Efficacy and Safety of the MDM2-p53 Antagonist Brigimadlin (BI 907828) in Patients with Advanced Biliary Tract Cancer: A Case Series.

Background: In patients with advanced biliary tract cancer (BTC), first-line chemotherapy plus immunotherapy has improved outcomes; however, second-line options that reflect the disease's molecular heterogeneity are still needed. One emerging target is MDM2, amplified in ~5-8% of BTC cases. Methods: This is a subset analysis of two ongoing Phase Ia/Ib trials assessing patients treated with brigimadlin (BI 907828; a highly potent, oral MDM2-p53 antagonist) ± ezabenlimab (PD-1 inhibitor) ± BI 754111 (anti-LAG-3; n = 1). Results: Results from 12 patients with BTC are shown (monotherapy: n = 6/combination: n = 6). Six patients achieved partial response (monotherapy: n = 2/combination: n = 4), four had stable disease; responses were durable. Brigimadlin had a manageable safety profile. Seven patients had dose reductions due to adverse events, but no treatment-related adverse events led to treatment discontinuation. Conclusion: Brigimadlin demonstrated anti-tumor activity in patients with advanced MDM2-amplified BTC, and warrants further investigation.

Biliary tract carcinoma (BTC) is a cancer that affects the bile ducts which are part of the digestive system. Usually, the first treatment for advanced BTC (ie cannot be removed surgically and/or has spread) is chemotherapy in combination with immunotherapy. However, if chemotherapy does not work, or stops working, there are few treatment options available in second-line. Accordingly, intensive research is ongoing to try and find effective drugs. One potential medicine, called brigimadlin (or BI 907828), is a tablet that activates a molecule in tumor cells called p53. The normal function of p53 is to kill cells when they first start to become cancerous. However, if p53 is turned off by genetic mutations, or other mechanisms, then cancer can develop. Although p53 is rarely mutated in BTC tumors, it is inactivated by another molecule called MDM2 which is usually present at abnormally high levels in BTC. Brigimadlin prevents interaction between MDM2 and p53. This activates p53 and causes the cancer to die. Two clinical trials are currently assessing brigimadlin in a range of cancers, including BTC, with the aim of identifying a safe dose that can be examined in more detail in larger trials. So far, 12 patients with BTC have been treated. The patients' tumors significantly shrank in six of these patients and remained stable in a further four patients. Side effects were as expected and could be tolerated by pausing treatment or lowering the dose. These results show that brigimadlin should be tested further in patients with advanced BTC.

Brightline-2: a phase IIa/IIb trial of brigimadlin (BI 907828) in advanced biliary tract cancer, pancreatic ductal adenocarcinoma or other solid tumors.

Mouse double minute 2 homolog (MDM2) is a key negative regulator of the tumor suppressor p53. Blocking the MDM2-p53 interaction, and restoring p53 function, is therefore a potential therapeutic strategy in MDM2-amplified, TP53 wild-type tumors. MDM2 is amplified in several tumor types, including biliary tract cancer (BTC), pancreatic ductal adenocarcinoma (PDAC), lung adenocarcinoma and bladder cancer, all of which have limited treatment options and poor patient outcomes. Brigimadlin (BI 907828) is a highly potent MDM2-p53 antagonist that has shown promising activity in preclinical and early-phase clinical studies. This manuscript describes the rationale and design of an ongoing phase IIa/IIb Brightline-2 trial evaluating brigimadlin as second-line treatment for patients with advanced/metastatic BTC, PDAC, lung adenocarcinoma, or bladder cancer.

Brightline-2: a phase IIa/IIb trial of brigimadlin (BI 907828) in advanced BTC, PDAC, or other solid tumorsIn some types of cancer, including cancers of the bile duct, pancreas, bladder and lung, the number of copies of a gene called MDM2 is abnormally increased (MDM2 amplification). MDM2 usually regulates p53, a protein that stops cancer cells from growing uncontrollably. When MDM2 is amplified, the cell makes too much of the MDM2 protein, which prevents p53 from stopping cancer growth. Blocking the interaction between MDM2 and p53 may allow p53 to do its job again and stop cancer cells from growing.Brightline-2 is a clinical trial that is currently in progress. This trial is assessing the efficacy and safety of an investigational drug, brigimadlin (or BI 907828), in patients with selected advanced or metastatic cancers. To be included, patients must have advanced biliary tract cancer, pancreatic ductal adenocarcinoma, bladder cancer, or lung adenocarcinoma. The tumor must show amplification of MDM2 when tested by a laboratory. Patients will take a 45 mg tablet of brigimadlin by mouth, once every 3 weeks. In this trial, researchers are investigating the ability of the drug to shrink tumors, the side effects of the drug, and the impact of the drug on a patients' quality of life.The goal of this trial is to assess the potential of brigimadlin as a new treatment option for patients with advanced biliary tract cancer, pancreatic ductal adenocarcinoma, bladder cancer, or lung adenocarcinoma.Clinical Trial Registration: NCT05512377 (ClinicalTrials.gov).

The MDM2-p53 Antagonist Brigimadlin (BI 907828) in Patients with Advanced or Metastatic Solid Tumors: Results of a Phase Ia, First-in-Human, Dose-Escalation Study.

Brigimadlin (BI 907828) is an oral MDM2-p53 antagonist that has shown encouraging antitumor activity in vivo. We present phase Ia results from an open-label, first-in-human, phase Ia/Ib study investigating brigimadlin in patients with advanced solid tumors (NCT03449381). Fifty-four patients received escalating doses of brigimadlin on day 1 of 21-day cycles (D1q3w) or days 1 and 8 of 28-day cycles (D1D8q4w). Based on dose-limiting toxicities during cycle 1, the maximum tolerated dose was selected as 60 mg for D1q3w and 45 mg for D1D8q4w. The most common treatment-related adverse events (TRAE) were nausea (74.1%) and vomiting (51.9%); the most common grade ≥3 TRAEs were thrombocytopenia (25.9%) and neutropenia (24.1%). As evidence of target engagement, time- and dose-dependent increases in growth differentiation factor 15 levels were seen. Preliminary efficacy was encouraging (11.1% overall response and 74.1% disease control rates), particularly in patients with well-differentiated or dedifferentiated liposarcoma (100% and 75% disease control rates, respectively). SIGNIFICANCE: We report phase Ia data indicating that the oral MDM2-p53 antagonist brigimadlin has a manageable safety profile and shows encouraging signs of efficacy in patients with solid tumors, particularly those with MDM2-amplified advanced/metastatic well-differentiated or dedifferentiated liposarcoma. Further clinical investigation of brigimadlin is ongoing. See related commentary by Italiano, p. 1765. This article is highlighted in the In This Issue feature, p. 1749.

Broad spectrum of regorafenib activity on mutant KIT and absence of clonal selection in gastrointestinal stromal tumor (GIST): correlative analysis from the GRID trial.

BACKGROUND: In the phase 3 GRID trial, regorafenib improved progression-free survival (PFS) independent of KIT mutations in exons 9 and 11. In this retrospective, exploratory analysis of the GRID trial, we investigated whether a more comprehensive KIT mutation analysis could identify mutations that impact treatment outcome with regorafenib and a regorafenib-induced mutation pattern. METHODS: Archived tumor samples, collected at any time prior to enrollment in GRID, were analyzed by Sanger sequencing (n = 102) and next-generation sequencing (FoundationONE; n = 47). Plasma samples collected at baseline were analyzed by BEAMing (n = 163) and SafeSEQ (n = 96). RESULTS: In archived tumor samples, 67% (68/102) had a KIT mutation; 61% (62/102) had primary KIT mutations (exons 9 and 11) and 12% (12/102) had secondary mutations (exons 13, 14, 17, and 18). At baseline, 81% of samples (78/96) had KIT mutations by SafeSEQ, including the M541L polymorphism (sole event in 6 patients). Coexisting mutations in other oncogenes were rare, as were mutations in PDGFR, KRAS, and BRAF. Regorafenib showed PFS benefit across all primary and secondary KIT mutational subgroups examined. Available patient-matched samples taken at baseline and end of treatment (n = 41; SafeSEQ), revealed heterogeneous KIT mutational changes with no specific mutation pattern emerging upon regorafenib treatment. CONCLUSION: These data support the results of the GRID trial, and suggest that patients may benefit from regorafenib in the presence of KIT mutations and without the selection of particular mutation patterns that confer resistance. The study was not powered to address biomarker-related questions, and the results are exploratory and hypothesis-generating.

Phase 1 dose-escalation and pharmacokinetic study of regorafenib in paediatric patients with recurrent or refractory solid malignancies.

BACKGROUND: This phase 1 study evaluated safety, pharmacokinetics (PK), maximum tolerated dose (MTD), and antitumour activity of regorafenib in paediatric patients with solid tumours. PATIENTS AND METHODS: Patients (aged 6 months to <18 years) with recurrent/refractory solid tumours received oral regorafenib once daily for 3 weeks on/1 week off. The starting dose (60 mg/m2) was derived from an adult physiology-based PK model and scaled to children; dose escalation was followed by safety expansion of the MTD cohort. Treatment-emergent adverse events (TEAEs) were evaluated using National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. Regorafenib PK was evaluated using a population PK model. RESULTS: Forty-one patients (median age 13 years) received regorafenib (four cohorts: 60-93 mg/m2). Five of 23 evaluable patients experienced dose-limiting toxicities (Grade 4 thrombocytopenia, Grade 3 maculopapular rash, pyrexia, hypertension, and exfoliative dermatitis [each n = 1]). The MTD was defined as 82 mg/m2. The most common Grade ≥3 drug-related TEAE was thrombocytopenia (10%). The incidence and severity of hypertension, diarrhoea, fatigue, hypothyroidism, and hand-foot skin reaction were lower than reported in adults. Regorafenib exposure increased with dose, with substantial overlap because of moderate-to-high interpatient variability. One patient with rhabdomyosarcoma experienced an unconfirmed partial response; 15 patients had stable disease, five for >16 weeks. CONCLUSIONS: The recommended phase 2 dose of single-agent regorafenib in paediatric patients with solid malignancies is 82 mg/m2. Regorafenib demonstrated acceptable tolerability and preliminary antitumour activity, supporting further investigation in paediatric patients. CLINICAL TRIAL NUMBER: NCT02085148.

Biomarkers Associated With Response to Regorafenib in Patients With Hepatocellular Carcinoma.

BACKGROUND & AIMS: In a phase 3 trial (RESORCE), regorafenib increased overall survival compared with placebo in patients with hepatocellular carcinoma (HCC) previously treated with sorafenib. In an exploratory study, we analyzed plasma and tumor samples from study participants to identify genetic, microRNA (miRNA), and protein biomarkers associated with response to regorafenib. METHODS: We obtained archived tumor tissues and baseline plasma samples from patients with HCC given regorafenib in the RESORCE trial. Baseline plasma samples from 499 patients were analyzed for expression of 294 proteins (DiscoveryMAP) and plasma samples from 349 patients were analyzed for levels of 750 miRNAs (miRCURY miRNA PCR). Tumor tissues from 7 responders and 10 patients who did not respond (progressors) were analyzed by next-generation sequencing (FoundationOne). Forty-six tumor tissues were analyzed for expression patterns of 770 genes involved in oncogenic and inflammatory pathways (PanCancer Immune Profiling). Associations between plasma levels of proteins and miRNAs and response to treatment (overall survival and time to progression) were evaluated using a Cox proportional hazards model. RESULTS: Decreased baseline plasma concentrations of 5 of 266 evaluable proteins (angiopoietin 1, cystatin B, the latency-associated peptide of transforming growth factor beta 1, oxidized low-density lipoprotein receptor 1, and C-C motif chemokine ligand 3; adjusted P ≤ .05) were significantly associated with increased overall survival time after regorafenib treatment. Levels of these 5 proteins, which have roles in inflammation and/or HCC pathogenesis, were not associated with survival independently of treatment. Only 20 of 499 patients had high levels and a reduced survival time. Plasma levels of α-fetoprotein and c-MET were associated with poor outcome (overall survival) independently of regorafenib treatment only. We identified 9 plasma miRNAs (MIR30A, MIR122, MIR125B, MIR200A, MIR374B, MIR15B, MIR107, MIR320, and MIR645) whose levels significantly associated with overall survival time with regorafenib (adjusted P ≤ .05). Functional analyses of these miRNAs indicated that their expression level associated with increased overall survival of patients with tumors of the Hoshida S3 subtype. Next-generation sequencing analyses of tumor tissues revealed 49 variants in 27 oncogenes or tumor suppressor genes. Mutations in CTNNB1 were detected in 3 of 10 progressors and VEGFA amplification in 1 of 7 responders. CONCLUSION: We identified expression patterns of plasma proteins and miRNAs that associated with increased overall survival times of patients with HCC following treatment with regorafenib in the RESORCE trial. Levels of these circulating biomarkers and genetic features of tumors might be used to identify patients with HCC most likely to respond to regorafenib. ClinicalTrials.gov number NCT01774344. NCBI GEO accession numbers: mRNA data (NanoString): GSE119220; miRNA data (Exiqon): GSE119221.

Addition of radium-223 to abiraterone acetate and prednisone or prednisolone in patients with castration-resistant prostate cancer and bone metastases (ERA 223): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Abiraterone acetate plus prednisone or prednisolone improves progression-free survival and overall survival in patients with metastatic castration-resistant prostate cancer. Radium-223 improves overall survival and delays the onset of symptomatic skeletal events in patients with castration-resistant prostate cancer and bone metastases. We assessed concurrent treatment with abiraterone acetate plus prednisone or prednisolone and radium-223 in such patients. METHODS: We did a randomised, double-blind, placebo-controlled, phase 3 trial at 165 oncology and urology centres in 19 countries. Eligible patients were aged 18 years or older, and had histologically confirmed, progressive, chemotherapy-naive, asymptomatic or mildly symptomatic castration-resistant prostate cancer and bone metastases, Eastern Cooperative Oncology Group performance status of 0 or 1, life expectancy of at least 6 months, and adequate haematological, renal, and liver function. Participants were randomly assigned (1:1) according to a permuted block design (block size 4) via interactive response technology to receive up to six intravenous injections of radium-223 (55 kBq/kg) or matching placebo once every 4 weeks. All patients were also scheduled to receive oral abiraterone acetate 1000 mg once daily plus oral prednisone or prednisolone 5 mg twice daily during and after radium-223 or placebo treatment. The primary endpoint was symptomatic skeletal event-free survival, which was assessed in the intention-to-treat population. Safety analyses were done in all patients who received at least one dose of any study drug. This trial is registered with ClinicalTrials.gov, number NCT02043678. Enrolment has been completed, and follow-up is ongoing. FINDINGS: Between March 30, 2014, and Aug 12, 2016, 806 patients were randomly assigned to receive radium-223 (n=401) or placebo (n=405) in addition to abiraterone acetate plus prednisone or prednisolone. The study was unblinded prematurely, on Nov 17, 2017, after more fractures and deaths were noted in the radium-223 group than in the placebo group (in an unplanned ad-hoc analysis), but all patients had completed radium-223 or placebo before this date. At the primary analysis (data cutoff Feb 15, 2018), 196 (49%) of 401 patients in radium-223 group had had at least one symptomatic skeletal event or died, compared with 190 (47%) of 405 patients in the placebo group (median follow-up 21·2 months [IQR 17·0-25·8]). Median symptomatic skeletal event-free survival was 22·3 months (95% CI 20·4-24·8) in the radium-223 group and 26·0 months (21·8-28·3) in the placebo group (hazard ratio 1·122 [95% CI 0·917-1·374]; p=0·2636). Fractures (any grade) occurred in 112 (29%) of 392 patients in the radium-223 group and 45 (11%) of 394 patients in the placebo group. The most common grade 3-4 treatment-emergent adverse events were hypertension (43 [11%] patients in the radium-223 group vs 52 [13%] patients in the placebo group), fractures (36 [9%] vs 12 [3%]) and increased alanine aminotransferase concentrations (34 [9%] vs 28 [7%]). Serious treatment-emergent adverse events occurred in 160 (41%) patients in the radium-223 group and 155 (39%) in the placebo group. Treatment-related deaths occurred in two (1%) patients in the radium-223 group (acute myocardial infarction and interstitial lung disease) and one (<1%) in the placebo group (arrhythmia). INTERPRETATION: The addition of radium-223 to abiraterone acetate plus prednisone or prednisolone did not improve symptomatic skeletal event-free survival in patients with castration-resistant prostate cancer and bone metastases, and was associated with an increased frequency of bone fractures compared with placebo. Thus, we do not recommend use of this combination. FUNDING: Bayer.

Identification and characterisation of carnostatine (SAN9812), a potent and selective carnosinase (CN1) inhibitor with in vivo activity.

Carnosinase 1 (CN1) has been postulated to be a susceptibility factor for developing diabetic nephropathy (DN). Although its major substrate, carnosine, is beneficial in rodent models of DN, translation of these findings to humans has been hampered by high CN1 activity in human serum resulting in rapid degradation of carnosine. To overcome this hurdle, we screened a protease-directed small-molecule library for inhibitors of human recombinant CN1. We identified SAN9812 as a potent and highly selective inhibitor of CN1 activity with a Ki of 11 nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30 mg/kg SAN9812 led to a sustained reduction in circulating CN1 activity in human CN1 transgenic (TG) mice. Simultaneous administration of carnosine and SAN9812 increased carnosine levels in plasma and kidney by up to 100-fold compared to treatment-naïve CN1-overexpressing mice. To our knowledge, this is the first study reporting on a potent and selective CN1 inhibitor with in vivo activity. SAN9812, also called carnostatine, may be used to increase renal carnosine concentration as a potential therapeutic modality for renal diseases linked to glycoxidative conditions.

Phase II Studies with Refametinib or Refametinib plus Sorafenib in Patients with RAS-Mutated Hepatocellular Carcinoma.

Purpose: Refametinib, an oral MEK inhibitor, has demonstrated antitumor activity in combination with sorafenib in patients with RAS-mutated hepatocellular carcinoma (HCC). Two phase II studies evaluated the efficacy of refametinib monotherapy and refametinib plus sorafenib in patients with RAS-mutant unresectable or metastatic HCC.Patients and Methods: Eligible patients with RAS mutations of cell-free circulating tumor DNA (ctDNA) determined by beads, emulsion, amplification, and magnetics technology received twice-daily refametinib 50 mg ± sorafenib 400 mg. Potential biomarkers were assessed in ctDNA via next-generation sequencing (NGS).Results: Of 1,318 patients screened, 59 (4.4%) had a RAS mutation, of whom 16 received refametinib and 16 received refametinib plus sorafenib. With refametinib monotherapy, the objective response rate (ORR) was 0%, the disease control rate (DCR) was 56.3%, overall survival (OS) was 5.8 months, and progression-free survival (PFS) was 1.9 months. With refametinib plus sorafenib, the ORR was 6.3%, the DCR was 43.8%, OS was 12.7 months, and PFS was 1.5 months. In both studies, time to progression was 2.8 months. Treatment-emergent toxicities included fatigue, hypertension, and acneiform rash. Twenty-seven patients had ctDNA samples available for NGS. The most frequently detected mutations were in TERT (63.0%), TP53 (48.1%), and ß-catenin (CTNNB1; 37.0%).Conclusions: Prospective testing for RAS family mutations using ctDNA was a feasible, noninvasive approach for large-scale mutational testing in patients with HCC. A median OS of 12.7 months with refametinib plus sorafenib in this small population of RAS-mutant patients may indicate a synergistic effect between sorafenib and refametinib-this preliminary finding should be further explored. Clin Cancer Res; 24(19); 4650-61. ©2018 AACR.

Antitumor effects of regorafenib and sorafenib in preclinical models of hepatocellular carcinoma.

The purpose of this study was to investigate the antitumor activity of regorafenib and sorafenib in preclinical models of HCC and to assess their mechanism of action by associated changes in protein expression in a HCC-PDX mouse model. Both drugs were administered orally once daily at 10 mg/kg (regorafenib) or 30 mg/kg (sorafenib), which recapitulate the human exposure at the maximally tolerated dose in mice. In a H129 hepatoma model, survival times differed significantly between regorafenib versus vehicle (p=0.0269; median survival times 36 vs 27 days), but not between sorafenib versus vehicle (p=0.1961; 33 vs 28 days). Effects on tumor growth were assessed in 10 patient-derived HCC xenograft (HCC-PDX) models. Significant tumor growth inhibition was observed in 8/10 models with regorafenib and 7/10 with sorafenib; in four models, superior response was observed with regorafenib versus sorafenib which was deemed not to be due to lower sorafenib exposure. Bead-based multiplex western blot analysis was performed with total protein lysates from drug- and vehicle-treated HCC-PDX xenografts. Protein expression was substantially different in regorafenib- and sorafenib-treated samples compared with vehicle. The pattern of upregulated proteins was similar with both drugs and indicates an activated RAF/MEK/ERK pathway, but more proteins were downregulated with sorafenib versus regorafenib. Overall, both regorafenib and sorafenib were effective in mouse models of HCC, although several cases showed better regorafenib activity which may explain the observed efficacy of regorafenib in sorafenib-refractory patients.

Phase I/II Study of Refametinib (BAY 86-9766) in Combination with Gemcitabine in Advanced Pancreatic cancer.

BACKGROUND: Activating KRAS mutations are reported in up to 90% of pancreatic cancers. Refametinib potently inhibits MEK1/2, part of the MAPK signaling pathway. This phase I/II study evaluated the safety and efficacy of refametinib plus gemcitabine in patients with advanced pancreatic cancer. METHODS: Phase I comprised dose escalation, followed by phase II expansion. Refametinib and gemcitabine plasma levels were analyzed for pharmacokinetics. KRAS mutational status was determined from circulating tumor DNA. RESULTS: Ninety patients overall received treatment. The maximum tolerated dose was refametinib 50 mg twice daily plus standard gemcitabine (1000 mg/m2 weekly). The combination was well tolerated, with no pharmacokinetic interaction. Treatment-emergent toxicities included thrombocytopenia, fatigue, anemia, and edema. The objective response rate was 23% and the disease control rate was 73%. Overall response rate, disease control rate, progression-free survival, and overall survival were higher in patients without detectable KRAS mutations (48% vs. 28%, 81% vs. 69%, 8.8 vs. 5.3 months, and 18.2 vs. 6.6 months, respectively). CONCLUSION: Refametinib plus gemcitabine was well tolerated, with a promising objective response rate, and had an acceptable safety profile and no pharmacokinetic interaction. There was a trend towards improved outcomes in patients without detectable KRAS mutations that deserves future investigation.

Analysis of circulating DNA and protein biomarkers to predict the clinical activity of regorafenib and assess prognosis in patients with metastatic colorectal cancer: a retrospective, exploratory analysis of the CORRECT trial.

BACKGROUND: Tumour mutational status is an important determinant of the response of metastatic colorectal cancer to targeted treatments. However, the genotype of the tissue obtained at the time of diagnosis might not accurately represent tumour genotype after multiple lines of treatment. This retrospective exploratory analysis investigated the clinical activity of regorafenib in biomarker subgroups of the CORRECT study population defined by tumour mutational status or plasma protein levels. METHODS: We used BEAMing technology to identify KRAS, PIK3CA, and BRAF mutations in DNA obtained from the plasma of 503 patients with metastatic colorectal cancer who enrolled in the CORRECT trial. We quantified total human genomic DNA isolated from plasma samples for 503 patients using a modified version of human long interspersed nuclear element-1 (LINE-1) quantitive real-time PCR. We also measured the concentration of 15 proteins of interest-angiopoietin 2, interleukin 6, interleukin 8, placental growth factor, soluble TIE-1, soluble VEGFR1, VEGF-A, VEGF-C, VEGF-D, VEGF-A isoform 121, bone morphogenetic protein 7, macrophage colony-stimulating factor, stromal cell-derived factor-1, tissue inhibitor of metalloproteinase 2, and von Willebrand factor-in plasma samples from 611 patients. We did correlative analyses of overall survival and progression-free survival in patient subgroups based on mutational status, circulating DNA concentration, and protein concentrations. The CORRECT trial was registered with ClinicalTrials.gov, number NCT01103323. FINDINGS: Tumour-associated mutations were readily detected with BEAMing of plasma DNA, with KRAS mutations identified in 349 (69%) of 503 patients, PIK3CA mutations in 84 (17%) of 503 patients, and BRAF mutations in 17 (3%) of 502 patients. We did not do correlative analysis based on BRAF genotype because of the low mutational frequency detected for this gene. Some of the most prevalent individual hot-spot mutations we identified included: KRAS (KRAS G12D, 116 [28%] of 413 mutations; G12V, 72 [17%]; and G13D, 67 [16%]) and PIK3CA (PIK3CA E542K, 27 [30%] of 89 mutations; E545K, 37 [42%]; and H1047R, 12 [14%]). 41 (48%) of 86 patients who had received anti-EGFR therapy and whose archival tumour tissue DNA was KRAS wild-type in BEAMing analysis were identified as having KRAS mutations in BEAMing analysis of fresh plasma DNA. Correlative analyses suggest a clinical benefit favouring regorafenib across patient subgroups defined by KRAS and PIK3CA mutational status (progression-free survival with regorafenib vs placebo: hazard ratio [HR] 0·52, 95% CI 0·35-0·76 for KRAS wild-type; HR 0·51, 95% CI 0·40-0·65 for KRAS mutant [KRAS wild type vs mutant, pinteraction=0·74]; HR 0·50, 95% CI 0·40-0·63 for PIK3CA wild-type; HR 0·54, 95% CI 0·32-0·89 for PIK3CA mutant [PIK3CA wild-type vs mutant, pinteraction=0·85]) or circulating DNA concentration (progression-free survival with regorafenib vs placebo: HR 0·53, 95% CI 0·40-0·71, for low circulating DNA concentrations; HR 0·52, 95% CI 0·40-0·70, for high circulating DNA concentrations; low vs high circulating DNA, pinteraction=0·601). With the exception of von Willebrand factor, assessed with the median cutoff method, plasma protein concentrations were also not associated with regorafenib activity in terms of progression-free survival. In univariable analyses, the only plasma protein that was associated with overall survival was TIE-1, high concentrations of which were associated with longer overall survival compared with low TIE-1 concentrations. This association was not significant in multivariable analyses. INTERPRETATION: BEAMing of circulating DNA could be a viable approach for non-invasive analysis of tumour genotype in real time and for the identification of potentially clinically relevant mutations that are not detected in archival tissue. Additionally, the results show that regorafenib seems to be consistently associated with a clinical benefit in a range of patient subgroups based on mutational status and protein biomarker concentrations. FUNDING: Bayer HealthCare Pharmaceuticals.

Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase.

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).