Toward the harmonization of immune monitoring in clinical trials: Quo vadis?

The goals of harmonization are to identify crucial protocol choices that influence the outcome of an applied assay, and to standardize those choices across laboratories throughout the entire field for optimal performance and consistent results. Recent activities in the immunotherapy field indicate that there is an increasing awareness of the necessity of such harmonization and assay validation [4‐8]. In parallel to intra-laboratory standardization, leading to standard operating procedures (SOP) for a given work setting, large comparative studies between laboratories with different protocols are required to identify and standardize crucial aspects in the protocols used and provide external validation. Such studies bear various logistical, organisational, and financial challenges. A well-coordinated centralized effort is necessary to accomplish this goal. Proficiency panels conducted by community-spanning organizations are a suitable answer.

Over the last 2 years, two non-profit organizations in the cancer immunotherapy field, the Cancer Vaccine Consortium (CVC) of the Sabin Vaccine Institute and the Cancer Immunotherapy (CIMT) monitoring panel have independently initiated large international proficiency panels. Their findings are now being published in two “technical focus articles” in this issue of CII [9, 10]. Both studies collected specific protocol information from participating laboratories and identified various protocol variables, which clearly can influence the assay outcome. Some of them appear to be “common sense” decisions, but are still neglected by a part of the laboratories in the immunotherapy field. Most importantly, the findings reported are concordant in both studies, and no contradiction in results is imminent. This is particularly relevant when taking into account that both panel initiatives chose a different experimental design. The need to increase the awareness for harmonization initiatives and external validation of assay performance is one of the general common findings of both initiatives. Specific recommendations arising for the ELISPOT from both CVC and CIMT panels are based on the importance of accurate and optimal number of viable cells added per well, which determine the sensitivity of the assay. These recommendations include a resting-phase of cells, the use of automated cell counters allowing the determination of apoptotic and dead cell fractions, and an optimal number of cells per well. Another crucial variable is the unspecific stimulation of cells, which can prevent detection of low frequency T cell response and demands the use of autologous APC as well as critical selection of serum sources. Appropriate plate evaluation strategies and training status of personnel further influence the assay outcome. These findings offer first harmonization guidelines that could instantly be accepted by a broad user base. Further assay optimization can be accomplished by conducting proficiency panels that specifically focus on the incorporation of identified crucial assay variables.

Historically, the first inter-laboratory testing reports for ELISPOT were published 7 years ago [11]. Subsequently, some guidelines resulting from a workshop for assay performance were published about 2 years later [12]. An important proficiency panel program was initiated by the NIAID (National Institute of Allergy and Infectious Diseases), Division of AIDS, for the HIV vaccine field, which served as a first model for the CVC panel program [4]. All these initiatives have certainly attracted attention of the scientific community but have so far not led to broadly accepted standards for immune monitoring. However, through the recent proficiency panels of the CIMT and CVC, which are described in this issue of CII, a chance is given to harmonize assay criteria within widely applicable guidelines.

It is no co-incidence that both proficiency panels were conducted by community-wide organizations. The history of unsuccessful individual laboratory or small-scale efforts has lead to the idea of joining forces and of establishing non-profit organizations that can serve as facilitators of the required initiatives in the United States and Europe. These organizations have large networks of members throughout the immunotherapy community. They (1) give regular updates on the scientific progress, (2) identify the bottlenecks and the various barriers to future development and (3) can facilitate the dialogue between the different stakeholders such as regulatory authorities, pharmaceutical and biotechnology companies, academic institutions, patients’ advocacy groups and the public. Without these organizations the described proficiency panels would not have been possible.

The CVC as part of the Sabin Vaccine Institute initiated an Assay Working Group, which has conducted two large-scale ELISPOT proficiency panels over the last 2 years with access for laboratories world-wide. These served not only as external validation programs for their participants (36 laboratories in panel I, and 29 laboratories in panel II), but also yielded first harmonization steps and guidelines to standardize and validate this assay as a reliable and widely acceptable tool for monitoring of immune responses in clinical trials.

The CIMT monitoring panel is a part of the Association for Immunotherapy of Cancer in Germany. It has until now initiated and completed two phases of an inter-laboratory testing project with focus on ELISPOT and tetramer staining (12 laboratories in panel phase I and 13 laboratories in panel phase II). The initiative is open for all labs with focus on immunomonitoring. A two-step approach was established that facilitates the systematic search for protocol variables that are predictive for high performance in the assay.

The breadth and size of both programs have the potential to help shape immune assay standardization and validation, and serve the ultimate goal of assay harmonization. Collaboration between both groups has proven meaningful for this first round of testing and is planned to increase for future activities.

The conducted proficiency panels represent the largest inter-laboratory testing projects in this field to date. They provide first harmonization guidelines for ELISPOT assay and Tetramer staining. In addition, they demonstrate that international collaboration on this scale driven by non-profit organizations is feasible and forms the basis for expanded future initiatives. Consequently, new Proficiency Panels are now being started or are already under way. They include for CVC: ELISPOT, Tetramer, ICS and CFSE-labelling, and for CIMT: Elispot, Tetramer, ICS. Presentations during the coming annual meetings of both organizations will focus on recent developments and new technologies in the field. First guidelines for validation and training beyond this publication as well as for assay acceptance are in preparation. Additional organisational structures are now being established to offer the possibility of regular participation in different panels to the increasing number of laboratories world-wide that are seeing the value in assay harmonization. Aims are to find standards for training of staff, requirements for handling and test performance and for generating, measuring and reporting of data in broadly available technologies.

With the larger picture in mind, harmonization of these assays may provide the immunotherapy community with valuable surrogate markers for developing new therapies in clinical trials. This may ultimately help improving the clinical success of immunotherapies and bring them to patients more expeditiously.