Benefits and risks of adjuvant treatment with zoledronic acid in stage II/III breast cancer. 10 years follow-up of the AZURE randomized clinical trial (BIG 01/04).

Adjuvant bisphosphonates improve disease outcomes in postmenopausal early breast cancer (EBC) but the long-term effects are poorly described. The AZURE trial (ISRCTN79831382) was designed to determine whether adjuvant zoledronic acid (ZOL) improves disease outcomes in EBC. Previous analyses showed no effect on overall outcomes but identified benefits in postmenopausal women. Here we present the long-term risks and benefits of adjuvant ZOL with 10-years follow-up. PATIENTS AND METHODS: 3360 patients with stage II/III breast cancer were included in an academic, international, phase III, randomized, open label trial. Patients were followed up on a regular schedule until 10 years. Patients were randomized on a 1:1 basis to standard adjuvant systemic therapy +/- intravenous ZOL 4 mg every 3-4 weeks x6, and then at reduced frequency to complete 5 years treatment. The primary outcome was disease free survival (DFS). Secondary outcomes included invasive DFS (IDFS), overall survival (OS), sites of recurrence, skeletal morbidity and treatment outcomes according to primary tumor amplification of the transcription factor, MAF. Pre-planned subgroup analyses focused on interactions between menopausal status and treatment effects. RESULTS: With a median follow up of 117 months [IQR 70.4-120.4), DFS and IDFS were similar in both arms (HRDFS  = 0.94, 95%CI = 0.84-1.06, p = 0.340; HRIDFS  = 0.91, 95%CI = 0.82-1.02, p = 0.116). However, outcomes remain improved with ZOL in postmenopausal women (HRDFS  = 0.82, 95%CI = 0.67-1.00; HRIDFS  = 0.78, 95%CI = 0.64-0.94). In the 79% of tested women with a MAF FISH negative tumor, ZOL improved IDFS (HRIDFS  = 0.75, 95%CI = 0.58-0.97) and OS HROS  = 0.69, 95%CI = 0.50-0.94), irrespective of menopause. ZOL did not improve disease outcomes in MAF FISH + tumors. Bone metastases as a first DFS recurrence (BDFS) were reduced with ZOL (HRB-DFS  = 0.76, 95%CI = 0.63-0.92, p = 0.005). ZOL reduced skeletal morbidity with fewer fractures and skeletal events after disease recurrence. 30 cases of osteonecrosis of the jaw in the ZOL arm (1.8%) have occurred. CONCLUSIONS: Disease benefits with adjuvant ZOL in postmenopausal early breast cancer persist at 10 years of follow-up. The biomarker MAF identified a patient subgroup that derived benefit from ZOL irrespective of menopausal status.

Tetracycline-regulated overexpression of glycosyltransferases in Chinese hamster ovary cells.

The glycosylation patterns of recombinant therapeutic glycoproteins can be engineered by overexpression of glycosyltransferases in the host cells used for glycoprotein production. Most prior glycosylation engineering experiments have involved constitutive expression of cloned glycosyltransferases. Here we use tetracycline-regulated expression of two glycosyltransferases, N-acetylglucosaminlytransferases III and V (GnTIII and GnTV) to manipulate glycoform biosynthesis in Chinese hamster ovary (CHO) cells and to study the effect of glycosyltransferase overexpression on this host. The amount of GnTIII and GnTV in these cells, and the glycosylation patterns of several cellular glycoproteins, could be controlled simply by manipulating the concentration of tetracycline in the culture medium. Using this system, it was found that overexpression of either GnTIII or GnTV to high levels led to growth inhibition and was toxic to the cells, indicating that this may be a general feature of glycosyltransferase overexpression. This phenomenon has not been reported previously, probably due to the widespread use of constitutive promoters, and should be taken into account when designing vectors for glycosylation engineering. The growth inhibition effect sets an upper limit to the level of glycosyltransferase overexpression, and may thereby also limit the maximum extent of in vivo modification of poorly accessible glycosylation sites. Also, such inhibition implies a bound on constitutive glycosyltransferase expression which can be cloned.

Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity.

The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline-regulated expression of beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region-associated, bisected complex oligosaccharides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.