS- Peptide CD4 T-Zellen stimulieren können. Epitop-flankierende AS können aber eine wichtige Rolle bei der CD4 T-Zell-Stimulation spielen, da der TCR auch AS außerhalb der MHC-Bindungssequenz erkennen kann. SCHLUSSFOLGERUNGEN: Bei der Untersuchung von CD8 T-Zell-Antworten empfehlen wir zum Screening oder als Verlaufskontrolle 15-AS-Peptid-Pools. Zur Quantifizierung halten wir kurze Peptide für überlegen. Sollen sowohl CD4 als auch CD8 T-Zell-Antworten untersucht werden, können 15-AS-Peptid-Pools verwendet werden. Eine interessante Alternative dazu stellt die Kombination aus kurzen und langen Peptiden dar.BACKGROUND: Human Cytomegalovirus (HCMV) primary infection as well as reactivation is still a severe clinical problem for immunodeficient patients. T-cell mediated immunity plays an important role in controlling the disease. Therefore the investigation of the cellular immunity is fundamental. While peptides and peptide pools for stimulation of T-cells are a helpful tool, it still is not known how they should be configured for best results. The aim of this work is to define peptides and peptide pools to detect specific T-cell answers. Therefore peptides out of the virus proteins pp65 and IE1 were chosen. It is important to clarify whether the peptides should meet different demands for simultaneous investigations of CD4 and CD8 T-cells and in which way epitope flanking amino acids affect the efficiency of the peptides. In addition we clarified whether a pp65 mixture composed of 9mers is feasible and if its stimulative abilities are comparable to a mixture of 15mers. METHODS: 15mers and 9mers, or 10mers respectively, were used for the stimulation of PBMC of 11 donors with a known reactivity towards epitopes out of the virus proteins pp65 and IE1. In part, the peptides have been synthesized independently with a Solid Phase Peptide Synthesis. The resulting T-cell responses were identified by flow cytometric detection of intracellular interferon gamma. RESULTS: Short peptides stimulate CD8 T-cells more effeciently than corresponding longer ones. A pp65 peptide pool containing overlapping 9mers was as effective as the established pp65 peptide pool made of 15mers. We extended defined short peptides by adding single amino acids at the C- as well as at the N-terminus. The addition of the amino acids had disparate impact on the efficacy of T-cell stimulation depending on the added amino acid, whether it was attached C- or N-terminal and on the individual equipment of HLA molecules, peptidases and other not yet known factors. For screening of CD4 T-cell responses pools with 15mers and 12 overlapping amino acids are at least equally effective as recombinant proteins. If shorter peptides are used, some CD4 T-cell responses may be lost. Epitope flanking amino acids may play an important role in stimulating CD4 T-cells because the TCR is able to identify amino acids beyond the MHC binding sequence. Additionally 9mers showed that they are in principle able to stimulate CD4 T-cells. CONCLUSION: For investigating CD8 T-cell responses we recommend pools with 15mers for screening or course observation. For quantification of the CD8 T-cell response short peptides are superior. For investigating CD4 and CD8 T-cell responses, pools with 15mers are a possible choice. An alternative would be the combination of short and long peptides

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma

Abstract Background Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested) and for metastatic melanoma of 96% (146/151 tested) in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. Methods In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. Results Sequence analysis of nine independent overlapping clones (length 3100–5600 bp) represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. Conclusion The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.</p

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma-8

<p><b>Copyright information:</b></p><p>Taken from "The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma"</p><p>Bmc Cancer [Electronic Resource] 2006;6():8-8.</p><p>Published online 11 Jan 2006</p><p>PMCID:PMC1351261.</p><p>Copyright © 2006 Trefzer et al; licensee BioMed Central Ltd.</p>molecular weight standard; 2: SMMUpos; 3: SMMUneg

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma-5

<p><b>Copyright information:</b></p><p>Taken from "The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma"</p><p>Bmc Cancer [Electronic Resource] 2006;6():8-8.</p><p>Published online 11 Jan 2006</p><p>PMCID:PMC1351261.</p><p>Copyright © 2006 Trefzer et al; licensee BioMed Central Ltd.</p>by the universal M13 reverse primer in the reverse orientation. Since the range of sequenced DNA was short (a few hundred base pairs), a sequence from near the end of this run was used to design a second primer, which primed the sequence from this region and moved another few hundred nucleotides further. From the end of this run the third primer was designed and the circle was repeated until the entire insert had been covered

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma-4

<p><b>Copyright information:</b></p><p>Taken from "The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma"</p><p>Bmc Cancer [Electronic Resource] 2006;6():8-8.</p><p>Published online 11 Jan 2006</p><p>PMCID:PMC1351261.</p><p>Copyright © 2006 Trefzer et al; licensee BioMed Central Ltd.</p

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma-2

<p><b>Copyright information:</b></p><p>Taken from "The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma"</p><p>Bmc Cancer [Electronic Resource] 2006;6():8-8.</p><p>Published online 11 Jan 2006</p><p>PMCID:PMC1351261.</p><p>Copyright © 2006 Trefzer et al; licensee BioMed Central Ltd.</p>15 and FN15 while PET does not

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma-7

<p><b>Copyright information:</b></p><p>Taken from "The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma"</p><p>Bmc Cancer [Electronic Resource] 2006;6():8-8.</p><p>Published online 11 Jan 2006</p><p>PMCID:PMC1351261.</p><p>Copyright © 2006 Trefzer et al; licensee BioMed Central Ltd.</p>ker: Lane 2: Fibronectin expression in SMMUpos; Lane 3: Fibronectin expression in SMMUneg; Lane 4: β-actin expression in SMMUpos; Lane 5: β-actin expression in SMMUneg