Critical evaluation of molecular tumour board outcomes following 2 years of clinical practice in a Comprehensive Cancer Centre

Recently, molecular tumour boards (MTBs) have been integrated into the clinical routine. Since their benefit remains debated, we assessed MTB outcomes in the Comprehensive Cancer Center Ostbayern (CCCO) from 2019 to 2021. Methods and results In total, 251 patients were included. Targeted sequencing was performed with PCR MSI-evaluation and immunohistochemistry for PD-L1, Her2, and mismatch repair enzymes. 125 treatment recommendations were given (49.8%). High-recommendation rates were achieved for intrahepatic cholangiocarcinoma (20/30, 66.7%) and gastric adenocarcinoma (10/16, 62.5%) as opposed to colorectal cancer (9/36, 25.0%) and pancreatic cancer (3/18, 16.7%). MTB therapies were administered in 47 (18.7%) patients, while 53 (21.1%) received alternative treatment regimens. Thus 37.6% of recommended MTB therapies were implemented (47/125 recommendations). The clinical benefit rate (complete + partial + mixed response + stable disease) was 50.0% for MTB and 63.8% for alternative treatments. PFS2/1 ratios were 34.6% and 16.1%, respectively. Significantly improved PFS could be achieved for m1A-tier-evidence-based MTB therapies (median 6.30 months) compared to alternative treatments (median 2.83 months; P = 0.0278). Conclusion The CCCO MTB yielded a considerable recommendation rate, particularly in cholangiocarcinoma patients. The discrepancy between the low-recommendation rates in colorectal and pancreatic cancer suggests the necessity of a weighted prioritisation of entities. High-tier recommendations should be implemented predominantly

Identification of Disparities in Personalized Cancer Care—A Joint Approach of the German WERA Consortium

(1) Background: molecular tumor boards (MTBs) are crucial instruments for discussing and allocating targeted therapies to suitable cancer patients based on genetic findings. Currently, limited evidence is available regarding the regional impact and the outreach component of MTBs; (2) Methods: we analyzed MTB patient data from four neighboring Bavarian tertiary care oncology centers in Würzburg, Erlangen, Regensburg, and Augsburg, together constituting the WERA Alliance. Absolute patient numbers and regional distribution across the WERA-wide catchment area were weighted with local population densities; (3) Results: the highest MTB patient numbers were found close to the four cancer centers. However, peaks in absolute patient numbers were also detected in more distant and rural areas. Moreover, weighting absolute numbers with local population density allowed for identifying so-called white spots—regions within our catchment that were relatively underrepresented in WERA MTBs; (4) Conclusions: investigating patient data from four neighboring cancer centers, we comprehensively assessed the regional impact of our MTBs. The results confirmed the success of existing collaborative structures with our regional partners. Additionally, our results help identifying potential white spots in providing precision oncology and help establishing a joint WERA-wide outreach strategy

Regulation und Funktion des Onkogens MYC durch die nicht-steroidalen Antirheumatika Diclofenac und Diflunisal

Ein veränderter Zellmetabolismus ist wesentliches Charakteristikum von Tumorzellen und trägt zu Proliferation und Progression bei. Das in etwa 30% aller Tumore dysregulierte Onkogen MYC gilt als einer der zentralen Regulatoren des Glukose- und Glutamin-Stoffwechsels. Die Deregulation dieser metabolischen Wege stellt somit eine vielversprechende Zielstruktur für die Tumortherapie dar. Nicht-steroidale Antirheumatika (NSAIDs) werden schon seit längerem mit anti-tumoraler Aktivität in Zusammenhang gebracht, wobei bisher COX-spezifische Effekte als Ursache gezeigt wurden. Im Rahmen der vorliegenden Arbeit wurde die Regulation von MYC durch die NSAIDs Diclofenac und Diflunisal im Vergleich zu Acetylsalicylsäure analysiert. Dabei wurde zunächst die Proteinexpression von MYC in einer Melanomzelllinie (MelIm), einer Prostatakarzinomzelllinie (PC3), einer Histiozytomzelllinie (U937) und einer Leukämiezelllinie (Jurkat) mit Hilfe von Western-Blot untersucht. Hierbei konnte sowohl bei steigenden Diclofenac- als auch bei steigenden Diflunisalkonzentrationen eine Hemmung der Proteinexpression von MYC in allen Zelllinien festgestellt werden. Eine Behandlung der Zellen mit Acetylsalicylsäure (ASS) zeigte dies jedoch nicht. Die Effekte auf Proteinebene sollten anschließend auch durch intrazelluläre Färbung von MYC mit Hilfe von direkt fluoreszenz-markierten Antikörpern untersucht werden. Zu diesem Zweck wurde ein Färbeprotokoll mit verschiedenen anti-MYC Antikörpern in MelIm und U937 etabliert. Dabei konnte in beiden Zelllinien MYC mittels intrazellulärer Färbung nachgewiesen werden. Als Vergleich wurden nicht-proliferierende Monozyten verwendet, die keine MYC-Expression zeigten. In weiteren Experimenten ist zu untersuchen, ob nach Behandlung mit NSAIDs immunzytochemisch eine veränderte Expression nachgewiesen werden kann. Nachdem auf Proteinebene eine Hemmung der Expression von MYC festgestellt werden konnte, wurde auch die Regulation auf transkriptioneller Ebene untersucht. Zu diesem Zweck wurden verschiedene Reporterkonstrukte kloniert, die unterschiedlich lange Bereiche um den Hauptpromotor von MYC enthielten. Nach Transfektion in die Zelllinie MelIm und Inkubation in An- und Abwesenheit von Diclofenac und Diflunisal wurde die Promotoraktivität mittels Luziferase-Assays getestet. Diclofenac supprimierte die Transkription signifikant, während Diflunisal keinen Einfluss zeigte. Die drei unterschiedlich großen Promotorregionen ließen keinen signifikanten Unterschied erkennen. Da MYC entscheidend die Regulation von Proliferation und Apoptose beeinflusst, wurde des Weiteren analysiert, inwieweit Diclofenac und Diflunisal Apoptose in den untersuchten Zelllinien auslösen. Mit Hilfe von durchflusszytometrischen Analysen wurde die Vitalität von MelIm und U937 nach Behandlung mit Diclofenac und Diflunisal getestet. Es konnte gezeigt werden, dass sich Diclofenac und Diflunisal bei den behandelten Zelllinien unterschiedlich verhalten. Während bei MelIm nur durch die Kombination beider NSAIDs in den höchsten Konzentrationen wesentlich Apoptose induziert werden konnte, verringerten in U937 bereits die einzelnen NSAIDs in den höchsten Konzentrationen signifikant die Vitalität der Zellen. Die Kombination von Diclofenac und Diflunisal führte in beiden Zelllinien zur deutlich verstärkten Apoptose. Die dargestellten Arbeiten deuten an, dass Diclofenac und Diflunisal das Onkogen MYC auf unterschiedliche Weise beeinflussen. Während Diclofenac auf transkriptioneller Ebene und Proteinebene agiert, erreicht Diflunisal nur eine deutliche Hemmung der Proteinexpression. Die Tatsache, dass ASS keinen Effekt zeigt, lässt außerdem annehmen, dass es sich hierbei um Substanz-spezifische und COX-unabhängige Regulationsmechanismen handelt. Insgesamt stellt die Beeinflussung von MYC durch NSAIDs einen vielversprechenden Ansatz für eine spezifische Tumortherapie dar, sodass Diclofenac als interessante Substanz zur Unterstützung einer Tumortherapie anzusehen ist

New aspects of an old drug: Diclofenac targets MYC and glucose metabolism in tumor cells

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies

Effects of diclofenac on tumor cells.

<p>n.d.: not done; statistical analysis was performed with Student's t-test, n.s.: no significant change; ***p<0.001; **p<0.01; *p<0.05.</p

Contribution of MYC to suppression of proliferation by diclofenac.

<p>P493-6 cells were incubated first for 24 h in the presence of tetracycline to suppress MYC expression. Cells were then treated with diclofenac for 24 h. Proliferation (A) was determined by ³[H]-thymidine incorporation, while lactate (B) was determined in cell culture supernatants. Results represent the mean +/− standard deviation of 3 independent experiments. MYC protein expression was analyzed in cell lysates of P493-6 with or without tetracycline treatment for 24 h (C). Cells were then treated with diclofenac for an additional 2 h and 24 h, respectively, and MYC expression was determined in cell lysates by western blotting. One representative western blot is shown.</p

Diclofenac decreases glucose consumption and modulates lactate accumulation.

<p>(A) Glucose levels were determined in MelIm supernatants after 24 h and 48 h of incubation with diclofenac. “Medium” represents the glucose concentration in the culture medium without cells. Lactate was determined in cell culture supernatants of MelIm after 24 h and 48 h with or without diclofenac,(B) ASA or NS-398 (C). Lactate was determined in cell culture supernatants of B16 after 48 h (D). Lactate levels were determined in supernatants of U937 (E) after incubation with increasing concentrations of diclofenac for 48 h. Results represent the mean +/− standard deviation of 3 independent samples. Diclofenac reduced mitochondrial activity in both cell lines after 24 h (F). A reduction of about 40% was observed in MelIm at a concentration of 0.2 mM and in U937 at a concentration of 0.1 mM. Basal mitochondrial activity (ROUTINE), oligomycin inhibited respiration and capacity of electron transfer system (complex I to IV) were diminished by diclofenac. Results represent the mean+/−SD of 3 and 5 independent experiments, respectively, for MelIm and U937.</p

<i>In vitro</i> and <i>in vivo</i> effects of diclofenac on proliferation of B16 murine melanoma cells.

<p>(A) Proliferation of B16 cells was determined in the presence or absence of diclofenac after 24 h incubation. Results represent the mean +/− standard deviation of 4 independent experiments. (B) MYC expression was determined in B16 after incubation with diclofenac for 2 h and 24 h by western blotting. (C/D) For the analysis of in vivo effects of diclofenac on tumor growth, 1×10⁵ B16 cells were injected subcutaneously into C57/BL6 mice. At a tumor volume of 50–80 mm³ (day 14), mice received diclofenac (15 mg/kg, n = 7) or saline (control, n = 7) via intraperitoneal injection every other day. The tumor weight was determined on day 23 after termination of the experiment (D). Statistical analysis was performed with unpaired, two-tailed Student's t-test, ***p<0.001; **p<0.01; *p<0.05.</p