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Indian Journal of Cancer, Vol. 44, No. 1, January-March, 2007, pp. 17-24 Review Article Initiating tumor banking for translational research: MD Anderson and Liverpool experience Mishra A, Pandey A, Shaw R Department of Otolaryngology at King George's Medical University Lucknow, India and currently a Visiting Faculty and International UICC (American Cancer society) Fellow at the Department of Head and Neck Surgery, UT MD Anderson Cancer Center, Houston, USA Code Number: cn07004 Abstract The ultimate progress in the cancer diagnosis and therapy has only been possible with the ongoing translational research that is likely to play a very important role in future as well. Hence the importance of such translation from bedside to bench and visa versa cannot be over-emphasized. Accordingly it has become more important to collect tumor samples along with the clinical information in a systematic manner to perform a good basic science research in future. With a population of over a billion and a heavy burden of cancer, India has the 'biggest' potential to establish the 'largest' tumor bank across the globe. Establishing a tumor bank involves money and manpower that may not be feasible across most of the centers in India. Taking into the considering the model of tumor banking of the two leading institutions of the world (MD Anderson Cancer Center, USA and University Hospital Aintree, Liverpool UK), this article presents the salient tips for a center in India to get started with tumor banking with minimal investment. Furthermore a simplified form of ethical consent is presented for the centers to adapt unanimously.Keywords: Tissue-banking, tumor-banking Tissue sample of tumors is the most important rate-limiting factor for carrying out any translational research. Such samples need to be properly processed, stored and carefully tagged with its clinical behavior. Hence a cancer research center needs to maintain a proper tissue bank from where serial stored samples along with the relevant clinical records can be obtained for a translational cancer research in future. Till date the concept of tissue banking in India is mainly concentrated in a few major cancer research institutes such as the Tata Memorial Hospital. Most of the cancer centers across the country do not have the concept of tumor banking. With over a billion population and an enormous cancer burden, such a concept would definitely unfold the enormous potential for translational cancer research in India. Considering the existing protocol of tumor banking at MD Anderson Cancer Center (USA) and University Hospital Aintree (Liverpool) we suggest some guidelines to initiate this concept in less well-equipped institutes of India. Ideally it has to be defined beforehand whether such a repository is a freestanding entity, virtual or a part of institution and moreover the standard operating procedures manual (SOPs) should be framed accordingly. Further discussion would highlight our views for starting this the concept as a freestanding entity with meager resources across the country. MD Anderson Cancer Center Protocol The head and neck tissue banking was started approximately 15 years ago at MD Anderson Cancer Center, Houston Texas and till date the bank has a total collection of approximately 25000 tissue specimen. This subsequently proved to be a model for Central MD Anderson Tissue Bank. The salient features of the protocol are as follows:
Developing Tissue Banking in India A very important technical aspect of concern is the temperature for storing the tissue specimen. Many scientists believe that storing the tissues at lower temperatures helps in preserving the integrity of the biopsy specimen for long-term,[1],[2] however there is no general consensus on this. Even RNA studies have been performed on paraffin block tissues but the sensitivity decreases with increasing temperature of storage. Hence the more lower the storage-temperature, the better. Ideally the specimen should be preserved in large liquid nitrogen cylinder, which unfortunately may not be available in the majority of centers across the country. In most medical colleges the departments of pathology and surgery are located in different buildings and hence a significant delay occurs in tissue transportation. In such situations it seems ideal to maintain a deep freeze refrigerator for small tissue collections in the respective departments. Several options for storing the tissues in a deep freeze refrigerator as a less expensive methodology can be considered based on UKBTS guidelines.[3] The biopsy tissue must be placed in an ambient temperature of -20oC within 48h of retrieval. This does not only enhance bacteriostasis but is known to facilitate long-term storage for six months. Alternatively if such storage can be maintained at -40oC then long-term storage can be maintained for a period of three years. It is worth mentioning that as per UKBTS guidelines,[3] the tissue if preserved in high concentration glycerol may be stored at 0oC to -10oC for up to two years. Considering the above methods it may be possible to store the tissue in heavy glycerine at -40oC for a very long time. It has been a convention to store tissue at less that -80oC for long term, but considering the practicalities in India, this glycerol-preservation seems to be the best alternative in initiating the process. Such 'compromised tissues' may not be well suited for RNA studies as such. It is not infrequent to have frequent short-time power failures in the absence of uninterrupted power backup in our facility. To withstand this, we suggest the use of cryogel bags around the storage racks inside the freezer to maintain the desired temperature in a 10 degree celsius range as advocated[4] for a prolonged time. However in case of prolonged electrical failure the freezer should be backed-up with a UPS / generator facility. Also adequate backup capacity for low-temperature units must be maintained in anticipation of possible equipment failure. It is desirable to have satisfactory temperature (less than 22 degree Celsius), lighting and sufficient space for air circulation at the place where mechanical freezers are installed. Adequate ventilation is critical in liquid-N2 repositories and where dry ice is used to ensure that sufficient oxygen levels are maintained. The most important issue in tissue banking is freely given informed patient consent. Tissue banks of human specimens for research must adhere to all central / state government and local regulations. Research on human specimen involves no interaction with patient and the risks are primarily from loss of confidentiality.[4] Accordingly the processes and procedures for storage of human specimens for research should be available for review by an IRB to assure that they are appropriate to protect human subjects. For prospective collection of human biological materials the disclosure should include details of procedures (drawing blood, buccal swab), risks / benefits involved (bruising at site, access to records by others, no prospect of direct benefit) and guarded 'right-to-withdraw' because active participation ends at the time of collection, while sample would remain but links would be destroyed and genetic information would exist in the databases. Consent can be for a specific research use or for future unspecified uses. If the use is unspecified, an IRB review of the research must be conducted to assure that the use is consistent with the original consent.[4] An example of the customized consent form based on a project 'Cytogenetic analysis of oral squamous cell carcinoma'is depicted in [Table - 2]. This has been adapted from the 'University Hospital Aintree, Liverpool' protocol, where such tumor banking has recently been initiated. [Table - 3] depicts a sample of the relevant information necessary to be conveyed to the patient before consenting. Ideally all patient consent forms must be updated annually to be in compliance with IRB regulations. Another important aspect is maintenance of clinical records and linking it to the specimen. Confidentiality of clinical records needs to be maintained specially in terms of identity of the subject. However the HIPAA 'Privacy rule' 1996 establishes a category of health information referred to as protected health information (PHI) which may be disclosed only under certain conditions.[4] This is with an aim to permit researchers to review PHI in medical records or elsewhere to prepare a research protocol. Such a de-identified data would exclude all the possible subject identifiers and would be released after a non-disclosure agreement by the researcher. The Records need to be retained for a minimum of 10 years after the date of distribution / expiration of sample. Ideally the electronic records should be backed-up regularly on a network or remote server or on a CD / floppy. The continuance, success and long term utility of a tumor bank depends upon its quality assurance (QA) and intermittent quality control checks. The QA programme of a tissue bank involves planning, implementation, documentation, assessment and improvement to ensure that the processing or specimen / item is of a standard quality. Quality control check on the other hand involves technical activities to measure the attributes and performance of a certain process or item against defined quality standards. It is important for a quality assessment program to maximally adapt to current-good-manufacturing-practices (cGMP)-regulatory guidelines as per its limitations. This would include secured area with limited access, trained personnel for required procedure, provision of internal QA audits by external agents, maintenance of extensive paper trail for all materials and equipments, documentation of equipment maintenance procedures as well as for all events that fall outside SOPs.[4] It is important to note that ISO9001 is another system standard created through the international organization for standardization (ISO). ISO is similar to cGMP, but is more recognizable in international settings. Hence this may not initially suit a small repository of a developing country. All the tumor banks must be subjected to regular audits for example a weekly check of freezer temperature logs and a more complete review on a monthly or quarterly basis that includes random checks of SOPs for expiration and possibly pulling and checking random sets of specimens against the inventory list. The consideration of safety is of paramount importance for those persons dealing with human samples in tumor banks. Apart from the general safety measures against fire, electrical and physical hazards as applicable elsewhere, special precautions need to be undertaken to protect the employees against blood-borne pathogens and diseases spread through skin-to-skin contact such as scabies and tuberculosis (specially in India). Vaccination against hepatitis should be encouraged. Accordingly every tumor bank should define the safety priorities and thereby develop a comprehensive safety programme in the light of national, state, regional and local regulations for health protection. It is preferable to establish a safety committee for planning, implementing and monitoring overall safety programme through a safety officer, which in turn collaborates with local area supervisors to establish safety-training-programmes. In the absence of such safety committee the legal responsibility of a hazard lies with the person designated or the overall in-charge of the tumor bank. Hence training for all the employees is mandatory in every domain (biohazards, chemical hazards, radiological hazards) and that too should be documented and updated each year. It is very important to note that all human specimens irrespective of state are to be treated as infectious since prions (C-J disease) may still be active with fixed tissues processed to paraffin blocks. Hence the use of apron coats, long pants, covered shoes, plastic gloves, glasses and masks protect the employee from biohazard as well as avoid chemical and thermal burns from both heat and cold (liquid N 2 and dry ice). All chemicals should have material safety data sheets (MSDS) available for reference for employees who potentially will come into contact with these chemicals. MSDS are available from manufacturers. Storage of food / beverages should be strongly prohibited in laboratory areas and the same applies to mouth pipetting / suctioning. After handling potential hazardous material, hands and other exposed skin surfaces need to be thoroughly washed. A written emergency plan should be established to address the prevention, containment, cleanup and waste disposal in case of chemical spills. In general, some basic security systems such as alarms are desirable and a responsible person should take necessary action in a time frame to minimize the damage to collected material / specimen. Electricity safety can be assured by proper grounding of electric equipments, using electrical base plugs in good condition, proper use of fuses and surge-protectors as well as ensuring that water sources (sinks) are away from electrical devices. Fire safety can be ensured by storing flammable agents in fire cabinets, purchasing refrigerators/ freezers that are noncombustible, use of different types of fire-extinguisher-cylinders and strictly prohibiting smoking. Carefully analyzing the work environment and correctly applying ergonomics thereafter can enhance physical safety. This would also significantly reduce the visual and musculoskeletal discomfort. Considering the overall security it is important to limit access of unauthorized personnel in the workplace. For initiating tumor banking following 'material-protocol' may be adapted:
The 'linking' of clinical records with the stored specimen through the use of simple Microsoft access software seems to be most appropriate initially. This preliminary level of tumor banking is applicable to most of the medical colleges across India. The training of post-graduate residents for tissue collection, processing, storage and retrieval would be much easier and definitely desirable since with limited resources, it would be difficult to employ separate staff for the tumor bank. This would further minimize the hazards associated with the program. In places where storage in liquid nitrogen cylinders is feasible, it is important to note that the vapor-phase avoids safety hazards inherent in the liquid-phase storage. It may not be possible to bar-code the aliquots and hence appropriate coding for example including the patient's initial, date of collection or some alternate method should be adapted. It is important to use proper labels / stickers and ink to last for at least 10 years. Certain recently built cancer centers across the country have an advantage that the pathology facility exists in close vicinity to the operating theatres in the same hospital premises. This facilitates the tissue specimen from all the departments to be processed at a single place only, thus reducing the expenses incurred for manpower and equipment, that otherwise need to be maintained at the respective departments. However if the geographic distance is substantial, the postoperative tissue specimen can be temporarily stored for transportation in either small liquid-nitrogen cylinders or a tight thermacol box with frozen cryogel bags. Alternatively if the geographical distance is not much the fresh specimen may be transported in a sterile container on wet ice from surgery to pathology / repository. This still facilitates the concept of tissue processing at only one specific place, thus reducing the additional costs. To adapt the MD Anderson model of tissue banking it is essential to have the following:
Acknowledgment The work done (main author: AM) has been supported by a UICC American Cancer Society International Fellowship for Beginning Investigators. The author would like to further acknowledge Dr. Adel El-Naggar MD, Professor, Department of Pathology and Dr. GL Clayman MD Professor, Department of Head and Neck Surgery, UT MD Anderson Cancer Center, Houston for their expert inputs.References
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