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Summary Background We evaluated the efficacy and safety of tepotinib, a potent and highly selective oral MET inhibitor, plus gefitinib in patients with epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) with MET overexpression (immunohistochemistry [IHC]2+ or IHC3+) or MET amplification having acquired resistance to EGFR inhibition. Methods In this open-label, phase 1b/2, multicentre, randomised trial (the INSIGHT study), we enrolled adult patients (≥18 years) with advanced or metastatic NSCLC, and Eastern Cooperative Oncology Group performance status of 0 or 1, from academic medical centres and community clinics in six Asian countries. In phase 1b, patients received oral tepotinib 300 mg or 500 mg plus gefitinib 250 mg once daily. In phase 2, patients with EGFR-mutant, T790M-negative NSCLC MET overexpression or MET amplification were randomly assigned (initially in a 1:1 ratio and then 2:1 following a protocol amendment) to tepotinib plus gefitinib at the recommended phase 2 dose or to standard platinum doublet chemotherapy. Randomisation was done centrally via an interactive voice-response system. The primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included overall survival (OS) and safety. Subgroup analyses were preplanned in patients with high MET overexpression (IHC3+) or MET amplification (mean gene copy number ≥5 or MET to centromere of chromosome 7 ratio ≥2). Efficacy and patient characteristics were assessed on an intention-to-treat basis and safety was assessed for all patients who received at least one dose of study medication. Low recruitment led to early termination of phase 2, so all analyses are considered to be exploratory. This study is registered with ClinicalTrials.gov , NCT01982955 , and the European Union Drug Regulating Authorities Clinical Trials Database, Eudra-CT 2016-001604-28. Findings From Dec 23, 2013, to May 25, 2017, 18 patients were enrolled in phase 1b (n=6 in the 300 mg tepotinib group; n=12 in the 500 mg tepotinib group) and 55 patients in phase 2 (n=31 in the tepotinib plus gefitinib group; n=24 in the chemotherapy group). No dose-limiting toxicities were observed in phase 1b, so tepotinib 500 mg was used as the recommended phase 2 dose. In phase 2, survival outcomes were similar between groups: median PFS was 4·9 months in the tepotinib plus gefitinib group (90% CI 3·9–6·9) versus 4·4 months in the chemotherapy group (90% CI 4·2–6·8; hazard ratio [HR] 0·67, 90% CI 0·35–1·28). Median OS was 17·3 months in the tepotinib plus gefitinib group (12·1–37·3) versus 18·7 months in the chemotherapy group (15·9–20·7; HR 0·69, 0·34–1·41). PFS and OS were longer with tepotinib plus gefitinib than with chemotherapy in patients with high (IHC3+) MET overexpression (n=34; median PFS 8·3 months [4·1–16·6] vs 4·4 months [4·1–6·8]; HR 0·35, 0·17–0·74; median OS 37·3 months [90% CI 24·2–37·3] vs 17·9 months [12·0–20·7]; HR 0·33, 0·14–0·76) or MET amplification (n=19; median PFS 16·6 months [8·3–not estimable] vs 4·2 months [1·4–7·0]; HR 0·13, 0·04–0·43; median OS 37·3 months [90% CI not estimable] vs 13·1 months [3·25–not estimable]; HR 0·08, 0·01–0·51). The most frequent treatment-related grade 3 or worse adverse events were increased amylase (5 [16%] of 31 patients) and lipase (4 [13%]) concentrations in the tepotinib plus gefitinib group and anaemia (7 [30%] of 23 patients) and decreased neutrophil count (3 [13%]) in the chemotherapy group. Interpretation Despite early study termination, in a preplanned subgroup analysis, our findings suggest improved anti activity for tepotinib plus gefitinib compared with standard chemotherapy in patients with EGFR-mutant NSCLC and MET amplification, warranting further exploration. Funding Merck KGaA.

M7583 is a potent, highly selective, covalent BTK inhibitor in development. In this phase I, first-in-human, open label, multicenter dose-escalation trial, M7583 was given at 80 mg (threedays)/160 mg (full 28-day cycle), then 300 mg/day, 600 mg/day, 900 mg/day, and 300 mg twice daily to 18 patients (median age 63years) with refractory/resistant, stage III/IV B-cell malignancies who failed prior therapy (NCT02825836). No dose-limiting toxicities were reported. Treatment-emergent adverse events (AEs) occurred in 89% of patients, treatment-related AEs in 78%, and treatment-related grade ≥3 AEs in 17%. Common AEs were diarrhea (33%), fatigue (22%), and vomiting (17%). M7583 was rapidly absorbed and exposure was dose-proportional. BTK occupancy was >95% in the 300 mg twice daily and 900 mg/day cohorts. Objective response rate was 50% and disease control rate 78%, supporting a favorable benefit:risk profile. Fasted doses up to 900 mg once daily and 300 mg twice daily were well tolerated and may be tested in future clinical studies.

9575 Background: In the phase II VISION study (NCT02864992) tepotinib had promising efficacy (response rate of 40–50% & median duration of response >1 y) and tolerable safety in pts with advanced NSCLC with METex14 skipping (3–4% of NSCLC), who are typically elderly with poor prognosis. Pt reported outcomes (PROs) of HRQoL are described here. Methods: Pts with advanced NSCLC positive for METex14 skipping by tissue or liquid biopsy received oral tepotinib 500 mg once daily; PROs were assessed using QLQ-LC13 (lung cancer symptoms), EORTC QLQ-C30 (Global health status [GHS] & 5 functional scales), and EQ-5D-5L (VAS). Questionnaires were completed at baseline (BL) and every 6 weeks (Wk); results were scored from 0–100 (minimal clinically important difference [MCID] ≥10 points). Mean change from BL was analyzed at Wk 12 (predefined analyses). Results: By 19 Jul 19 cut-off, 130 pts across treatment lines were enrolled (median age 74.2 y), with PROs available for 129. Questionnaire completion rates were 90.1% at Wk 12. Symptom burden at BL was moderate for advanced NSCLC; mean change from BL for PROs are shown in the table (better functioning: lower QLQ-LC13 or higher QLQ-C30 scores). For the QLQ-LC13 symptoms, mean changes from BL indicated a meaningful improvement in coughing, with a median time to improvement (2.8 months) paralleling the onset of objective response (within first 3 months), and a numerical improvement in dyspnea (–2.3 at Wk 12) and chest pain (–4.2 at Wk 12). QLQ-C30 values remained stable over treatment as did EQ-5D-5L scores (higher=better): mean (standard deviation, SD) change from BL score (60 [20.4]) was 6 (18.6) at Wk 6 and 5 (20.9) at Wk 12. Conclusions: In this first analysis of PROs in pts with advanced NSCLC with METex14 skipping with a moderate symptom burden, treatment with tepotinib led to a clinically meaningful improvement in coughing symptoms, while maintaining HRQoL. Coupled with the efficacy and safety profile, the predefined HRQoL analysis from the VISION study supports tepotinib as a promising treatment option for this elderly population with METex14+ NSCLC. Clinical trial information: NCT02864992 . [Table: see text]

9556 Background: Preliminary tepotinib data showed durable activity in pts with NSCLC with METex14 skipping prospectively identified by liquid (L+) or tissue (T+) biopsy. Having met target enrollment of ≥60 L+ pts & ≥60 T+ pts, we report primary data. Methods: VISION Cohort A enrolled pts with advanced EGFR/ALK wt, METex14 skipping NSCLC (asymptomatic brain metastases [BM] allowed), who received oral tepotinib 500 mg QD. On-study treatment decisions were based on investigator assessment (INV) of response. Primary endpoint was objective response rate (ORR) by independent review committee (IRC) analyzed in 3 primary ITT sets: L+ and/or T+, L+, T+. 2ary endpoints included ORR by INV, duration of response (DOR), disease control rate (DCR), PFS, OS, & safety. Preplanned analyses were performed in pts with BM at baseline (BL). BL/on-treatment ctDNA plasma samples (L+) were analyzed using a 73-gene NGS panel (Guardant360). Deep molecular responses (MR), defined as > 75% depletion of METex14, were compared with objective responses (OR). Results: By data cut-off (1 Oct 19) 151 pts received tepotinib (safety set); 99 L+/T+, 66 L+, 60 T+ pts comprised the 3 ITT sets with ≥6-month [m] follow-up. Across treatment lines (n = 44 1L, n = 55 ≥2L), primary ORR & mPFS [95% CI] in 99 L+/T+ pts were 43% [34–54] & 8.6 m [6.9–11.0] by IRC and 56% [45–66] & 9.5 m [6.7–13.5] by INV. ORR was similar in L+ or T+ pts (table) or in T+L− pts (n = 25): 40% [21–61] by IRC and 48% [28–69] by INV. Only 2 pts were T−L+. Outcomes were also comparable in pts with BM (n = 11): IRC ORR 55% [23–83] & mPFS 10.9 m [8.0–ne]. 34/51 pts (67%) with matched BL/on-treatment L+ samples had deep MR strongly associated with clinical response: 32/34 pts (94%) with MR had disease control (INV), including 29/34 pts (85%) with OR; 2/34 pts had progressive disease. Further biomarker data will be presented. Grade ≥3 treatment-related adverse events (TRAEs) were reported by 37/151 pts (25%). 13 pts (9%) discontinued due to TRAEs. Conclusions: Tepotinib is a promising targeted therapy with durable clinical activity and manageable toxicity in pts with METex14 skipping NSCLC L+ or T+, including pts with BM. High ORR & DCR in pts with ctDNA molecular responses support that MET inhibition in METex14+ tumor cells can lead to clinical benefit. Clinical trial information: NCT02864992 . [Table: see text]

Background MET exon 14 skipping (METex14), reported in 3–4% of NSCLC patients (pts), is sensitive to MET inhibition. We report interim data from Cohort A of the phase II VISION study (NCT02864992) of tepotinib, an oral potent highly selective MET inhibitor in NSCLC pts with METex14 skipping. Methods Pts with advanced wt EGFR/ALK NSCLC and METex14 skipping identified by liquid (L+) or tumor (T+) biopsy enrolled at 124 sites in 11 countries (35 in Asia: Japan [from Dec 2016], Republic of Korea, Taiwan [from Oct 2018]) received tepotinib 500 mg once daily until progression, intolerable toxicity or withdrawal. Primary endpoint is objective response rate (ORR) by independent review (IRC). Secondary endpoints include investigator-assessed ORR (INV), duration of response (DOR), PFS and safety (CTCAE 4.0). Pts evaluable for ORR have ≥2 post-baseline assessments or have discontinued for any reason. Predefined analysis sets include pts who are L+ or T+. Results As of 14 June 2019, 1071 of 5102 prescreened pts were from Asia, 28 had been enrolled (17 Japan, 8 Korea, 3 Taiwan). At data cutoff (18 Feb 19) for efficacy and safety analyses, 87 pts had received tepotinib, treatment was ongoing in 47. In evaluable pts, ORR was 45–55% (Table). mDOR was >1 year in all groups (12.4–17.1 months) and >50% of pts were event free at 12 months (55–70%). mPFS was 9.5–12.2 months. 14 pts were from Asia (all from Japan), treatment was ongoing in 8. Of 11 evaluable pts from Japan, 6 were L+, 9 were T + (with overlap). In L+ pts, confirmed ORR (95% CI) was 66.7% (22.3, 95.7) by IRC and 83.3% (35.9, 99.6) by INV. In T+ pts, it was 33.3% (7.5, 70.1) by IRC, 55.6% (21.2, 86.3) by INV. Any/grade 3 treatment-related adverse events (TRAEs) reported by ≥ 15% of 87 treated pts were peripheral edema (48.3/8.0%), nausea (23.0/0%), diarrhea (20.7/1.1%). No TRAEs were grade 4/5. The safety profile was similar in pts from Japan.Table62OTableL +T +IRCInv n = 47IRC n = 51Inv n = 51n = 48ORR,* n (%)24 (50.0)26 (55.3)23 (45.1)28 (54.9)95% CI35.2, 64.840.1, 69.831.1, 59.740.3, 68.9mDOR, months 95% CI12.417.115.714.35.8, ne7.1, ne9.0, ne5.7, ne12-month event-free rate 95% CI58%55%70%59%30, 7828, 7640, 8732, 79n = 57n = 58PFS events22242322mPFS, months 95% CI9.59.510.812.26.7, ne5.3, 21.16.9, ne6.3, ne12-month event-free rate39%42%49%55%95% CI21, 5625, 5831, 6439, 69CI, confidence interval; ne, not estimable.*ORR: confirmed complete response/partial response. Patients evaluable for ORR have ≥2 post-baseline assessments or have discontinued for any reason. Conclusions Tepotinib has durable clinical activity and an acceptable safety profile in NSCLC pts with METex14 skipping detected by liquid or tissue biopsy. Clinical trial identification NCT02864992. Editorial acknowledgement Medical writing assistance (funded by Merck KGaA, Darmstadt, Germany) was provided by Matthew deSchoolmeester, PhD of Bioscript Science (Macclesfield, UK). Legal entity responsible for the study Merck KGaA. Funding Merck KGaA. Disclosure K. Park: Advisory / Consultancy: AstraZeneca, Boehringer Ingelheim, Clovis; Elli Lilly; Hanmi; ONO; Roche; Novartis. E. Felip: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck KGaA; Advisory / Consultancy, Speaker Bureau / Expert testimony: AbbVie; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy: Blueprint Medicines; Research grant / Funding (institution): EMD Serono; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy: Celgene, Guardant Health; Speaker Bureau / Expert testimony: MSD, Novartis, Pfizer, Roche, Takeda. R. Veillon: Research grant / Funding (institution): Takeda, AbbVie, Merck KGaA, BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Speaker Bureau / Expert testimony, Research grant / Funding (institution): Roche; Speaker Bureau / Expert testimony: DMS; Advisory / Consultancy: Pfizer, Novartis. A. Cortot: Research grant / Funding (institution): Merck KGaA; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Takeda. J. Mazieres: Advisory / Consultancy: Roche, BMS, MSD, AstraZeneca, Pfizer, Novartis. H. Sakai: Speaker Bureau / Expert testimony, Research grant / Funding (institution): BMS, Ono, Chugai, MSD, Lilly, AstraZeneca; Research grant / Funding (institution): Merck KGaA, Regeneron, Taiho. N. Reinmuth: Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Takeda; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony: AbbVie; Advisory / Consultancy: Merck KGaA; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer. J-Y. Han: Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Research grant / Funding (institution): Roche; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Takeda; Advisory / Consultancy: Lilly; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy: Novartis; Research grant / Funding (institution): ONO. M. Morise: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Chugai; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Pfizer; Honoraria (self), Travel / Accommodation / Expenses: Eli Lilly; Honoraria (self), Travel / Accommodation / Expenses: MSD; Honoraria (self), Research grant / Funding (institution): Ono; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Taiho, Boehringer Ingelheim, Novartis, EMD, Kissei. T. Tokito: Honoraria (self): MSD, Chu-gai, Ono, Boehringer Ingelheim, Pfizer; Honoraria (self), Advisory / Consultancy: AstraZeneca. R. Bruns: Full / Part-time employment: Merck KGaA. J. Scheele: Full / Part-time employment: Merck KGaA. J. Straub: Full / Part-time employment: Merck KGaA. X. Le: Advisory / Consultancy: AstraZeneca, Lilly. P.K. Paik: Research grant / Funding (self), Research grant / Funding (institution): EMD Serono; Advisory / Consultancy, Research grant / Funding (institution): Celgene; Advisory / Consultancy: AbbVie, BMS, Lilly, Takeda. All other authors have declared no conflicts of interest.

Background Promising clinical activity has been reported in patients with MET-altered NSCLC treated with tepotinib, an oral, selective, potent MET tyrosine kinase inhibitor. To support clinical development, we aimed to further characterize the safety profile of tepotinib in patients with cancer. Methods In this analysis from 5 phase I and II studies of tepotinib administered 500 mg once daily, adverse events (AEs), graded by NCI CTCAE v4.03, were pooled and summarized for all patients and for the subgroup of Asian patients. Results As of 28 Sep 2018, 228 patients (81 [35.5%] Asian, 59 [25.9%] NSCLC, 121 [53.1%] HCC) had received tepotinib 500 mg/day for a median of 2.7 months (range 0–21.5); in the ongoing phase II VISION study (NCT02864992), median was 4.1 months. The most common treatment-related AE of any grade was peripheral edema occurring in 33.8% of patients overall and in 24.7% of Asian patients. Other common treatment-related AEs were diarrhea, fatigue, and nausea (Table). Dose reduction due to an AE regardless of causality occurred in 33 (14.5%) patients and discontinuation in 49 (21.5%) patients; peripheral edema led to a dose reduction in 12 (5.3%) patients and discontinuation in 7 (3.1%) patients. In the Asian population, 13 (16.0%) patients had a dose reduction and 13 (16.0%) patients discontinued due to an AE; peripheral edema led to a dose reduction in 4 (4.9%) Asian patients and to no discontinuations. Two AEs leading to death were considered by the investigator to be potentially treatment-related and occurred in patients with HCC (upper gastrointestinal hemorrhage [in an Asian patient] and hypoglycemic coma).Table67PTableAll patients (N = 228)Asian patients (N = 81)Any GradeGrade ≥3Any GradeGrade ≥3Treatment-related AE (≥10%), n (%)172 (75.4)52 (22.8)64 (79.0)26 (32.1)Peripheral edema77 (33.8)8 (3.5)20 (24.7)0Diarrhea45 (19.7)4 (1.8)27 (33.3)4 (4.9)Fatigue34 (14.9)3 (1.3)14 (17.3)3 (3.7)Nausea29 (12.7)09 (11.1)0Decreased appetite27 (11.8)012 (14.8)0Blood creatinine increased18 (7.9)1 (0.4)11 (13.6)0Hypoalbuminemia16 (7.0)1 (0.4)10 (12.3)0Amylase increased13 (5.7)4 (1.8)9 (11.1)2 (2.5) Conclusions Tepotinib demonstrated an acceptable safety profile in patients with advanced cancer, with a similar profile in Asian patients and in the overall population. Further characterization of the clinical relevance of peripheral edema is ongoing. Clinical trial identification NCT01014936, NCT01832506, NCT01988493, NCT02115373, NCT02864992. Editorial acknowledgement Medical writing assistance (funded by Merck Healthcare KGaA, Darmstadt, Germany) was provided by Jen Lewis, PhD of Bioscript Science (Macclesfield, UK). Legal entity responsible for the study Merck KGaA. Funding Merck KGaA. Disclosure K. Yamazaki: Honoraria (self): Chugai; Honoraria (self): Takeda; Honoraria (self): Yakult; Honoraria (self): Daiichi‐Sankyo; Honoraria (self): Merck Serono; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Bayer; Honoraria (self): Eli Lilly; Honoraria (self): Taiho; Research grant / Funding (institution): BMS; Research grant / Funding (institution): Sanofi. P.K. Paik: Advisory / Consultancy: Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy: Lilly; Honoraria (self), Advisory / Consultancy: AbbVie; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Celgene; Research grant / Funding (institution), Travel / Accommodation / Expenses: EMD Serono; Honoraria (self): Takeda. R. Veillon: Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Novartis; Speaker Bureau / Expert testimony, Research grant / Funding (institution): BMS; Speaker Bureau / Expert testimony, Research grant / Funding (institution): Roche; Research grant / Funding (institution): Takeda; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): Merck. T. Decaens: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Honoraria (self), Travel / Accommodation / Expenses: Bayer; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy: Ipsen; Honoraria (self), Travel / Accommodation / Expenses: Gilead; Honoraria (self), Travel / Accommodation / Expenses: AbbVie; Honoraria (self): Sanofi; Advisory / Consultancy: BTG; Advisory / Consultancy: Sirtex; Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Travel / Accommodation / Expenses: MSD; Travel / Accommodation / Expenses: Merck Serono; Research grant / Funding (institution): ArQule; Research grant / Funding (institution): Genoscience Pharma. S. Faivre: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Blueprint; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Bayer Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Eli Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Ipsen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck Serono; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis. G.S. Falchook: Licensing / Royalties: Wolters Kluwer; Advisory / Consultancy, Travel / Accommodation / Expenses: Fujifilm; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: EMD Serono; Travel / Accommodation / Expenses: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Millennium; Speaker Bureau / Expert testimony: Total Health Conferencing; Research grant / Funding (institution): 3-V Biosciences; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): ADC Therapeutics; Research grant / Funding (institution): Aileron; Research grant / Funding (institution): American Society of Clinical Oncology; Research grant / Funding (institution): Amgen; Research grant / Funding (institution): ARMO; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): BeiGene; Research grant / Funding (institution): Bioatla; Research grant / Funding (institution): Biothera; Research grant / Funding (institution): Celldex; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Ciclomed, Curegenix, Curis, DelMar, eFFECTOR, Eli Lilly, Exelixis, Fujifilm, Genmab, GlaxoSmithKline, Hutchison MediPharma, Ignyta, Incyte, Jacobio, Jounce, Kolltan, Loxo, MedImmune, Millennium, Merck, miRNA Therapeutics, National Institutes of Health, No. J. Scheele: Full / Part-time employment: Merck KGaA. R. Bruns: Full / Part-time employment: Merck KGaA. K. Berghoff: Full / Part-time employment: Merck KGaA. All other authors have declared no conflicts of interest.

Background In patients with EGFR-mutant NSCLC, MET amplification (METamp) is a resistance mechanism to EGFR tyrosine kinase inhibitors (TKI). In the randomized phase Ib/II INSIGHT study (NCT01982955) that halted full enrolment due to low recruitment, tepotinib (TEP), a potent, selective MET TKI, plus gefitinib (GEF) improved progression-free survival (PFS) and objective response rate (ORR) vs chemotherapy (CTx) in patients with EGFR-mutant NSCLC and resistance to 1st-line EGFR TKIs due to METamp (≥6-month follow-up). We now present long-term survival outcomes (≥18-month follow-up) for this predefined analysis. Methods Patients were randomized to TEP 500 mg + GEF 250 mg orally once daily (until progressive disease, intolerable toxicity or other withdrawal) or platinum + pemetrexed IV (≤6 x 21-day cycles). METamp was defined as GCN ≥5 and/or MET/CEP7 ratio ≥2. Primary endpoint was investigator-assessed (INV) PFS. Secondary endpoints included overall survival (OS), ORR, PFS by independent review (IRC), safety. Results From 04/24/15 to 06/12/17, 55 patients enrolled in the INSIGHT study and 19 were METamp (TEP+GEF 12; CTx 7); this predefined subgroup is analysed here. Median GCN was 8.8 (range 4.8–29.5); 18 patients had GCN ≥5, 13 had MET/CEP7 ratio ≥2. At data cutoff (12/12/18), median treatment duration (weeks [range]) for TEP+GEF was 49 (4.6–110.9; 3 patients still ongoing for ≥24 months), pemetrexed was 18.0 (5.9–60.4), cisplatin 12.0 (6.6–25.1) or carboplatin 12.8 (5.9–19.7), all CTx patients discontinued. TEP+GEF compared with CTx improved PFS (INV mPFS 21.2 vs 4.2 months, HR [90% CI] 0.13 [0.04, 0.43]; IRC mPFS 19.3 vs 5.5 months; 0.18 [0.06, 0.61]) and OS (mOS 37.3 vs 13.1 months, 0.08 [0.01, 0.51]), as well as ORR (INV 66.7 v 42.9%; OR 2.67 [0.37, 19.56]; IRC 75.0 vs 42.9%; OR 4.00 [0.51, 31.38]). Grade ≥3 treatment-related AEs in METamp patients (≥15% in either arm, TEP+GEF vs CTx) were amylase or lipase increased (both 33.3% vs 0%), anemia, neutrophil or WBC count decreased (all 0 vs 28.6%). Conclusions TEP+GEF improved survival of patients with EGFR TKI-resistant NSCLC due to METamp. TEP + osmertinib is currently being investigated in patients with METamp, EGFR-mutant NSCLC with acquired EGFR TKI resistance (NCT03940703). Clinical trial identification NCT01982955. Editorial acknowledgement Medical writing assistance was provided by Lisa Jolly, Bioscript, Macclesfield, UK, and funded by Merck KGaA, Darmstadt, Germany. Legal entity responsible for the study Merck KGaA, Darmstadt, Germany. Funding Merck KGaA, Darmstadt, Germany. Disclosure K. Park: Advisory / Consultancy: AstraZeneca, Boehringer Ingelheim, Clovis; Elli Lilly; Hanmi; ONO; Roche; Novartis. R.A. Soo: Honoraria (self): BMS, Celgene, Ignyta, Lilly, Merck, Novartis, Pfizer, Roche, Taiho, Takeda, Yuhan; Honoraria (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Research grant / Funding (institution): Boehringer Ingelheim. R. Bruns: Full / Part-time employment: Merck KGaA. J. Straub: Full / Part-time employment: Merck KGaA. A. Johne: Full / Part-time employment: Merck KGaA. J. Scheele: Full / Part-time employment: Merck KGaA. J.C-H. Yang: Honoraria (self), Advisory / Consultancy: Boehringer Ingelheim, Eli Lilly, Bayer, Roche/Genentech/Chugai, MSD, Merck Serono, Pfizer, Novartis, Celgene, Merrimack, Yuhan Pharmaceuticals, BMS, Ono pharmaceutical, Daiichi Sankyo and AstraZeneca, Takeda Oncology, Blueprint Medicines, Hansoh Pharmaceu. Y-L. Wu: Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): Boehringer Ingelheim ; Advisory / Consultancy: Merck; Honoraria (self): Eli Lilly, Pierre Fabre, Pfizer, Sanofi. All other authors have declared no conflicts of interest.

Background MET amplification (amp) is a resistance mechanism to EGFR tyrosine kinase inhibitors (TKIs). Tepotinib (TEP), an oral, selective MET TKI, has shown promising antitumor activity in combination with gefitinib (GEF) in patients with MET+ EGFR-mutant NSCLC and acquired resistance to 1st-line EGFR TKIs in a phase 1b/2 study (NCT01982955). In the phase 1b part of the study, 4/6 patients with METamp had an objective response with TEP+GEF (67%). Methods In the phase 2 part of the study, Asian patients with advanced MET+ (MET2+ or 3+ by immunohistochemistry and/or METamp by in-situ hybridization) EGFR-mutant T790M- NSCLC and acquired resistance to 1st-line EGFR TKIs were randomized to receive oral TEP 500 mg + GEF 250 mg once daily or platinum + pemetrexed chemotherapy (CTx). The primary endpoint was investigator-assessed PFS. Secondary endpoints included PFS by independent review committee (IRC), overall response rate (ORR), and safety. Data from a minimum follow-up of 6-months are presented (cut-off 26 Mar 2018) for the pre-planned analysis of patients with METamp, defined as gene copy number (GCN) ≥5 and/or MET/CEP7 ratio ≥2. Results Low recruitment halted enrolment into phase 2. From 24 Apr 2015 to 12 Jun 2017, 55/156 planned patients were enrolled; 19 had METamp (TEP+GEF, 12; CTx, 7) and are included in this analysis. Median age of METamp patients was 60.4 years (range 42-74), 6 were men, 13 were never smokers, 5 had ECOG PS 0 and 14 had PS 1. Median GCN was 8.8 (range 4.8-29.5); 18 patients had GCN ≥5, 13 had MET/CEP7 ratio ≥2. Median TEP+GEF treatment duration was 37 weeks (range 4.6-91.9) and 4 patients are still receiving treatment, ongoing for ≥18 months. Investigator-assessed PFS improved with TEP+GEF vs CTx (mPFS 21.2 vs 4.2 months; HR 0.17 [90% CI 0.05, 0.57]), as well as IRC-assessed PFS (19.8 vs 5.5 months; HR 0.25 [90% CI 0.07, 0.85]). ORR also improved with TEP+GEF (investigator: 66.7% vs 42.9%; OR 2.67 [90% CI 0.37, 19.56] and IRC: 75.0% vs 42.9%; OR 4.00 [90% CI 0.51, 31.38]). Disease control was achieved in 11 (91.7%) patients receiving TEP+GEF vs 5 (71.4%) patients receiving CTx. Related grade ≥3 treatment-emergent adverse events (TEAEs) were reported in 6 (50.0%) patients receiving TEP+GEF and 5 (71.4%) patients receiving CTx. The most common related TEAEs in the TEP+GEF arm were diarrhea (TEP+GEF 50.0% vs CTx 14.3%) and amylase increased (TEP+GEF 41.7% vs CTx 0%) and in the CTx arm were anemia (TEP+GEF 0% vs CTx 57.1%), white blood cell count decreased (TEP+GEF 8.3% vs CTx 57.1%), neutrophil count decreased (TEP+GEF 0% vs CTx 57.1%), and nausea (TEP+GEF 8.3% vs CTx 42.9%). Conclusions Promising antitumor activity with TEP+GEF was reported in patients with METamp, EGFR-mutant T790M- NSCLC and acquired resistance to 1st-line EGFR TKIs. No new safety signals were observed. METamp can be considered a biomarker for tepotinib that should be further explored. Citation Format: James Chih-Hsin Yang, Jianying Zhou, Dong-Wan Kim, Azura Rozila Ahmad, Ross Andrew Soo, Rolf Bruns, Josef Straub, Andreas Johne, Jürgen Scheele, Keunchil Park, Yi-Long Wu. Tepotinib + gefitinib vs chemotherapy in MET-amplified EGFR-mutant non-small cell lung cancer (NSCLC): Predefined subgroup analysis of a Phase Ib/II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT193.

9005 Background: MET exon 14 skipping ( METex14) mutations - reported in 3~4% of NSCLC patients (pts) - are activating, sensitive to MET inhibition and can be conveniently detected using liquid biopsy (LBx). We report data from an ongoing single-arm phase II study of tepotinib, a highly selective MET inhibitor, in NSCLC pts with METex14 mutations identified by LBx or tumor biopsy (TBx) (NCT02864992). Methods: Pts with advanced WT EGFR/ALK NSCLC, prospectively enrolled via either LBx (≥60 pts) or TBx (≥60 pts, overlap anticipated) central RNA-based METex14 mutation testing, receive tepotinib 500 mg QD until progression, intolerable toxicity or withdrawal. Primary endpoint: objective response rate (ORR) by independent review (IRC). Secondary endpoints: ORR by investigator assessment (INV) and safety. Results: To date, 85 pts have been enrolled (55 LBx pts and 52 TBx pts). At data cut-off (16 Oct 2018), in 35 evaluable LBx pts (≥2 post-baseline assessments or discontinuation for any reason), ORR was 51.4% by IRC and 63.9% by INV. In 41 evaluable TBx pts, ORR was 41.5% by IRC and 58.5% by INV. Median duration of response (mDoR) and ORR by line of treatment are shown in the table. Any grade treatment-related adverse events (TRAEs) reported by ≥10% of 69 pts evaluable for safety were peripheral edema (47.8%), diarrhea (18.8%), nausea (15.9%), asthenia (10.1%). No TRAEs were grade 4 or led to death. TRAEs led to permanent discontinuation in 2 (2.9%) pts (1 ILD, 1 diarrhea & nausea). Conclusions: Tepotinib has promising activity with a long DoR across treatment lines in NSCLC pts with METex14 mutations detected by LBx or TBx. The safety profile was favorable. Recruitment is ongoing. Clinical trial information: NCT02864992. [Table: see text]

Somatic adrenal stem cells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01–0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified.In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3β-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished inPbx1haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficientTpit−/−mice was significantly wider compared with wild-type adrenals (Tpit−/−259±10.7 vsTpit+/−100±12.3%;PTpit−/−0.45±0.16 vsTpit+/−0.13±0.04%;PP=0.33 and 0.09%), providing indirect evidence for a stem cell ‘arrest’ inTpit−/−mice and the role of ACTH in adrenocortical stem cell modulation and differentiation.

Rap1, which is closely related to ras, plays a key role in T-cell receptor (TCR)-signaling. TCR-stimulation without costimulation leads to constitutively activated rap1, which may mediate T-cell anergy via inhibition of ras-dependent induction of extracellular signal-regulated kinases (ERK). This activation is mediated by a second protein kinase b-Raf. Rap1-GTP is thought to activate ERK in a ras-independent manner by binding b-raf. Generally, T cells do not express b-raf while they express the adaptor protein raf-1, which is usually sequestered by rap1 leading to inhibition of ras-mediated ERK activation. In this study, we demonstrate that in rap1-deficient T cells, signaling by the ERK and p38 kinases is increased following activation by different stimuli leading to increased intracellular accumulation and secretion of cytokines. In addition, in a hypersensitivity model rap1-deficient mice demonstrated reduced contact dermatitis compared to wildtype mice, demonstrating the impact of rap1-deficiency on the inflammatory response in vivo.

Background: Bruton's tyrosine kinase (BTK) has a key role in B-cell receptor mediated pathways that are implicated in the pathogenesis of several B-cell malignancies. Inhibition of BTK has been shown to block several B-cell functions, including proliferation, antigen presentation, antibody production and cell migration. Small molecule BTK-inhibitors (BTKi) have been approved for the treatment of several B-cell malignancies, including resistant/refractory chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström's macroglobulinemia (WM). M7583 is a potent and highly selective BTKi, which is currently being investigated in a two-part, phase I/II trial (NCT02825836) in patients with refractory/resistant B cell malignancies. Methods: In the dose-escalation part of the study (Part 1), patients with refractory/resistant B cell malignancies received once-daily (QD) M7583 in 28-day cycles in ascending dose cohorts following an adaptive Bayesian design; the starting dose was 80 mg for 3 days followed by 160 mg (80/160 mg QD). The dose-expansion part of the study (Part 2) will investigate M7583 in patients with diffuse large B cell lymphoma (DLBCL; activated B-cell subtype) or MCL who have failed 1-3 lines of previous therapy. Here, we present safety and tumor response outcomes as assessed by the investigators according to Cheson/CLL/Owen criteria for patients in Part 1. Results: In total, 25 patients were screened for inclusion in Part 1, as of 20th May 2018 18 had received treatment with M7583 in 5 dose cohorts (80/160 mg, n=3; 300 mg QD, n=3; 600 mg QD, n=5; 300 mg BID, n=3; 900 mg QD, n=4). Treatment is ongoing in 11 patients. Patients were mostly male (72%, n=13), aged 43-80 years (median 63 years) and had a diagnosis of MCL (n=8), WM (n=4), DLBCL (n=3), marginal zone lymphoma (n=2), or small lymphocytic lymphoma (n=1). Treatment-emergent adverse events (TEAEs) occurred in 89% (n=16/18) of patients; the most common TEAE was diarrhea (n=6/18; 33%). No dose-limiting toxicities were reported. Grade ≥3 TEAEs occurred in 10 patients (56%), events in 3 (17%) were considered related to treatment. Six patients (33%) had serious TEAEs and 2 patients (11%) had treatment-related serious TEAEs. The maximum tolerated dose was not reached. Patients received M7583 for up to 20 months (median 7.9 [range 0.5-20] months). Preliminary study data show the objective response rate was 50% (9 of 18 subjects). Disease control was observed in 14 of 18 subjects (disease control rate 78%): 2 subjects had a complete response (CR), 7 subjects had a partial response (PR), 1 subjects had a minor response (MR) and 4 subjects had stable disease (SD). Responses were observed across all doses: 80/160 mg QD, 1 PR, 2 SD; 300 mg QD, 2 PR, 1 MR; 600 mg QD, 1 CR, 2 PR; 300 mg BID, 2 PR, 1 SD; 900 mg QD, 1 CR, 1 SD. Both the 300 mg BID and 900 mg QD doses met the criteria for the optimal biological dose, therefore, these doses represent potential recommended phase 2 doses in support of further development. Conclusions: Clinical benefit was observed with M7583 across the doses investigated and was well tolerated in patients with refractory/resistant B cell malignancies. These outcomes indicate a favorable benefit:risk profile for M7583. Disclosures Jurczak: TG therapeutics: Research Funding; Roche: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding; Morphosys: Research Funding; Nordic Nanovector: Research Funding; Epizyme: Research Funding; Celgene: Research Funding; Beigene: Research Funding; Bayer: Research Funding; Afimed: Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Sandoz-Novartis: Consultancy; Acerta: Consultancy, Research Funding; AstraZeneca: Consultancy; European Medicines Agency: Consultancy; Servier: Consultancy, Honoraria, Research Funding. Rule:Celltrion: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees. Townsend:Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Sarholz:Merck KGaA: Employment. Scheele:Merck KGaA: Employment. Gribben:TG Therapeutics: Honoraria; Wellcome Trust: Research Funding; Medical Research Council: Research Funding; Roche: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria; Abbvie: Honoraria; Cancer Research UK: Research Funding; Unum: Equity Ownership; Kite: Honoraria; NIH: Research Funding; Pharmacyclics: Honoraria. Zinzani:MSD: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra Zeneca: Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Auron-Misheil-Therapy (AMT) is currently under development as an anticancer treatment. The aim of the present study was the identification of possible effects and properties of AMT with regard to human skin. The study consisted of three experimental phases. Phase 1 assessed the effects of AMT on the viability of 2-D evaluated and accredited cell cultures of three cell types of human skin, namely: keratinocytes, melanocytes and fibroblasts. Three separate assays were used in this phase. Phase 2 was designed to clarify the effects of AMT on cell viability to investigate the mode of action of AMT. Two possible modes of action were investigated: proliferation inhibition and apoptosis. There was one assay to assess proliferation, and two independent assays to assess apoptosis. The third phase assessed the effects of AMT on two different types of 3-D skin models, an ex vivo model and a de novo reconstituted model. In the phase 1 tests, reduction of cell viability by AMT was demonstrated in all three cell types. In phase 2, the cell proliferation assay showed decreased proliferation rates in the presence of AMT in three out of four cell populations. In phase 3, histochemical investigations with 3-D models indicated that AMT induces desquamation, most likely as the result of apoptosis of epidermal cells. The experiments generally showed that AMT does not harm human skin at concentrations up to 3%.

Background: The Guanine Triphosphatase (GTPase) Rap1A has been implicated in cell proliferation and migration in different tissues. Its role in the production, adhesion and migration of bone marrow-derived hematopoietic cells is however incompletely understood. Methods: We analysed mice lacking one or both Rap1A alleles and measured their adhesion function and their hematopoietic repopulation capacity using competitive bone marrow transplantation in the CD45.1/CD45.2 transplant model. We further employed flow cytometry and shear stress-dependent flow chamber adhesion assays for phenotypic characterization and analysis of adhesion functions. Results: Rap1A-/- mice showed decreased numbers of lymphocytes and hematopoietic colony forming progenitors in the blood, while the numbers of granulocytes, monocytes or platelets were not significantly altered compared to controls. Chemokine CXCL12-induced adhesion of lineage marker depleted (Lin-) bone marrow cells to Vascular Cell Adhesion Molecule (VCAM)-1 and to Intercellular Adhesion Molecule (ICAM)-1 was significantly reduced in Rap1A-/- mice. However, competitive repopulation by Rap1A deficient bone marrow cells was not inferior to wild type bone marrow 9 months after transplantation. No outgrowth of Mesenchymal Stromal Cells (MSCs) was observed from bone marrow of Rap1A-/- mice, whereas Rap1A+/- MSCs displayed reduced expression of adhesion molecules involved in progenitor homing and impaired adhesion to primary endothelial cells and VCAM-1 under shear stress in vitro, as compared to their wild-type counterparts. Conclusions: Rap1A regulates adhesion of immature hematopoietic cells and mesenchymal stromal cells, but appears to be redundant for hematopoietic engraftment.

Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1-/- cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.

The E2a-Pbx1 oncoprotein of human pre-B cell leukemia prevents differentiation and maintains continued cell division in cultured myeloid progenitors. Previously, estrogen-dependent forms of E2a-Pbx1 were generated that immortalized neutrophil (ECoM-G cells) or monocyte (ECoM-M cells) progenitors and that permitted their terminal differentiation upon estrogen withdrawal. Here, representational difference analysis (RDA) and Affymetrix array analysis are used to identify changes in gene expression that accompany the early differentiation of these cells. The promoters of these genes, whose expression changes upon E2a-Pbx1 inactivation, integrate the biochemical mechanism through which E2a-Pbx1 arrests differentiation and maintains cell division. Inactivation of E2a-Pbx1 caused the 10- to 80-fold up regulation of a small subset of myeloid differentiation genes (MRP8, Cnlp, NB1, Bactenecin, YM1, Stefin 1, Lipocortin, Lactoferrin, gp91 phox and Ly6-G) and a 10-fold down regulation of the TLE1 corepressor gene, as well as of a group of genes expressed in dividing cells (c-Myc, Nucleophosmin, Spermidine synthase, NOP56, Hnrpa1). Transcription of 97% of cellular genes, including 300 other transcription factor genes (21 Hox genes) and other myeloid genes, varied less than 3-fold, with most varying less than 50%. Therefore, E2a-Pbx1 prevents transcription and maintains the cell cycle by a specific rather than a global transcriptional mechanism. Monocyte progenitors were distinguished by persistent expression of IRF8 and of a category of other genes characterized as "interferon-stimulated" (ISG15, ISG20, Ifit1, Ifi202a, Ifi203, IfiS204, Ifi204-related, IRF7 and Ly6-E.1), as well as by the upregulation of the Lrg21 bZip transcription factor gene during late differentiation. The synchronous expression of stage-specific and cell cycle genes regulated by E2a-Pbx1 in these cell lines comprises a model system in which analysis of their promoters can be used as a starting point to backtrack to the transcriptional mechanisms of oncogenesis by E2a-Pbx1.

Background: Approximately 3-4% of non-small cell lung cancers (NSCLCs) harbor activating mutations of the MET proto-oncogene that cause exon 14 skipping (METex14) and accumulation of c-Met lacking a juxtamembrane domain. Such tumors are sensitive to nonselective kinase inhibitors with activity against c-Met, so selective c-Met inhibitors may have greater efficacy and/or tolerability. This single-arm phase II trial (NCT02864992) is investigating the efficacy and safety of the potent and selective c-Met inhibitor tepotinib in patients (pts) with NSCLC harboring METex14. Methods: Eligible patients (pts) are adults with stage IIIB/IV NSCLC and METex14 mutations identified in tumor and/or circulating tumor DNA (ctDNA) in plasma (liquid biopsy) by a central laboratory. Pts with EGFR-activating mutations, ALK rearrangements, or >2 lines of prior therapy are excluded. Pts receive tepotinib 500 mg QD on a 21-day cycle until disease progression, intolerable toxicity, or withdrawal from treatment for other reasons. Primary endpoint: objective response rate (ORR). Secondary endpoints: progression-free and overall survival, safety, pharmacokinetics, and quality of life. Recruitment of up to 120 (minimum 60 tumor, 60 ctDNA METex14-positive) pts in Europe, USA, and Japan is planned. Results: Five pts (age 55-77 years; 4 stage IV, 1 stage IIIB; all male; 4 Caucasian, 1 Asian) have been enrolled. Pts have currently completed 0-8 cycles of tepotinib therapy. Most adverse events observed to date have been grade 1/2 in severity, mostly unrelated to tepotinib treatment. One grade ≥3 tepotinib-related AE has occurred: grade 3 increased serum amylase. One pt has died due to his disease. Of the four pts with post-baseline tumor evaluations, two have confirmed partial response, one unconfirmed partial response, and one (with only one post-baseline assessment) stable disease. Conclusions: Initial data suggest that tepotinib 500 mg QD has promising activity in METex14 NSCLC. The safety profile of tepotinib appears favorable. Recruitment to the trial is ongoing. Citation Format: Enriqueta Felip, Remi Veillon, Santiago Viteri, Jürgen Scheele, Rolf Bruns, Paul K. Paik. Phase II trial of the c-Met inhibitor tepotinib in patients with advanced non-small cell lung cancer harboring MET exon 14-skipping mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A097.

8547 Background: Patients (pts) with NSCLC that is initially responsive to EGFR tyrosine kinase inhibitors (EGFR TKI) typically develop resistance, often associated with aberrant c-Met activity. Dual inhibition of EGFR and c-Met is therefore a rational option to treat c-Met+ EGFR TKI-resistant NSCLC. Tepotinib is a highly selective c-Met inhibitor with good tolerability and promising activity against solid tumors. We report final data from a phase Ib trial of tepotinib + gefitinib in pts with c-Met+/EGFR-mutant NSCLC conducted in Asia. Methods: Eligible pts were adults with locally advanced/metastatic NSCLC and ECOG PS 0–1. Tumors had to express EGFR with an activating mutation be resistant to EGFR TKI therapy and be c-Met+ by IHC. Pts received tepotinib 300 or 500 mg QD combined with gefitinib 250 mg QD (T300G250 or T500G250). The primary objective was to determine the recommended phase II dose (RP2D) of tepotinib in combination with gefitinib; secondary objectives included pharmacokinetics (PK), safety, and antitumor activity. Results: 18 pts were enrolled (median age 65 [41–78]; 8 male); 6 received T300G250, 12 T500G250. No dose-limiting toxicities were observed, and tepotinib 500 mg QD was confirmed as the RP2D. 17 pts experienced treatment-related treatment-emergent adverse events (TRTEAEs), mostly grade ≤2 and most commonly diarrhea (12), rash (8), and amylase increase (6). Grade ≥3 TRTEAEs were increased amylase (n = 4), increased lipase (3), neutropenia (1), and hyperglycemia (1). The best overall response was partial response in 6 pts; 4/7 pts with IHC 3+ tumors responded (all treated with T500G250) vs 2/11 with IHC 2+ tumors. Response durations of pts with PR were 4.2–12.5 months. 4/18 pts (IHC 2+, n = 3) had stable disease. 8 pts experienced progression free survival > 5 months, 3 pts > 10 months. PK were as expected from previous studies. Conclusions: Tepotinib in combination with gefitinib was well tolerated. The RP2D of tepotinib for use in combination with gefitinib in NSCLC is 500 mg QD. T500G250 showed signs of activity against c-Met+ tumors. A phase II trial is randomizing ≈156 pts with c-Met+/T790M– tumors who have failed first-line EFGR TKI 2:1 to tepotinib + gefitinib or pemetrexed + cisplatin/carboplatin. Clinical trial information: NCT02864992.

e20541 Background: Approximately 3-4% of lung adenocarcinomas express a truncated form of c-Met (c-Metex14) due to mutation-induced exon 14 skipping. c-Metex14 accumulates on the cell surface and is constitutively active with the ability to drive NSCLC. Data suggest that lung adenocarcinomas harboring c-METex14 are sensitive to c-Met kinase inhibitors. The highly selective c-Met inhibitor tepotinib is well tolerated and active at an oral dose of 500 mg QD. This single-arm phase II trial (NCT02864992) is investigating the efficacy and safety of tepotinib in patients (pts) with advanced lung adenocarcinoma harboring METex14. Methods: Adults with stage IIIB/IV lung adenocarcinoma who have failed 1 or 2 lines of systemic therapy, including a platinum doublet-containing regimen, are eligible. Tumors must harbor mutations that are known to cause exon 14 skipping, confirmed by a central laboratory, but not activating EGFR mutations or ALK rearrangements. Pts receive tepotinib 500 mg QD until disease progression, intolerable toxicity, or withdrawal from treatment for other reasons. The primary endpoint is objective response rate. Secondary endpoints include progression-free and overall survival, safety, pharmacokinetics, and quality of life. Recruitment of 60 patients in Europe, USA, and Japan is planned. Results: Four pts (age 64–77 years; 3 stage IV, 1 stage IIIB, all Caucasian males) have been enrolled. All had received two prior chemotherapy regimens including a platinum doublet. Pts have currently completed 1–5 cycles of tepotinib therapy. The majority of adverse events observed to date have been grade 1/2 in severity; grade 3 disease-related dyspnea, pulmonary embolism, and pleural effusion were observed in one patient and grade 3 tepotinib-related elevated serum amylase in another. Of the 3 pts with post-baseline tumor evaluations, two have had an unconfirmed partial response and the third (with only one post-baseline assessment) stable disease. Conclusions: These initial data suggest that the efficacy of tepotinib 500 mg QD is comparable to that of less selective c-Met inhibitors in pts with c-METex14 NSCLC (ORR > 40%). Tepotinib is also well tolerated. Recruitment to the trial is ongoing. Clinical trial information: NCT02864992.

e14101 Background: BTK is a cytoplasmic tyrosine kinase expressed in cells of hematopoietic origin that is involved in innate and adaptive immunity. BTK inhibition blocks B-cell functions including proliferation, antigen presentation, antibody production, and cell migration. The B-cell receptor pathway is implicated in the pathogenesis of B-cell malignancies such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and B-cell chronic lymphocytic leukemia (CLL). Small molecule BTKis are a new class of agent against B-cell malignancies; ibrutinib has recently been approved for the treatment of resistant/refractory CLL, MCL, and Waldenström’s macroglobulinemia (WM). This ongoing phase I/II clinical trial (NCT02825836) is designed to determine the safety, tolerability, PK, BTK occupancy, and preliminary antitumor activity of the highly selective BTKi M7583 in patients (pts) with refractory/resistant B cell malignancies. Methods: The trial is in two parts: a dose-escalation part for which pts with refractory/resistant B cell malignancies are eligible; and a dose-expansion part for which pts with DLBCL (ABC subtype) or MCL who have failed 1-3 prior lines of therapy are eligible. Pts in the dose-escalation part will be enrolled to sequential dose escalation, with ascending once-daily M7583 doses starting at 80 mg (3 initial days)/160 mg (full 28-day cycle) and increasing according to an adaptive Bayesian design. Results: Three pts have been enrolled to treatment with M7583 80/160 mg (Table). All 3 patients have had an objective response or stable disease and relevant clinical benefit.Conclusions: M7583 appears to have a favorable benefit:risk profile, with an efficacy signal at a dose of 80 mg (3 initial days)/160 mg (full cycle) with objective response or clinical benefit. Clinical trial information: NCT02825836. [Table: see text]

4087 Background: The incidence of hepatocellular carcinoma (HCC), a leading cause of cancer death, is increasing with the increasing incidence of chronic liver disease. Sorafenib, the only approved systemic therapy for advanced HCC, provides modest improvement in overall survival. Preclinical studies suggest c-Met is a valid target in HCC, but non-selective TKIs with c-Met inhibitory activity have not shown efficacy in trials, possibly due to lack of c-Met inhibition. Tepotinib (MSC2156119J) is a highly selective c-Met inhibitor that has favorable safety and promising activity, particularly against c-Met+ solid tumors. We report the final results of a phase Ib trial of tepotinib in patients (pts) with advanced HCC. Methods: Pts were Asian adults with confirmed HCC of BCLC Stage C, Child-Pugh Class A liver function without encephalopathy, and ECOG PS 0–2. Pts received tepotinib 300, 500 (the RP2D) or 1,000 mg/day on a 21-day cycle. c-Met expression status was retrospectively determined by IHC. Results: 27 pts were enrolled (median age 57 [38-69]; male 23; ECOG PS 0/1 11/16); 7 received tepotinib 300 mg/day, 14 500 mg/day, and 6 1,000 mg/day (3 with dose reduction). No DLTs were observed. 22 pts experienced treatment-related treatment-emergent adverse events (TRTEAEs), most commonly diarrhea (n = 10), nausea (8), elevated AST (7), and elevated ALT (6). 9 pts had grade ≥3 TRTEAEs, including elevated AST (3) and elevated ALT (3). Best overall response (BOR) was partial response (PR) in 2 pts, one of whom received tepotinib 500 mg (response duration 16.1 months) and one 1,000 mg (4.4 months); both had c-Met+ tumors. A further 8 pts had a BOR of stable disease (SD), 1 pt non-complete response (CR)/non-progressive disease (PD), and 14 pts had PD (2 pts not evaluable). Five pts had progression free survival > 8 months. PK were as expected from previous studies. Conclusions: Tepotinib at doses of up to 1,000 mg/day was well tolerated by Asian pts with advanced HCC and a maximum tolerated dose was not reached. Antitumor activity was observed, particularly in pts with c-Met+ tumors. The ongoing phase II part of this study is comparing the efficacy and safety of first-line tepotinib and sorafenib in pts with c-Met+ HCC. Clinical trial information: NCT01988493.

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP- dependent kinase creates the more acidic species.

We previously described the isolation of a variant subline of HL-60 cells that does not differentiate in response to nitric oxide (NO)-generating agents or to cGMP analogs [7]. The variant cells have normal guanylate cyclase activity and normal NO-induced increases in the intracellular cGMP concentration. We now show that the variant cells have normal cGMP-dependent protein kinase (G-kinase) activity, both by an in vitro and in vivo assay, and using two-dimensional gel electrophoresis we have identified six G-kinase substrates in the parental cells. Of these six proteins, we found considerably less phosphorylation of one of the proteins in the variant cells than in parental cells, both in vitro and in intact cells, and by 35S-methionine/35S-cysteine incorporation we found much less of this protein in the variant cells than in parental cells. The protein is a shared substrate of cAMP-dependent protein kinase (A-kinase); since cAMP analogs still induce differentiation of the variant cells, it appears that the NO/cGMP/G-kinase and cAMP/A-kinase signal transduction pathways share some but not all of the same target proteins in inducing differentiation of HL-60 cells.

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce .NO and citrulline from L-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2-3 x 10(6) M-1 s-1) and another slow (2-4 x 10(4) M-1 s-1). The relative proportions of the two forms vary with conditions. For the heme domain, fast forms dominate except in the presence of both tetrahydrobiopterin and Arg. In the holoenzyme, slow forms dominate except when both reagents are absent. Geminate recombination is substantial, approximately 50%, only when fast forms predominate. Stopped-flow mixing found dissociation constants near 0.3 s-1. These data imply an equilibrium constant such that very little CO should bind at physiological conditions unless large CO concentrations are present locally.

E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.

We devised enzyme-based methods to measure fmol amounts of GDP and GTP and applied these methods to measure absolute amounts of Ras-bound GDP and GTP in NIH 3T3 fibroblasts. We found that parental NIH 3T3 cells contained 509 and 1.3 fmol of Ras-bound GDP and GTP, respectively, per mg of cellular protein and that stable transfectants of NIH 3T3 cells overexpressing wild-type Ha-Ras contained 7008 and 21.3 fmol of Ras-bound GDP and GTP, respectively, per mg of cellular protein; thus, in both cell types < 0.3% of Ras was in the active GTP-bound state. In contrast, NIH 3T3 cells overexpressing an activated form of Ha-Ras contained 5013 and 2049 fmol of Ras-bound GDP and GTP, respectively, per mg of protein, yielding 29% of Ras in the GTP-bound state. Since intracellular Ras is probably all in a guanine-nucleotide bound state, this method allows one to calculate the number of Ras molecules in each cell: parental NIH 3T3 cells and the Ha-Ras overproducing cells contain approximately 20,000 and approximately 275,000 Ras molecules per cell, respectively. We also incubated parental NIH 3T3 cells with 32PO4 and determined the radioactivity in Ras-bound GDP and GTP and the specific activity of cytosolic GDP and GTP; these experiments indicated that Ras-bound GTP may not be in equilibrium with the total cytosolic GTP pool.

Sodium nitroprusside and sodium nitrite, which generate nitric oxide and increase the intracellular cGMP concentration, and 8-bromo-cGMP, a membrane-permeable cGMP analog, induce myelomonocytic differentiation of HL-60 cells (Boss, G. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7174-7178). We have selected HL-60 cells resistant to nitroprusside-induced differentiation as assessed by acquisition of the OKM-1 antigen, reduction of nitro blue tetrazolium, and morphologic maturation. The variant cells were also resistant to differentiation induced by sodium nitrite and two cGMP analogs but still differentiated in response to other inducing agents such as dimethyl sulfoxide and cAMP analogs and showed the same changes in c-myc and c-fos expression in response to the latter drugs as occurred in parental cells. We studied the early steps of the NO/cGMP signal transduction pathway in the variant cells and found that basal and nitroprusside-stimulated guanylate cyclase activity was similar in parental and variant cell extracts and that nitroprusside increased the intracellular cGMP concentration to the same extent in parental and variant cells. As part of these studies we found that HL-60 cells expressed only alpha 2 and beta 2 guanylate cyclase mRNA; the abundance of these two mRNA species was similar in parental and variant cells. Neither nitroprusside nor 8-bromo-cGMP changed the intracellular calcium concentration in parental or variant cells. The data suggest that the defect in the variant cells is after guanylate cyclase activation in the NO/cGMP transduction pathway and that the cGMP and cAMP transduction pathways operate independently in inducing differentiation of HL-60 cells.

Murine erythroleukemia (MEL) cells deficient in cAMP-dependent protein kinase (A-kinase) activity are impaired in chemically induced differentiation (Pilz, R. B., Eigenthaler, M., and Boss, G. R. (1992) J. Biol. Chem. 267, 16161-16167). We identified by two-dimensional polyacrylamide gel electrophoresis two low molecular weight proteins (referred to as pp 21-1 and 21-2) that were phosphorylated when parental MEL cells, but not A-kinase-deficient MEL cells, were treated with the membrane-permeable cAMP analog 8-bromo-cAMP. We showed that pp 21-1 and 21-2: (a) were direct A-kinase substrates; (b) bound GTP; and (c) belonged to the ras superfamily of proteins. The only ras-related proteins that are clearly A-kinase substrates both in vitro and in vivo are Rap 1A and 1B while H- and K-Ras can be A-kinase substrates in vitro; we showed by immunological methods, phosphopeptide mapping, and migration on two-dimensional gels that pp 21-1 and 21-2 were not identical to one of these four proteins. We found a 3-fold increase of 32PO4 incorporation into pp 21-2 in hexamethylene bisacetamide-treated parental MEL cells which was not secondary to an increase in pp 21-2 protein but appeared secondary to increased phosphorylation of pp 21-2 by A-kinase. Thus, pp 21-1 and 21-2 are either new ras-related proteins or are previously identified ras-related proteins not known to be A-kinase substrates, and increased phosphorylation of pp 21-2 occurs during differentiation of MEL cells.

Auron-Misheil-Therapy (AMT) is currently under development as an anticancer treatment. The aim of the present study was the identification of possible effects and properties of AMT with regard to human skin. The study consisted of three experimental phases. Phase 1 assessed the effects of AMT on the viability of 2-D evaluated and accredited cell cultures of three cell types of human skin, namely: keratinocytes, melanocytes and fibroblasts. Three separate assays were used in this phase. Phase 2 was designed to clarify the effects of AMT on cell viability to investigate the mode of action of AMT. Two possible modes of action were investigated: proliferation inhibition and apoptosis. There was one assay to assess proliferation, and two independent assays to assess apoptosis. The third phase assessed the effects of AMT on two different types of 3-D skin models, an ex vivo model and a de novo reconstituted model. In the phase 1 tests, reduction of cell viability by AMT was demonstrated in all three cell types. In phase 2, the cell proliferation assay showed decreased proliferation rates in the presence of AMT in three out of four cell populations. In phase 3, histochemical investigations with 3-D models indicated that AMT induces desquamation, most likely as the result of apoptosis of epidermal cells. The experiments generally showed that AMT does not harm human skin at concentrations up to 3%.

Auron-Misheil-Therapy (AMT) is a defined but unique combination of approved pharmaceuticals. It consists of insulin, chlorpheniramine and an aqueous camomile extract, and it has been successfully applied clinically in late-stage cancer patients. The purpose of this study was to elucidate the anti-tumor efficacy of AMT in a validated murine renal cell carcinoma animal model (RENCA). There were two independent studies; each animal group consisted of 16 mice. During a 6-week pretreatment period, vehicle (group A) and AMT (1.6 mg/kg/d) (group B) were administered once daily in a 5 days/week schedule either intramuscularly or subcutaneously. Tumor challenge at day 0 was followed by a 3-week treatment period (either vehicle or AMT once daily intramuscularly for 21 days consecutively). In study 2 the AMT dosage was increased up to 4-fold by doubling individual doses and switching to a twice daily schedule. The injections were all intramuscular. With the exception of group D, a six-week pretreatment period preceded the tumor challenge at day 0. Tumor challenge was followed by a 3-week treatment period (vehicle, AMT at either 3.2 mg/kg/d) (group A) or 6.4 mg/kg/d (group B), or AMT0, an AMT preparation which does not stimulate IL-6 secretion (6.4 mg/kg/d, group C) continuously for 21 days. AMT administration for group D (6.4 mg/kg/d) was limited to the treatment period from day 1 to 21. All mice were sacrificed 21 days after tumour transplantation. AMT administration was safe and well tolerated, and significantly reduced primary tumor volume in pretreated animals. The effective route of application was intramuscular, with dose escalation resulting in an improved anti-tumor effect. This is the first demonstration of a significant anti-tumorigenic effect of AMT in a validated tumor model.

SummaryLeukocyte adhesion deficiency-III (LAD-III) also called leukocyte adhesion deficiency-1/variant (LAD1v) is a rare congenital disease caused by defective integrin activation of leukocytes and platelets. Patients with LAD-III present with non-purulent infections and increased bleeding symptoms. We report on a novel integrin-dependent platelet dys-function in two brothers with LAD-III syndrome caused by a homozygous mutation 1717C>T in the FERMT3 gene leading to a premature stop codon R573X in the focal adhesion protein kindlin-3. Stimulation of patients´ platelets with all used agonists resulted in a severely decreased binding of soluble fibrinogen indicating a defect in inside-out activation of the integrin αIIbβ3 (GPIIb/IIIa). Patients´ platelets did not respond to the α2β1-integrin agonist aggretin-A at all. Our data on granula secretion indicate for the first time that the thrombin receptor PAR-4 but not PAR-1 may be important in integrin-triggered granule secretion in response to thrombin. In contrast, collagen mediated platelet granule secretion was not affected in LAD-III-patients. Thus, integrin-signalling may be not essential in collagen-induced granule secretion. The patients’ peripheral blood mononuclear cells showed a severe loss of adhesion capacity to VCAM-1 and to endothelial cells compared to cells from healthy donors. Rap-1 activation after PMA stimulation could be observed in controls´ but not in patients´ cells. After haematogenesis stem cell transplantation (HSCT) the brothers showed no symptoms of bleeding or immunodeficiency and the integrin-dependent platelet and leukocyte functions normalised.

This pilot study of Auron Misheil Therapy (AMT) in women with advanced cervical cancer was an open-label, single arm study to collect initial safety, efficacy, and quality of life data. Fifteen women with stage IIIb or IVa cervical cancer were given twice daily intramuscular injections of AMT (insulin, chlorpheniramine and camomile extract) for 3 months. Objective tumor response was evaluated using CT scans and analyzing the data according to the WHO RECIST criteria. Clinical Benefit Response (CBR) was assessed using a composite score comprising Karnovsky performance status, pain intensity and body weight. Safety and tolerability parameters were monitored. Quality of life was evaluated using the European Organization for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC C-30). Eight out of 15 patients were rated as clinical responders (CBR) at 12 weeks. One patient had a partial response and 11 stable disease (WHO RECIST criteria). AMT was well tolerated. An initial analysis showed improvement in quality of life (EORTC C-30). Promising response rates, early indications of improved quality of life, and no significant safety issues mean that the second, randomized phase of the trial can be initiated with a longer treatment duration. Patients with advanced cervical cancer showed positive clinical responses to Auron Misheil Therapy. The treatment was well tolerated, with indications of improved quality of life.

Auron-Misheil-Therapy (AMT) consisting of aqueous camomile extract supplemented with calcium, vitamins, the antihistamine chlorpheniramine and human insulin is under development as anti-cancer treatment. AMT was preclinically investigated in tumour cell lines and tumour xenografts to guide clinical phase I/II studies. AMT was tested against 56 human tumour cell lines, in a clonogenic assay in 98 patient-derived xenografts and in in vivo studies. AMT showed in vitro cytotoxic activity with highest susceptibility in cervical cancer, glioblastoma and colon cancers. In the clonogenic assay, anti- cancer activity of AMT was most active in cervical and uterine tumours, in colon cancer, glioblastoma, leukaemia, melanoma and pancreatic cancer. In vivo, AMT showed slight activity in tumour xenograft models of colon and mammary cancer. It also showed immune stimulatory effects by induction of IL-6- and TNF-alpha secretion in human PBMCs. The immune stimulatory potential of AMT, together with slight anti-tumour efficacy observed in the present study, indicates a role of AMT in tumour therapy.

Acute myeloid leukemia (AML) shows malignant behavior through the ability of immature cells to circulate in blood and to invade peripheral tissues. Whereas binding of human AML cells to endothelial cells (ECs) through E-selectin has been shown to occur using classical adhesion assays, little is known about the ability of endothelial P-selectin to support this process. We therefore characterized the ability of AML blasts and KG-1 cells to bind to endothelial selectin type ligands. Flow cytometry revealed that, in addition to various integrin adhesion receptors, AML cells regularly express the P-selectin glycoprotein ligand (PSGL)-1, a ligand for P- and E-selectin on ECs. In parallel flow chambers, AML cells both rolled and adhered to TNF-alpha pretreated human umbilical vein endothelial cells (HUVECs). Pretreatment of HUVECs with anti-P- or anti-E-selectin function blocking antibodies significantly reduced both, rolling and subsequent arrest of primary AML cells. Intravital microscopy of i.v. injected fluorescence-labeled KG-1 cells into P-selectin deficient or wild type mice confirmed a significant role of endothelial P-selectin in the binding of human primary AML cells to ECs also in vivo. Thus, the currently available data suggest a role of P- and E-selectin in coordinated circulation of AML cells. Thus, P- or E-selectin mediated adhesion of AML cells may provide a target for the development anti-leukemic therapies.

Ras-associated protein 1 (Rap1), a small GTPase, attracted attention because of its involvement in several aspects of cell adhesion, including integrin- and cadherin-mediated adhesion. Yet, the role of Rap1 genes and of Rap1 effectors for angiogenesis has not been investigated. Human umbilical vein endothelial cells (HUVECs) express Rap1a and Rap1b mRNA. To determine the contribution of Rap1 activity for angiogenesis, we overexpressed Rap1GAP1, a GTPase-activating protein that inhibits Rap1 activity. Overexpression of Rap1GAP1 significantly blocked angiogenic sprouting and tube-forming activity of HUVECs as well as migration and integrin-dependent adhesion. Silencing of Rap1a, Rap1b, or both significantly blocked HUVECs sprouting under basal and basic fibroblast growth factor-stimulated conditions and reduced HUVEC migration and integrin-dependent adhesion. We found that Rap1a and Rap1b are essential for the conformational activation of β1-integrins in endothelial cells. Furthermore, silencing of Rap1a and Rap1b prevented phosphorylation of tyrosine 397 in focal adhesion kinase (FAK) and vascular endothelial growth factor-induced Akt1-activation. Rap1a−/−-deficient and Rap1a+/− heterozygote mice displayed reduced neovascularization after hind limb ischemia compared with wild-type mice. Silencing of RAPL significantly blocked the Rap1-induced sprouting of HUVECs, suggesting that the angiogenic activity of Rap1 is partly mediated by RAPL. Our data demonstrate a critical role of Rap1 in the regulation of β1-integrin affinity, adhesion, and migration in endothelial cells and in postnatal neovascularization.

The 21-kD protein Ras of the low-molecular-weight GTP-binding (LMWG) family plays an important role in transduction of extracellular signals. Ras functions as a ‘molecular switch’ in transduction of signals from the membrane receptors of many growth factors, cytokines, and other second messengers to the cell nucleus. Numerous studies have shown that in multiple malignant tumors and hematopoietic malignancies, faulty signal transduction via the Ras pathway plays a key role in tumorigenesis. In this work, a non-radioactive assay was used to quantify Ras activity in hematologic malignancies. Ras activation was measured in six different cell lines and 24 patient samples, and sequence analysis of N- and K-ras was performed. The 24 patient samples comprised of seven acute myelogenous leukemia (AML) samples, five acute lymphocytic leukemia (ALL) samples, four myeloproliferative disease (MPD) samples, four lymphoma samples, four juvenile myelomonocytic leukemia (JMML) samples, and WBC from a healthy donor. The purpose of this study was to compare Ras activity determined by percentage of Ras-GTP with the mutational status of the Ras gene in the hematopoietic cells of the patients. Mutation analysis revealed ras mutations in two of the seven AML samples, one in codon 12 and one in codon 61; ras mutations were also found in two of the four JMML samples, and in one of the four lymphoma samples (codon 12). We found a mean Ras activation of 23.1% in cell lines with known constitutively activating ras mutations, which was significantly different from cell lines with ras wildtype sequence (Ras activation of 4.8%). Two of the five activating ras mutations in the patient samples correlated with increased Ras activation. In the other three samples, Ras was probably activated through “upstream” or “downstream” mechanisms. Electronic supplementary material The online version of this article (doi:10.1007/s00277-008-0593-6) contains supplementary material, which is available to authorized users.