Defective Mismatch Repair and Cisplatin Resistance in Ovarian Cancer Cells

The development of resistance to anticancer drugs poses one of the major obstacles to improving the survival from malignant disease. Most clinically effective anticancer agents act upon DNA. Resistance has been shown to arise by mechanisms that reduce the amount of drug reaching the DNA. However, drug resistance developing through alterations in the capacity of tumour cells to repair or respond to DNA damage, may be as important. Microsatellite instability (RER+) is generally taken as an indication of defective DNA mismatch repair. Ovarian and breast carcinoma cell lines, selected for resistance to cisplatin and doxorubicin, were shown to display an RER+ phenotype as shown by multiple mutations at microsatellite loci in comparison to the parental line. The ovarian carcinoma cell line A2780 was shown to be RER- by microsatellite analysis of random sub-clones from the cell line. A2780/cp70, a cisplatin resistant cell line derived from A2780, displayed an RER+ phenotype when subject to the same analysis. Ten independently derived, cisplatin resistant, A2780 cell lines were developed by selection with increasing concentrations of cisplatin to a final concentration of 15muM. These were shown to display stable resistance to cisplatin of between 4 fold (as determined by clonogenic assay) and 7 fold (s determined by MTT assay), when compared to the parental line. The RER+ phenotype was detected in all but one of these lines. An association between the RER+ phenotype and cisplatin resistance was further strengthened by the finding of microsatellite instability in resistant A2780 cell lines selected by a single exposure to 15muM cisplatin. Mutations in genes involved in mismatch repair have been shown to be responsible for microsatellite instability. The biochemical basis of the RER+ phenotype in drug resistant A2780 derived cell lines was determined by in vitro repair assay. An inability to correct DNA mismatches, when repair was directed from 3' to the lesion, was observed, and hMutLalpha was identified as the defective component of mismatch repair by in vitro complementation. Further studies revealed the absence of hMLH1 and hPMS2 proteins, which constitute hMutLalpha, within the RER+ resistant cell lines. This arises from the failure of these cells to express hMLH1 mRNA. No defect in the expression of the other known mismatch repair genes was observed. P53-dependent DNA damage responses were characterised in the RER+, drug resistant cell lines. A correlation was observed between RER+ status and loss of radiation induced G1 arrest, reduced expression of the CIP 1 gene and decreased levels of apoptosis after cisplatin exposure. This association between DNA mismatch repair and the DNA damage response pathway suggests that the mismatch repair system might act as a sensor for DNA damage. This supports the hypothesis that mismatch repair may be involved in determining the cellular sensitivity to cisplatin; defects in such repair leading to cisplatin resistance

Phase II study of TP300 in patients with advanced gastric or gastro-oesophageal junction adenocarcinoma

Background: TP300, a recently developed synthetic camptothecin analogue, is a highly selective topoisomerase I inhibitor. A phase I study showed good safety and tolerability. As camptothecins have proven active in oesophago-gastric adenocarcinomas, in this phase II study we assessed the efficacy and safety of TP300 in patients with gastric or gastro-oesophageal junction (GOJ) adenocarcinomas. Methods: Eligible patients had metastatic or locally advanced gastric or Siewert Types II or III GOJ inoperable adenocarcinoma. Patients were chemotherapy naïve unless this had been administered in the perioperative setting. TP300 was administered as a 1-h intravenous infusion every 3 weeks (a cycle) for up to 6 cycles at a starting dose of 8 mg/m2 with intra-patient escalation to 10 mg/m2 from cycle 2 in the absence of dose-limiting toxicity. Tumour responses (RECIST 1.1) were assessed every 6 weeks. Toxicity was recorded by NCI-CTCAE version 3.0. Using a modified two-stage Simon design (Stage I and II), a total of 43 patients were to be included providing there were 3 of 18 patients with objective response in Stage I of the study. Results: In Stage I of the study 20 patients (14 males, 6 females), median age 67 years (range 40 − 82), performance status ECOG 0/1, with GC [14] or GOJ carcinoma [6] were enrolled. Of the 16 evaluable patients, 11 received the planned dose increase to 10 mg/m2 at cycle 2, 2 decreased to 6 mg/m2, and 3 continued on 8 mg/m2. There were no objective responses after 2 cycles of treatment. Twelve patients had stable disease for 1 − 5 months and 4 had progressive disease. Median progression free survival (PFS) was 4.1 months (CI [1.6 − 4.9]), median time to progression (TTP) was 2.9 months (CI [1.4 − 4.2]). Grade 3/4 toxicities (worst grade all cycles) included 7 patients (35 %) with neutropenia, 4 patients (20 %) with anaemia, 2 patients (10 %) with thrombocytopenia, and 3 patients (15 %) with fatigue. This study was terminated at the end of Stage I due to a lack of the required (3/18) responders. Conclusions: This study of TP300 showed good drug tolerability but it failed to demonstrate sufficient efficacy as measured by radiological response

Phase II study of TP300 in patients with advanced gastric or gastro-oesophageal junction adenocarcinoma

Background: TP300, a recently developed synthetic camptothecin analogue, is a highly selective topoisomerase I inhibitor. A phase I study showed good safety and tolerability. As camptothecins have proven active in oesophago-gastric adenocarcinomas, in this phase II study we assessed the efficacy and safety of TP300 in patients with gastric or gastro-oesophageal junction (GOJ) adenocarcinomas. Methods: Eligible patients had metastatic or locally advanced gastric or Siewert Types II or III GOJ inoperable adenocarcinoma. Patients were chemotherapy naïve unless this had been administered in the perioperative setting. TP300 was administered as a 1-h intravenous infusion every 3 weeks (a cycle) for up to 6 cycles at a starting dose of 8 mg/m2 with intra-patient escalation to 10 mg/m2 from cycle 2 in the absence of dose-limiting toxicity. Tumour responses (RECIST 1.1) were assessed every 6 weeks. Toxicity was recorded by NCI-CTCAE version 3.0. Using a modified two-stage Simon design (Stage I and II), a total of 43 patients were to be included providing there were 3 of 18 patients with objective response in Stage I of the study. Results: In Stage I of the study 20 patients (14 males, 6 females), median age 67 years (range 40 − 82), performance status ECOG 0/1, with GC [14] or GOJ carcinoma [6] were enrolled. Of the 16 evaluable patients, 11 received the planned dose increase to 10 mg/m2 at cycle 2, 2 decreased to 6 mg/m2, and 3 continued on 8 mg/m2. There were no objective responses after 2 cycles of treatment. Twelve patients had stable disease for 1 − 5 months and 4 had progressive disease. Median progression free survival (PFS) was 4.1 months (CI [1.6 − 4.9]), median time to progression (TTP) was 2.9 months (CI [1.4 − 4.2]). Grade 3/4 toxicities (worst grade all cycles) included 7 patients (35 %) with neutropenia, 4 patients (20 %) with anaemia, 2 patients (10 %) with thrombocytopenia, and 3 patients (15 %) with fatigue. This study was terminated at the end of Stage I due to a lack of the required (3/18) responders. Conclusions: This study of TP300 showed good drug tolerability but it failed to demonstrate sufficient efficacy as measured by radiological response

Phase I study of TP300 in patients with advanced solid tumors with pharmacokinetic, pharmacogenetic and pharmacodynamic analyses

Background: A Phase I dose escalation first in man study assessed maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and recommended Phase II dose of TP300, a water soluble prodrug of the Topo-1 inhibitor TP3076, and active metabolite, TP3011. <p/>Methods: Eligible patients with refractory advanced solid tumors, adequate performance status, haematologic, renal, and hepatic function. TP300 was given as a 1-hour i.v. infusion 3-weekly and pharmacokinetic (PK) profiles of TP300, TP3076 and TP3011 were analysed. Polymorphisms in CYP2D6, AOX1 and UGT1A1 were studied and DNA strand-breaks measured in peripheral blood mononuclear cells (PBMCs). <p/>Results: 32 patients received TP300 at 1, 2, 4, 6, 8, 10, 12 mg/m2. MTD was 10 mg/m2; DLTs at 12 (2/4 patients) and 10 mg/m2 (3/12) included thrombocytopenia and febrile neutropenia; diarrhea was uncommon. Six patients (five had received irinotecan), had stable disease for 1.5-5 months. TP3076 showed dose proportionality in AUC and Cmax from 1--10 mg/m2. Genetic polymorphisms had no apparent influence on exposure. DNA strand-breaks were detected after TP300 infusion. <p/>Conclusions: TP300 had predictable hematologic toxicity, and diarrhea was uncommon. AUC at MTD is substantially greater than for SN38. TP3076 and TP3011 are equi-potent with SN38, suggesting a PK advantage

A phase I pharmacokinetic and pharmacodynamic study of the oral mitogen-activated protein kinase kinase (MEK) inhibitor, WX-554, in patients with advanced solid tumours

Purpose: We performed a multi-centre phase I study to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the orally available small molecule mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, WX-554, and to determine the optimal biological dose for subsequent trials. Experimental design: Patients with treatment-refractory, advanced solid tumours, with adequate performance status and organ function were recruited to a dose-escalation study in a standard 3 + 3 design. The starting dose was 25 mg orally once weekly with toxicity, PK and PD guided dose-escalation with potential to explore alternative schedules. Results: Forty-one patients with advanced solid tumours refractory to standard therapies and with adequate organ function were recruited in eight cohorts up to doses of 150 mg once weekly and 75 mg twice weekly. No dose-limiting toxicities were observed during the study, and a maximum tolerated dose (MTD) was not established. The highest dose cohorts demonstrated sustained inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells following ex-vivo phorbol 12-myristate 13-acetate stimulation. There was a decrease of 70 ± 26% in mean phosphorylated (p)ERK in C1 day 8 tumour biopsies when compared with pre-treatment tumour levels in the 75 mg twice a week cohort. Prolonged stable disease (>6 months) was seen in two patients, one with cervical cancer and one with ampullary carcinoma. Conclusions: WX-554 was well tolerated, and an optimal biological dose was established for further investigation in either a once or twice weekly regimens. The recommended phase 2 dose is 75 mg twice weekly

A phase I trial of the γ-secretase inhibitor MK-0752 in combination with gemcitabine in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: The Notch pathway is frequently activated in cancer. Pathway inhibition by γ-secretase inhibitors has been shown to be effective in pre-clinical models of pancreatic cancer, in combination with gemcitabine. METHODS: A multi-centre, non-randomised Bayesian adaptive design study of MK-0752, administered per os weekly, in combination with gemcitabine administered intravenously on days 1, 8 and 15 (28 day cycle) at 800 or 1000 mg m-2, was performed to determine the safety of combination treatment and the recommended phase 2 dose (RP2D). Secondary and tertiary objectives included tumour response, plasma and tumour MK-0752 concentration, and inhibition of the Notch pathway in hair follicles and tumour. RESULTS: Overall, 44 eligible patients (performance status 0 or 1 with adequate organ function) received gemcitabine and MK-0752 as first or second line treatment for pancreatic cancer. RP2Ds of MK-0752 and gemcitabine as single agents could be combined safely. The Bayesian algorithm allowed further dose escalation, but pharmacokinetic analysis showed no increase in MK-0752 AUC (area under the curve) beyond 1800 mg once weekly. Tumour response evaluation was available in 19 patients; 13 achieved stable disease and 1 patient achieved a confirmed partial response. CONCLUSIONS: Gemcitabine and a γ-secretase inhibitor (MK-0752) can be combined at their full, single-agent RP2Ds

Circulating biomarkers during treatment in patients with advanced biliary tract cancer receiving cediranib in the UK ABC-03 trial

BACKGROUND: Advanced biliary tract cancer (ABC) has a poor prognosis. Cediranib, in addition to cisplatin/gemcitabine [CisGem], improved the response rate, but did not improve the progression-free survival (PFS) in the ABC-03 study. Minimally invasive biomarkers predictive of cediranib benefit may improve patient outcomes. METHODS: Changes in 15 circulating plasma angiogenesis or inflammatory-related proteins and cytokeratin-18 (CK18), measured at baseline and during therapy until disease progression, were correlated with overall survival (OS) using time-varying covariate Cox models (TVC). RESULTS: Samples were available from n=117/124 (94%) patients. Circulating Ang1&2, FGFb, PDGFbb, VEGFC, VEGFR1 and CK18 decreased as a result of the therapy, independent of treatment with cediranib. Circulating VEGFR2 and Tie2 were preferentially reduced by cediranib. Patients with increasing levels of VEGFA at any time had a worse PFS and OS; this detrimental effect was attenuated in patients receiving cediranib. TVC analysis revealed CK18 and VEGFR2 increases correlated with poorer OS in all patients (P< 0.001 and P=0.02, respectively). CONCLUSIONS: Rising circulating VEGFA levels in patients with ABC, treated with CisGem, are associated with worse PFS and OS, not seen in patients receiving cediranib. Rising levels of markers of tumour burden (CK18) and potential resistance (VEGFR2) are associated with worse outcomes and warrant validation

A Phase I/II trial of Oral SRA737 (a Chk1 Inhibitor) given in combination with low-dose gemcitabine in patients with advanced cancer

Purpose: This was a phase I/II trial of the novel checkpoint kinase 1 (Chk1) inhibitor SRA737 given in combination with gemcitabine. Its objectives were to establish the safety profile, recommended phase 2 dose (RP2D), pharmacokinetics profile, and clinical activity of SRA737. Patients and Methods: Patients with advanced solid tumors were enrolled into dose-escalation cohorts and treated in 28-day cycles with oral SRA737 on days 2, 3, 9, 10, 16 and 17, and intravenous gemcitabine on days 1, 8 and 15. Treatment was continued until progression. Each expansion cohort included up to 20 patients with specific genetically defined tumors. Results: The RP2D was determined to be 500 mg SRA737 combined with low-dose (250 mg/m2) gemcitabine. Of 143 enrolled patients, 77 were treated at doses of at least 500 mg SRA737 combined with 250 mg/m2 gemcitabine. Common toxicities of nausea, vomiting, fatigue and diarrhea were primarily mild to moderate, and rarely led to treatment discontinuation. Anemia, neutropenia and thrombocytopenia were grade ≥3 in 8.3% to 11.7% of patients treated at the RP2D. The objective response rate (ORR) was 10.8% overall and notably the ORR in anogenital cancer was 25%. Partial tumor responses were observed in anogenital cancer, cervical cancer, high-grade serous ovarian cancer, rectal cancer, and small cell lung cancer. Conclusions: SRA737 in combination with low-dose gemcitabine was well tolerated with lower myelotoxicity than has been seen at standard doses of gemcitabine or with other combinations of Chk1 inhibitors with gemcitabine. Tumor responses were observed in anogenital and other solid tumors

Two first-in-human studies of xentuzumab, a humanised insulin-like growth factor (IGF)-neutralising antibody, in patients with advanced solid tumours

BACKGROUND: Xentuzumab, an insulin-like growth factor (IGF)-1/IGF-2-neutralising antibody, binds IGF-1 and IGF-2, inhibiting their growth-promoting signalling. Two first-in-human trials assessed the maximum-tolerated/relevant biological dose (MTD/RBD), safety, pharmacokinetics, pharmacodynamics, and activity of xentuzumab in advanced/metastatic solid cancers. METHODS: These phase 1, open-label trials comprised dose-finding (part I; 3 + 3 design) and expansion cohorts (part II; selected tumours; RBD [weekly dosing]). Primary endpoints were MTD/RBD. RESULTS: Study 1280.1 involved 61 patients (part I: xentuzumab 10–1800 mg weekly, n = 48; part II: 1000 mg weekly, n = 13); study 1280.2, 64 patients (part I: 10–3600 mg three-weekly, n = 33; part II: 1000 mg weekly, n = 31). One dose-limiting toxicity occurred; the MTD was not reached for either schedule. Adverse events were generally grade 1/2, mostly gastrointestinal. Xentuzumab showed dose-proportional pharmacokinetics. Total plasma IGF-1 increased dose dependently, plateauing at ~1000 mg/week; at ≥450 mg/week, IGF bioactivity was almost undetectable. Two partial responses occurred (poorly differentiated nasopharyngeal carcinoma and peripheral primitive neuroectodermal tumour). Integration of biomarker and response data by Bayesian Logistic Regression Modeling (BLRM) confirmed the RBD. CONCLUSIONS: Xentuzumab was well tolerated; MTD was not reached. RBD was 1000 mg weekly, confirmed by BLRM. Xentuzumab showed preliminary anti-tumour activity