A Retrospective Analysis of Long-Term Prophylaxis with Berotralstat in Patients with Hereditary Angioedema and Acquired C1-Inhibitor Deficiency-Real-World Data.

Hereditary angioedema (HAE) and acquired C1-inhibitor deficiency (AAE-C1-INH) are orphan diseases. Berotralstat is a recently licensed long-term prophylaxis (LTP) and the first oral therapy for HAE patients. No approved therapies exist for AAE-C1-INH patients. This study is the first to report real-world clinical data of patients with AAE-C1-INH and HAE who received Berotralstat. All patients treated with Berotralstat were included in this retrospective, bi-centric study. Data was collected from patients' attack calendars and the angioedema quality of life (AE-QoL) and angioedema control test (AECT) questionnaires before treatment, and at 3, 6, and 12 months after treatment and was then analyzed. Twelve patients were included, 3 patients with AAE-C1-INH, 7 patients with HAE type I, and 2 patients with HAE-nC1-INH. One patient (HAE I) quit treatment. Berotralstat was associated with fewer attacks in all groups. After 6 months of treatment, a median decrease of attacks per month was noted for HAE type I patients (3.3 to 1.5) and AAE-C1-INH patients (2.3 to 1.0). No aerodigestive attacks were noted for AAE-C1-INH patients. For HAE-nC1-INH patients, a mean decrease from 3.8 to 1.0 was noted (3 months). For HAE I patients, the total AE-QoL lowered a mean of 24.1 points after 6 months, for HAE-nC1-HAE patients 8.0 points, and for AAE-C1-INH patients 13.7 points. AECT scores increased for HAE I patients (mean: 7.1), HAE-nC1-INH patients (9.0), and AAE-C1-INH patients (4.2) after 6 months. Patients with HAE, HAE-nC1-INH, and AAE-C1-INH treated with Berotralstat showed reduced angioedema attacks and improved AE-QoL and AECT scores.

Circulating immune response proteins predict the outcome following disease progression of osimertinib treated epidermal growth factor receptor-positive non-small cell lung cancer patients.

Background: Lung cancer patients with sensitizing epidermal growth factor receptor (EGFR) mutations treated with osimertinib will eventually develop progressive disease (PD). The survival following PD varies greatly between patients, and no effective treatment strategy has been established. Furthermore, at the moment, no easily accessible and precise biomarker exists that can predict the survival after PD. Methods: We analyzed blood samples drawn from non-small cell lung cancer patients harboring EGFR mutations that were treated with osimertinib. The levels of 92 circulating proteins were analyzed from plasma samples using a proximity extension assay (PEA). The results were evaluated with Gene Ontology (GO) enrichment analysis to reveal patterns of protein expression at progression while on osimertinib treatment. Results: We found that the expression of 7 proteins were significantly altered at PD, compared to a sample taken at osimertinib response. GO enrichment analysis demonstrated that most of the significant proteins were related to the immune system, specifically the adaptive immune response. Defining two groups of patients, based on the levels of circulating immune response proteins at PD, revealed significant differences in the overall survival (OS) after PD [hazard ratio (HR) =3.04; 95% confidence interval (CI): 1.24-7.45; P=0.0046]. Conclusions: In this study, we discover novel circulating biomarkers that can predict the OS after PD on osimertinib. These findings support the recent acknowledgement of the immune system's importance in osimertinib resistance.

Co-occurring MET Amplification Predicts Inferior Clinical Response to First-Line Erlotinib in Advanced Stage EGFR-Mutated NSCLC Patients.

BACKGROUND: Intrinsic resistance is a major obstacle in treatment of non-small cell lung cancer (NSCLC) patients with an activating mutation in the epidermal growth factor receptor (EGFR). We investigated co-occurring genetic alterations in circulating tumor DNA (ctDNA) as predictive markers of clinical response to first-line erlotinib. METHODS: Pretreatment plasma samples were collected from 76 patients with EGFR-mutated, advanced-stage NSCLC treated with first-line erlotinib. We isolated ctDNA from plasma for next-generation sequencing. RESULTS: Co-occurring oncogenic drivers were detected in 21% of pretreatment samples and correlated with decreased progression-free survival (PFS) (6.9 months vs 14.4 months; hazard ratio [HR], 2.088; 95% confidence interval [CI], 0.8119-5.370; P = .0355). Concurrent MET amplification was identified in 9 samples (12%), predicting inferior PFS (5.5 months vs 14.4 months; HR, 4.750; 95% CI, 0.5923-38.10; P = .0007) and overall survival (7.6 months vs 28.3 months; HR, 3.952; 95% CI, 0.8441-18.50; P = .0005). Co-occurring non-MET-amplification oncogenic alterations showed a tendency for shorter PFS (9.9 months vs 14.4 months; HR, 1.199; 95% CI, 0.3373-4.265; P = .7586). Clearing EGFR-mutated ctDNA during erlotinib treatment is a positive predictor of clinical outcomes. Among patients who cleared the EGFR mutation, 12% had a co-occurring oncogenic driver, with a tendency toward inferior PFS (8.7 months vs 16.1 months; HR, 1.703; 95% CI, 0.5347-5.424; P = .2508). CONCLUSION: Co-occurring MET amplification in pretreatment ctDNA samples predict inferior clinical response to first-line erlotinib in advanced-stage, EGFR-mutated NSCLC patients. Co-occurring oncogenic alterations were associated with inferior response and may be potential predictors of clinical outcome.

Etiology and predictors of cluster attacks of hereditary angioedema that recur despite pharmaceutical treatment.

Introduction: Hereditary angioedema (HAE) is a disease that leads to recurrent swelling of the skin and mucous membranes, including the upper airway tract. Apart from being deadly, these attacks can be debilitating, which leads to a poor quality of life in patients. Clinicians are occasionally confronted with patients who have recurrent attacks despite treatment with C1 esterase inhibitor concentrate or ß2-receptor antagonists. The goal of this study was to investigate repeated attacks that occur 48 hours to 7 days ("cluster attacks") after treatment, to determine why they occur and the factors that may be associated with them, and thus to prevent their occurrence. Methods: We conducted a multicenter mixed retrospective-prospective study with data acquired from all documented attacks in our patients with collective (n = 132) between 2015 and 2018. Results: Eighty-five percent (n = 132) of our total patient collective (N = 156) agreed to participate in the study. Nine percent of these patients (n = 12) had cluster attacks, with a total of 48 cluster attacks. The data procured from the patients were mixed retrospective-prospective. Approximately 72% of all the cluster attacks were caused by exogenous stimuli (41% due to psychological stress, 29% due to physical stimuli, and 2% due to menstruation). Cluster attacks occurred in 7% of the patients who received prophylactic therapy in comparison with 12.5% of patients who received on-demand therapy. Cluster attacks comprised 48.4% of all the attacks that patients with cluster-attacks (n= 9) experienced. In addition, the patients who were underdosing their C1 esterase inhibitor treatment had cluster attacks more often. A lower "time to repeated attack" was seen in the patients who received on-demand therapy compared with those who received prophylactic therapy. Discussion: The percentage of the patients who had attacks as a result of exogenous triggers was higher in the cluster-attack group (70.5%) compared with the general HAE population (30-42%). Repeated attacks, therefore, were strongly associated with external triggers. The patients who received prophylactic treatment and who experienced cluster attacks were highly likely to have been underdosing, which may explain the repeated attacks despite treatment. In the patients prone to cluster attacks, prophylaxis should be considered.

Clearing of circulating tumour DNA predicts clinical response to osimertinib in EGFR mutated lung cancer patients.

OBJECTIVES: Tyrosine kinase inhibitors (TKIs) are first line treatment choices for patients with epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC). However, responses vary among patients, therefore good biomarkers predicting better responses are required. EGFR mutations are detected in the blood from patients as circulating tumour DNA (ctDNA). Studies have shown that clearing ctDNA during first line TKI treatment predicts outcomes for first and second generation TKI treatments. We aimed to investigate the effects on outcome measures of ctDNA clearing in subsequent treatment lines to treatment with the third generation TKI osimertinib. METHODS: In total, 225 patients were included in a prospective, multicentre study, where consecutive blood samples were monitored for EGFR mutations during systemic treatment lines, using the Cobas® EGFR mutation test v2. This study focused on EGFR mutations in ctDNA of 82 systemically pre-treated patients receiving osimertinib. RESULTS: Clearing all EGFR mutations from the blood after osimertinib treatment, significantly predicted progression-free survival, objective response rates and disease control rates. Primary sensitising EGFR mutations were found in ctDNA in 70 % of patients, and were accompanied by the T790 M mutation in nearly two thirds of cases. The T790 M mutation was cleared in all cases, while the accompanying sensitising mutations did not necessarily clear. However, T790 M clearing without simultaneously clearing of the primary sensitising mutation did not predict clinical responses. Neither the detection of T790 M before osimertinib treatment, nor the presence of EGFR mutations at the time of osimertinib initiation predicted clinical outcomes. CONCLUSION: The clearing of EGFR mutations in ctDNA after osimertinib treatment initiation in patients with advanced NSCLC is useful as a positive predictor of clinical outcome.

Clearing of circulating tumour DNA predicts clinical response to first line tyrosine kinase inhibitors in advanced epidermal growth factor receptor mutated non-small cell lung cancer.

OBJECTIVES: Epidermal growth factor receptor (EGFR) mutations confer sensitivity to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). However, a subset of patients has limited or no response. We investigated the initial dynamics of EGFR mutations detected in circulating tumour DNA (ctDNA) during treatment as a predictive marker of outcome. METHODS: A total of 225 patients with advanced EGFR mutated NSCLC were included for consecutive blood sampling in this prospective multicentre study. Out of these, 146 patients received first line TKI and had a baseline blood sample available for EGFR mutation testing with the Cobas® EGFR mutation test V2. For examinations on clearing and clinical outcome, 98 patients who had detectable ctDNA at baseline and at least one follow-up blood sample were included. RESULTS: For patients with EGFR mutations present in plasma at baseline, clearing of mutations from the blood during first line TKI served as a positive predictor for objective response rate (p = 0.0008), progression-free survival (PFS) (p < 0.0001) and overall survival (OS) (p < 0.0001). This was seen both for patients who cleared the ctDNA within the first 7 weeks of treatment and patients who cleared the ctDNA at a slower pace. Baseline mutation presence was a negative predictor for PFS (p = 0.0069) and OS (p = 0.0340). CONCLUSION: The current study is the first to confirm, in a sizeable Caucasian cohort, that clearing of EGFR mutations predict outcome to first line TKI in patients with EGFR mutated NSCLC.

Characterization of glycolysis-related gene expression in malignant melanoma.

Malignant melanoma exhibits a distinct metabolic phenotype with high glycolytic activity. Previously, we have shown that glucose transporter isoform 1 (GLUT1) favors growth and metastasis of malignant melanoma. In this study, we investigated the expression of GLUT1 and the further glycolysis-related genes hexokinase 1 and 2 (HK1, HK2), lactate dehydrogenase A (LDH-A) and monocarboxylate transporters 1 and 4 (MCT1, MCT4) in eleven human melanoma cell lines under normoxic and hypoxic conditions. Furthermore, a set of 25 human malignant melanoma tissue samples was analyzed. Under hypoxic conditions, we could observe a significant upregulation of hypoxia-inducible factor 1 alpha (HIF-1a) target genes GLUT1, HK2 and LDH-A, but not MCT4. While under normoxic conditions the expression of glycolysis-related genes showed no correlation with origin or BRAF mutation status, GLUT1 expression was significantly elevated in metastatic and BRAF-V600E mutated melanoma cell lines under hypoxic conditions. Furthermore, GLUT1 expression in human melanoma tissue samples correlated significantly with HK1, LDH-A and MCT1 expression, confirming a glycolytic phenotype. Notably, Cyclin D1 expression, which is used as a prognostic marker for the outcome of melanoma patients, as it is associated with proliferation and invasiveness of melanoma, significantly correlated with GLUT1, HK1, LDH-A and MCT1 expression. In summary, our findings provide further evidence that enhanced glycolytic activity in melanoma favors disease progression and is an attractive therapeutic target for this highly aggressive tumor.

Circulating miR-30b and miR-30c predict erlotinib response in EGFR-mutated non-small cell lung cancer patients.

OBJECTIVES: MiR-30b, miR-30c, miR-221 and miR-222 are known to induce gefitinib resistance in lung cancer cell lines with activation of mutations in the epidermal growth factor receptor (EGFR). However, the role of these four microRNAs in tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC) patients is unknown. Thus, the aim of this study was to investigate the predictive value of miR-30b, miR-30c, miR-221 and miR-222 in plasma from EGFR-mutated lung cancer patients receiving erlotinib. MATERIALS AND METHODS: The cohort consisted of 29 EGFR-mutated lung cancer patients receiving erlotinib. Plasma levels of miR-30b, miR-30c, miR-221 and miR-222 were analyzed by qPCR from blood samples collected before treatment start. Plasma concentration of each microRNA was correlated to clinical outcome. RESULTS: Plasma concentrations of miR-30b and miR-30c could be determined in all 29 patients. Low plasma concentrations of miR-30b and miR-30c showed significant correlation with superior progression-free survival (PFS) (miR-30b: HR = 0.303 [0.123-0.747], p < 0.05; miR-30c: HR = 0.264 [0.103-0.674], p < 0.05). Low plasma concentrations of miR-30c were also significantly correlated with superior overall survival (OS) (HR = 0.30 [0.094-0.954], p < 0.041). CONCLUSION: High plasma concentrations of miR-30b and miR-30c predicted shorter PFS and OS. This implies that miR-30b and miR-30c could have clinical potential as biomarkers in EGFR-mutated lung cancer patients.

EGFR Gene Polymorphism Predicts Improved Outcome in Patients With EGFR Mutation-positive Non-small cell Lung Cancer Treated With Erlotinib.

BACKGROUND: Patients with advanced-stage non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutations are successfully treated with tyrosine kinase inhibitors (TKIs). However, treatment outcome varies significantly. Previously, we found the polymorphism 181946C>T (rs2293347) located in exon 25 of the EGFR gene to be a predictor of improved outcome. However, these data were based on a subgroup analysis. Furthermore, other minor studies have found conflicting data. Thus, the aim of this study was to demonstrate the association of 181946C>T with clinical outcome in an independent cohort of EGFR-mutated patients treated with erlotinib. PATIENTS AND METHODS: Seventy-five patients were prospectively enrolled. Blood samples were collected, and genotype for 181946C>T was determined by allele-specific polymerase chain reaction. Genotype was correlated with outcome. RESULTS: In 73 patients, 181946C>T was successfully measured. Patients harboring the 181946CT genotype had a significantly longer median progression-free survival compared with patients harboring the 181946CC genotype (49.9 months [95% confidence interval (CI), 5.9-93.9 months] versus 11.1 months (95% CI, 7.4-14.9 months); P = .020). Moreover, a significantly longer median overall survival of 65.6 months (95% CI, 11.0-120.3 months) versus 31.2 months (95% CI, 10.9-51.6 months) was found (P = .019). Both results remained significant in a multivariate analysis adjusting for potential confounders. CONCLUSION: We demonstrate that the 181946C>T polymorphism is a significant predictor of prolonged progression-free survival and overall survival in an independent cohort of EGFR mutation-positive patients treated with erlotinib. The polymorphism could be an important predictor of treatment response in these patients. A large multicenter cohort study involving other concurrent genetic alterations is warranted.

Effect of melanoma cells on proliferation and migration of activated hepatic stellate cells in vitro.

Melanoma is a highly aggressive tumor of the skin. The clinical outcome is determined by the presence or absence of metastases, and the liver is a common site of distant metastases. Hepatic metastasis is causing activation of hepatic stellate cells (HSC), which form the stroma of hepatic metastases and are increasingly recognized as a crucial component of the pro- metastatic liver microenvironment. Most studies have focused on the effects of HSC on (metastasizing) tumor cells. Here, we aimed to analyze functional in vitro effects of conditioned medium (CM) of twelve different human melanoma cell lines on LX2 cells and HSChtert cells, two well established human activated HSC cell lines. CM from melanoma cells significantly induced HSC proliferation and acted as chemoattractant for HSC in Boyden chamber assays. The CM effects significantly varied between different HSC as well as melanoma cells. Interestingly, CM from melanoma cell lines derived from melanoma metastases (WM239A, WM9, WM1158, WM1232, 451Lu and 1205Lu) had a stronger effect on proliferation of HSChtert cells than CM derived from primary melanoma tumors (SbCl2, WM3211, WM35, WM278, WM1366 and WM793). Moreover, we observed a significant correlation between the chemoattractive effects of CM from the different melanoma cells on HSChtert and LX2 cells. In contrast, the melanoma CM effects on the proliferation of the two HSC lines did not show a significant correlation. In summary, our data indicate that melanoma cells metastasizing to the liver have the potential to attract HSC and to induce HSC proliferation, respectively. Still, it appears that melanoma effects on HSC migration and proliferation are mediated via different soluble factors indicating the complexity of melanoma-HSC interaction. Furthermore, the intensity of at least some functional effects varies between different human tumor cells and HSC which may point to mechanisms explaining diverse hepatic metastasis in melanoma patients.