Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial.

Background: Bevacizumab is a recombinant humanised monoclonal antibody to vascular endothelial growth factor shown to improve survival in advanced solid cancers. We evaluated the role of adjuvant bevacizumab in melanoma patients at high risk of recurrence. Patients and methods: Patients with resected AJCC stage IIB, IIC and III cutaneous melanoma were randomised to receive either adjuvant bevacizumab (7.5 mg/kg i.v. 3 weekly for 1 year) or standard observation. The primary end point was detection of an 8% difference in 5-year overall survival (OS) rate; secondary end points included disease-free interval (DFI) and distant metastasis-free interval (DMFI). Tumour and blood were analysed for prognostic and predictive markers. Results: Patients (n=1343) recruited between 2007 and 2012 were predominantly stage III (73%), with median age 56 years (range 18-88 years). With 6.4-year median follow-up, 515 (38%) patients had died [254 (38%) bevacizumab; 261 (39%) observation]; 707 (53%) patients had disease recurrence [336 (50%) bevacizumab, 371 (55%) observation]. OS at 5 years was 64% for both groups [hazard ratio (HR) 0.98; 95% confidence interval (CI) 0.82-1.16, P = 0.78). At 5 years, 51% were disease free on bevacizumab versus 45% on observation (HR 0.85; 95% CI 0.74-0.99, P = 0.03), 58% were distant metastasis free on bevacizumab versus 54% on observation (HR 0.91; 95% CI 0.78-1.07, P = 0.25). Forty four percent of 682 melanomas assessed had a BRAFV600 mutation. In the observation arm, BRAF mutant patients had a trend towards poorer OS compared with BRAF wild-type patients (P = 0.06). BRAF mutation positivity trended towards better OS with bevacizumab (P = 0.21). Conclusions: Adjuvant bevacizumab after resection of high-risk melanoma improves DFI, but not OS. BRAF mutation status may predict for poorer OS untreated and potential benefit from bevacizumab. Clinical Trial Information: ISRCTN 81261306; EudraCT Number: 2006-005505-64.

DOC-MEK: a double-blind randomized phase II trial of docetaxel with or without selumetinib in wild-type BRAF advanced melanoma.

BACKGROUND: Treatment options for wild-type BRAF melanoma patients remain limited. Selumetinib, a MEK 1/2 inhibitor, suppresses pERK levels independent of BRAF and NRAS mutation status, and combination with docetaxel has demonstrated synergy in xenograft models. The aim of this study was to assess the efficacy and safety of selumetinib plus docetaxel as first-line treatment in patients with wild-type BRAF advanced melanoma. PATIENTS AND METHODS: In this double-blind multicentre phase II trial patients with wild-type BRAF melanoma were randomized (1:1) to docetaxel with selumetinib or placebo. Docetaxel 75 mg/m(2) was administered intravenously every 3 weeks up to six cycles. Selumetinib 75 mg or placebo was given orally twice daily until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). Tumour NRAS mutation status was analysed retrospectively and correlated with treatment outcomes. RESULTS: Eighty-three patients were randomized to docetaxel plus selumetinib (n = 41) or docetaxel plus placebo (n = 42). The PFS hazard ratio (HR) (selumetinib:placebo) was 0.75 [90% confidence interval (CI) 0.50-1.14; P = 0.130], with a median PFS of 4.23 months (90% CI 3.63-6.90) for docetaxel plus selumetinib and 3.93 months (90% CI 2.07-4.16) for docetaxel alone. There was no significant difference in overall survival. The objective response rate was 32% with selumetinib versus 14% with placebo (P = 0.059). In a retrospective subset analysis, NRAS mutation status did not affect significantly upon clinical outcomes in either arm. The combination of docetaxel and selumetinib could be administered effectively to patients with metastatic melanoma, although the combination was less well tolerated than docetaxel alone. CONCLUSIONS: The combination of docetaxel with selumetinib showed no significant improvement in PFS compared with docetaxel alone, although more patients showed a response to combination therapy. We found no evidence to support using tumour NRAS mutation as a basis for selecting patients for combined MEK inhibitor and chemotherapy. CLINICAL TRIAL: DOC-MEK (EudraCT no: 2009-018153-23).

Changes in tumour vessel density upon treatment with anti-angiogenic agents: relationship with response and resistance to therapy.

BACKGROUND: We examine how changes in a surrogate marker of tumour vessel density correlate with response and resistance to anti-angiogenic therapy. METHODS: In metastatic renal cancer patients treated with anti-angiogenic tyrosine kinase inhibitors, arterial phase contrast-enhanced computed tomography was used to simultaneously measure changes in: (a) tumour size, and (b) tumour enhancement (a surrogate marker of tumour vessel density) within individual lesions. RESULTS: No correlation between baseline tumour enhancement and lesion shrinkage was observed, but a reduction in tumour enhancement on treatment was strongly correlated with reduction in lesion size (r=0.654, P<0.0001). However, close examination of individual metastases revealed different types of response: (1) good vascular response with significant tumour shrinkage, (2) good vascular response with stabilisation of disease, (3) poor vascular response with stabilisation of disease and (4) poor vascular response with progression. Moreover, contrasting responses between different lesions within the same patient were observed. We also assessed rebound vascularisation in tumours that acquired resistance to treatment. The amplitude of rebound vascularisation was greater in lesions that had a better initial response to therapy (P=0.008). INTERPRETATION: Changes in a surrogate marker of tumour vessel density correlate with response and resistance to anti-angiogenic therapy. The data provide insight into the mechanisms that underlie response and resistance to this class of agent.

Sorafenib and dacarbazine as first-line therapy for advanced melanoma: phase I and open-label phase II studies.

METHOD: The safety of oral sorafenib up to a maximum protocol-specified dose combined with dacarbazine in patients with metastatic, histologically confirmed melanoma was investigated in a phase I dose-escalation study and the activity of the combination was explored in an open-label phase II study. RESULTS: In the phase I study, three patients were treated with sorafenib 200 mg twice daily (b.i.d.) plus 1000 mg m(-2) dacarbazine on day 1 of a 21-day cycle and 15 patients had the sorafenib dose escalated to 400 mg b.i.d. without reaching the maximum tolerated dose of the combination. In the phase II study (n=83), the overall response rate was 12% (95% CI: 6, 21): one complete and nine partial, with median response duration of 46.7 weeks. Stable disease was the best response in 37%; median duration was 13.3 weeks. Median overall survival (OS) was 37.0 weeks (95% CI: 33.9, 46.0). CONCLUSION: Oral sorafenib combined with dacarbazine had acceptable toxicity and some antineoplastic activity against metastatic melanoma.

A Phase Ib trial of CA4P (combretastatin A-4 phosphate), carboplatin, and paclitaxel in patients with advanced cancer.

BACKGROUND: The vascular disrupting agent combretastatin A4 phosphate (CA4P) causes major regression of animal tumours when given as combination therapy. METHODS: Patients with advanced cancer refractory to standard therapy were treated with CA4P as a 10-min infusion, 20 h before carboplatin, paclitaxel, or paclitaxel, followed by carboplatin. RESULTS: Combretastatin A4 phosphate was escalated from 36 to 54 mg m(-2) with the carboplatin area under the concentration curve (AUC) 4-5, from 27 to 54 mg m(-2) with paclitaxel 135-175 mg m(-2), and from 54 to 72 mg m(-2) with carboplatin AUC 5 and paclitaxel 175 mg m(-2). Grade 3 or 4 neutropenia was seen in 17%, and thrombocytopenia only in 4% of 46 patients. Grade 1-3 hypertension (26% of patients) and grade 1-3 tumour pain (65% of patients) were the most typical non-haematological toxicities. Dose-limiting toxicity of grade 3 hypertension or grade 3 ataxia was seen in two patients at 72 mg m(-2). Responses were seen in 10 of 46 (22%) patients with ovarian, oesophageal, small-cell lung cancer, and melanoma. CONCLUSION: The combination of CA4P with carboplatin and paclitaxel was well tolerated in the majority of patients with adequate premedication and had antitumour activity in patients who were heavily pretreated.

Epithelial ovarian cancer: a review of current management.

Epithelial ovarian cancer is the most lethal gynaecological cancer among women worldwide, with 6000 new cases diagnosed in the UK each year. Most women present with advanced disease, but, despite a good initial response to treatment, most relapse. The overall 5-year survival rate is 46%, although this drops to about 13% in women with advanced disease. Transvaginal ultrasound and the tumour marker CA125 are being investigated for screening in ongoing randomised trials. Treatment of ovarian cancer is dependent on clinical stage, and should always be managed within a multidisciplinary team. Most cases will require a pelvic clearance and adjuvant chemotherapy. Current guidelines by the National Institute of Clinical Excellence (NICE) recommend that first-line chemotherapy should include a platinum-based regimen with or without paclitaxel. Relapsed ovarian cancer is incurable; however, chemotherapy can improve quality of life and survival. Gene therapy, immunotherapy and signal transduction inhibitors are all potential future therapies, and are being investigated in ongoing clinical research. In this paper we review the literature on the epidemiology, pathology, clinical features and the current treatment options in epithelial ovarian cancer.

Use of monoclonal antibodies reactive with secretory epithelial cells for the immunocytochemical identification of plasma cells.

Six monoclonal antibodies (McAbs) were identified as plasma cell-reactive when screened on sections of human tonsil. They were all produced following immunisation of mice with cells of a human plasmacytoid line. Three of the antibodies also stained the cytoplasm (but not the surface) of blood B cells and were unreactive with other leucocytes; one McAb showed broad lymphocyte reactivity and two were completely unreactive with blood leucocytes; on testing with a panel of cell lines specificity for the plasmacytoid line was demonstrated by three of the McAbs. In spite of the marked restriction shown by the reactivity of these antibodies in tests on cells of haemopoietic origin, tests on other human tissues - including thyroid and pancreas - showed that a related antigen was present in the cytoplasm of secretory epithelial cells. The overall patterns of reactivity of the individual McAbs on various tissues and blood lymphocytes were different. Comparisons were made with the established McAb OKT10, which binds to plasma cells, early stem cells and activated lymphocytes; its binding to plasma cells was confirmed and it was shown that it did not stain secretory epithelia. The potent reactions obtained with the new McAbs suggest that antibodies to antigens associated with epithelial cell secretory apparatus provide potentially useful reagents for studying plasma cells.

Amino acid precursors of Myxococcus xanthus antibiotic TA.

The production of Myxococcus xanthus antibiotic TA was stimulated by addition of alanine, serine and glycine to Casitone medium. These three amino acids served as the major biosynthetic precursors of the antibiotic. Alanine and serine were incorporated via acetate. In Casitone medium supplemented with alanine and serine, 29 to 30 of the 34 carbon atoms of antibiotic TA were derived from these two amino acids. Both carbon atoms of glycine were incorporated into antibiotic TA by a mechanism not involving acetate as an intermediate. Antibiotic TA was split into two fragments by alkaline hydrolysis followed by periodate oxidation. Radioactive alanine was incorporated into both fragments, whereas glycine was incorporated only into the smaller, polar fragment.

Therapeutic opportunities in noncutaneous melanoma.

Recent evidence suggests that the biology of noncutaneous melanoma differs significantly from cutaneous melanoma and may provide therapeutic opportunity. The most frequent sites of origin of noncutaneous melanoma are the eye and mucosal surfaces. Although noncutaneous melanomas are an uncommon group of cancers (representing less than 10% of all melanomas) a greater understanding of their genetic and molecular abnormalites is being translated into novel treatment strategies. These developments are important because there is currently no effective systemic therapy for noncutaneous melanoma. Significant attention has been focused on the role of c-kit (KIT, CD117), a transmembrane receptor with tyrosine kinase activity. In vitro and ex vivo evidence suggests that c-kit is frequently expressed/over expressed/mutated in noncutaneous melanoma. Anti-tumour effects with c-kit inhibitors are seen in pre-clinical models. A variety of multitargeted kinase inhibitors which have activity against c-kit are currently in early phase clinical trials in metastatic ocular, mucosal and acral melanoma. The few case reports of significant clinical activity with targeted therapies provides hope that greater understanding of the biology of noncutaneous melanoma can be translated into effective treatment.

Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.

An analysis of myeloma plasma cell phenotype using antibodies defined at the IIIrd International Workshop on Human Leucocyte Differentiation Antigens.

Fresh bone marrow from 43 cases of myeloma and three cases of plasma cell leukaemia has been phenotyped both by indirect immune-rosetting and, on fixed cytospin preparations, by indirect immunofluorescence. Both clustered and unclustered B cell associated antibodies from the IIIrd International Workshop on Human Leucocyte Differentiation Antigens were used. The results confirm the lack of many pan-B antigens on the surface of myeloma plasma cells, i.e. CD19-23, 37, 39, w40. Strong surface reactivity is seen with CD38 antibodies and with one CD24 antibody (HB8). Weak reactions are sometimes obtained with CD9, 10 and 45R. On cytospin preparations CD37, 39 and w40 are sometimes weakly positive, and anti-rough endoplasmic reticulum antibodies are always strongly positive. Specific and surface-reacting antiplasma cell antibodies are still lacking.

An antigenic study of human plasma cells in normal tissue and in myeloma: identification of a novel plasma cell associated antigen.

A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (cIg). The BU11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens.

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.