An exploratory, open-label, randomized, multicenter study to investigate the pharmacodynamics of a glycoengineered antibody (imgatuzumab) and cetuximab in patients with operable head and neck squamous cell carcinoma.

BACKGROUND: In addition to inhibiting epidermal growth factor receptor (EGFR) signaling, anti-EGFR antibodies of the IgG1 'subtype' can induce a complementary therapeutic effect through the induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Glycoengineering of therapeutic antibodies increases the affinity for the Fc-gamma receptor, thereby enhancing ADCC. PATIENTS AND METHODS: We investigated the changes in immune effector cells and EGFR pathway biomarkers in 44 patients with operable, advanced stage head and neck squamous cell carcinoma treated with two preoperative doses of either glycoengineered imgatuzumab (GA201; 700 or 1400 mg) or cetuximab (standard dosing) in a neoadjuvant setting with paired pre- and post-treatment tumor biopsies. RESULTS: Significant antitumor activity was observed with both antibodies after just two infusions. Metabolic responses were seen in 23 (59.0%) patients overall. One imgatuzumab-treated patient (700 mg) achieved a 'pathological' complete response. An immediate and sustained decrease in peripheral natural killer cells was consistently observed with the first imgatuzumab infusion but not with cetuximab. The functionality of the remaining peripheral natural killer cells was maintained. Similarly, a pronounced increase in circulating cytokines was seen following the first infusion of imgatuzumab but not cetuximab. Overall, tumor-infiltrating CD3+ cell counts increased following treatment with both antibodies. A significant increase from baseline in CD3+/perforin+ cytotoxic T cells occurred only in the 700-mg imgatuzumab group (median 95% increase, P < 0.05). The most prominent decrease of EGFR-expressing cells was recorded after treatment with imgatuzumab (700 mg, -34.6%; 1400 mg, -41.8%). The post-treatment inflammatory tumor microenvironment was strongly related to baseline tumor-infiltrating immune cell density, and baseline levels of EGFR and pERK in tumor cells most strongly predicted therapeutic response. CONCLUSIONS: These pharmacodynamic observations and relationship with efficacy are consistent with the proposed mode of action of imgatuzumab combining efficient EGFR pathway inhibition with ADCC-related immune antitumor effects. CLINICAL TRIAL REGISTRATION NUMBER: NCT01046266 (ClinicalTrials.gov).

Glyco-engineered anti-EGFR mAb elicits ADCC by NK cells from colorectal cancer patients irrespective of chemotherapy.

BACKGROUND: The epidermal growth factor receptor (EGFR) is overexpressed in colorectal cancer (CRC), and is correlated with poor prognosis, making it an attractive target for monoclonal antibody (mAb) therapy. A component of the therapeutic efficacy of IgG1 mAbs is their stimulation of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells bearing the CD16 receptor. As NK cells are functionally impaired in cancer patients and may be further compromised upon chemotherapy, it is crucial to assess whether immunotherapeutic strategies aimed at further enhancing ADCC are viable. METHODS: CRC patients before, during and after chemotherapy were immunophenotyped by flow cytometry for major white blood cell populations. ADCC-independent NK cell functionality was assessed in cytotoxicity assays against K562 cells. ADCC-dependent killing of EGFR(+) A431 cancer cells by NK cells was measured with a degranulation assay where ADCC was induced by GA201, an anti-EGFR mAb glyco-engineered to enhance ADCC. RESULTS: Here, we confirm the observation that NK cells in cancer patients are dysfunctional. However, GA201 was able to induce robust NK cell-dependent cytotoxicity in CRC patient NK cells, effectively overcoming their impairment. CONCLUSIONS: These findings support the evaluation of the therapeutic potential of GA201 in combination with chemotherapy in CRC patients.

TuBaFrost: European virtual tumor tissue banking.

TuBaFrost is a consortium responsible for the task to create a virtual European human frozen tumor tissue bank, composed of high quality frozen tumor tissue collections with corresponding accurate diagnosis stored in European cancer centers and universities, searchable on the Internet, providing rules for access and use and a code of conduct to comply with the various legal and ethical regulations in European countries. Such infrastructure would enlarge tissue availability and accessibility in large amounts of specified or even rare tumor samples. Design of an infrastructure for European residual tissue banking with the described characteristics, clear focus points emerge that can be broken down in dedicated subjects: (1) standardization and quality assurance (QA) to avoid inter-institute quality variation; (2) law and ethics enabling exchange of tissue samples possible between institutes in the different European countries, where law and ethics are characterized by a strong variability; (3) rules for access, with sufficient incentives for collectors; (4) central database application containing innovations on search and selection procedures; (5) support when needed with histology images; and (6) Internet access to search and upload, with in addition a solid website giving proper information on the procedures, intentions and activities not only to the scientific community, but also to the general public. One consortium decision, part of the incentives for collectors, had major impact on the infrastructure; custodianship over the tissues as well as the tissues stay with the collector institute. Resulting in specimens that are not given to an organization, taking decisions on participation of requests, but instead the local collected tissues stay very easy to access by the collector and allows autonomous negotiation between collector and requestor on cooperation, coauthorship in publication or compensation in costs. Thereby, improving availability of large amounts of high quality samples of a highly specified or rare tumor types and contact opportunities for cooperation with other institutes.

Virtual microscopy in virtual tumor banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated virtual microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting biorepositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 6: virtual microscopy in virtual tumour banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated Virtual Microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting bio-repositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 1: Uniting local frozen tumour banks into a European network: an overview.

TuBaFrost is the consortium responsible for the creation of a virtual European human frozen tumour tissue bank: a collection of high quality frozen residual, accurately classified tumour tissue samples, which are stored in European cancer centres and universities. This virtual tissue bank, searchable on the internet, has rules for access and use, and a code of conduct to comply with the various legal and ethical regulations in European countries. The easy accessibility and the European scale of the bank will result in the availability of a large number of samples even of rarer tumour types. Standardisation of collection, storage and quality control throughout the network is achieved minimising inter-institutional variability. A website providing access to upload, search and request samples is a key tool of the tissue bank. The search engine makes use of virtual microscopy. An overview of the development of the European virtual frozen tissue bank infrastructure is described in this paper. The various key aspects are described in more detail in a series of articles to appear in this Journal.

TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network.

Tumour Bank Networking presents a great challenge for oncological research as in order to carry out large-scale, multi-centre studies with minimal intrinsic bias, each tumour bank in the network must have some fundamental similarities and be using the same standardised and validated procedures. The European Human Frozen Tumour Tissue Bank (TuBaFrost) has responded to this need by the promotion of an integrated platform of tumour banks in Europe. The operational framework for TuBaFrost has drawn upon the best practice of standard workflows and operating procedures employed by members of the TuBaFrost project and key initiatives worldwide.

TuBaFrost 4: access rules and incentives for a European tumour bank.

When designing infrastructure for a networked virtual tumour bank (samples remain at the collector institutes and sample data are collected in a searchable central database), it is apparent that this can only function properly after developing an adequate set of rules for use and access. These rules must include sufficient incentives for the tissue sample collectors to remain active within the network and maintain sufficient sample levels in the local bank. These requirements resulted in a key TuBaFrost rule, stating that the custodianship of the samples remains under the authority of the local collector. As a consequence, the samples and the decision to issue the samples to a requestor are not transferred to a large organisation but instead remain with the collector, thus allowing autonomous negotiation between collector and requestor, potential co-authorship in publications or compensation for collection and processing costs. Furthermore, it realises a streamlined cost effective network, ensuring tissue visibility and accessibility thereby improving the availability of large amounts of samples of highly specific or rare tumour types as well as providing contact opportunities for collaboration between scientists with cutting edge technology and tissue collectors. With this general purpose in mind, the rules and responsibilities for collectors, requestors and central office were generated.

TuBaFrost 3: regulatory and ethical issues on the exchange of residual tissue for research across Europe.

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.

TuBaFrost 5: multifunctional central database application for a European tumor bank.

Developing a tissue bank database has become more than just logically arranging data in tables combined with a search engine. Current demand for high quality samples and data, and the ever-changing legal and ethical regulations mean that the application must reflect TuBaFrost rules and protocols for the collection, exchange and use of tissue. To ensure continuation and extension of the TuBaFrost European tissue bank, the custodianship of the samples, and hence the decision over whether to issue samples to requestors, remains with the local collecting centre. The database application described in this article has been developed to facilitate this open structure virtual tissue bank model serving a large group. It encompasses many key tasks, without the requirement for personnel, hence minimising operational costs. The Internet-accessible database application enables search, selection and request submission for requestors, whereas collectors can upload and edit their collection. Communication between requestor and involved collectors is started with automatically generated e-mails.

Docetaxel and cisplatin as induction chemotherapy in patients with pathologically-proven stage IIIA N2 non-small cell lung cancer: a phase II study of the European organization for research and treatment of cancer (EORTC 08984).

The objective of this phase II study was to document activity and toxicity of docetaxel and cisplatin as induction chemotherapy in patients with stage IIIA N2 non-small cell lung cancer (NSCLC) before definitive local treatment. Forty-six chemotherapy-nai ve patients (median age 60 years) were included. Treatment consisted of 3 cycles of docetaxel (85 mg/m2 on day 1), followed by cisplatin (40 mg/m2/day on days 1 and 2) every 21 days. Grade 3-4 leukopenia and neutropenia occurred in 45.7% and 65.2% of the patients, respectively. Among 8 cases of febrile neutropenia (17.4%), one (2.2%) resulted in early death. Common grade 3-4 non-haematological toxicities were nausea (17.4%) and vomiting (13%). Eighty-five percent of the patients received three courses; six stopped prematurely due to toxicity, one due to protocol violation. Response rate was the primary endpoint of this study. Considering eligible patients (n=40), 18 responses (1 complete and 17 partial responses) were observed (response rate 45%; 95% Confidence interval (CI): 29.3%-61.5%). In stage IIIA-N2 NSCLC patients, docetaxel-cisplatin could be administered and demonstrated manageable toxicity with modest efficacy.

Extracellularly tumor-activated prodrugs for the selective chemotherapy of cancer: application to doxorubicin and preliminary in vitro and in vivo studies.

Oligopeptidic derivatives of anthracyclines unable to penetrate cells were prepared and screened for their stability in human blood and their reactivation by peptidases secreted by cancer cells. N-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin was selected as a new candidate prodrug. The NH2-terminal beta-alanine allows a very good blood stability. A two-step activation by peptidases found in conditioned media of cancer cells ultimately yields N-L-leucyl-doxorubicin. In vitro, when MCF-7/6 cancer cells are exposed to the prodrug, they accumulate about 14 times more doxorubicin than MRC-5 normal fibroblasts, whereas when exposed to doxorubicin the uptake is slightly higher in fibroblasts than in MCF-7/6 cells. This increased specificity of the prodrug over doxorubicin was confirmed in cytotoxicity assays using the same cell types. In vivo, the prodrug proved about nine times less toxic than doxorubicin in the normal mouse and also much more efficient in two different experimental chemotherapy models of human breast tumors.