The Use of Human Tissues in Research: A Summary

There have been several high-profile legal cases which have ended in the removal of human biospecimens from research teams. One case resulted in the destruction of more than 5 x 106 dried blood spots that were obtained from infants after a suit challenged the state of Texas’ right to store these blood spots for future research. All these cases have one thing in common: the argument whether participants were properly informed regarding how their samples will be used for research. Informed consent is being increasingly discussed not only by professionals but also by the public. These cases have also prompted discussions regarding the timing and type of consent required, use of samples, and more.

This article highlights the opinions of the following experts who all represent different viewpoints on informed consent:

• David S. Wendler (D): Advocate for rights of research donors

• Arthur L. Caplan (A): Bioethicist

• Michael Christman (M): President and Chief Executive Officer (CEO) for an independent non-profit biomedical research institution with a large biorepository

• Jack Moye Jr. (J): Researcher

Is consent necessary or do you prefer a presumed consent with an opt-out option with the idea that human biospecimens should be a common heritage that is used for the collective good?

David: The tissues are not just “a collective good”. Tissues are obtained from specific individuals with their use involving the interests of donors and participants. Some of the things to keep in mind include the risks involved when obtaining samples, privacy, contribution, and use of the sample in future research. The necessity of obtaining consent allows donors to decide if they are willing to face the risks involved, increase awareness of the possibility of new information, and the advantages of contributions.

Arthur: The efforts taken to obtain informed consent are doomed to failure as there are many programs that use open-ended informed consent forms that are incomplete and vague. There are also those that ask donors or participants to waive their commercial interest such as the use of blanket waivers. A more appropriate way would be through altruistic gifting. This means that the specimens are made a gift making it clear that commercial interest is forgone, the use of specimen is open-ended, and possession has been transferred to a third party. A presumed consent to gifting would make more sense as long as patients retain the ability to opt out of gifting.

Michael: Current specimens that are anonymous should be allowed for use by research without the need of consent as there identification of donors will not be required. While studies that use anonymous specimens are usually exempted from the institutional review board (IRB) review, proper regulations should be implemented to ensure that this is upheld. However, an exception should be made for the use of anonymous specimens in genomic research as there is a possibility of identification.

Jack: The concept of human biospecimens as a shared resource for the collective good is a fascinating idea that should be given more attention. A framework where human tissue is a common heritage of humanity that is to be used for the collective good can help prevent disputes about both specimens that are left over from clinical purposes and those obtained for research.
What type of informed consent is best: general permission, tiered consent, specific consent, or other?

David: Many studies have been conducted regarding individuals’ attitudes about consent. They have consistently observed that the majority of donors want to control if their samples are used for research. Most participants are also willing to contribute when asked. These studies have also found that most donors support one-time general consent with the understanding that future use will require a review and approval from the ethics review committee such as the IRB. A widespread support shows that the one-time general consent offers the choice most donors would make. It also offers an opportunity to decline from contributing for those unwilling to contribute or for those who want more specific control over their specimens.

Arthur: A tiered consent would outline the likely uses of the specimen, disposition of materials, policy regarding the sale of material to third parties, transfer of control, and availability of clinical findings that would be relevant to donors.

Michael: A consent menu that has multiple choices would be best. A study found that although 10 percent support the consenting menu, most prefer 48 percent of blanket consent while the rest (42 percent) prefer re-consenting when a new research project begins.

Jack: Specific consent would help provide assurances that both the participants and researchers are equal in the enterprise. It is also easily accomplished when samples are obtained for a specific project. However, it can become impractical if the samples are stored for long-term with undefined uses. Most participants that are based in the United States are willing to contribute their samples.

What about property rights to specimens? Should research participants share potential financial gain?

David: Generally, individuals should share the benefits to the project they contributed to. Failing to provide a fair level of benefit can be regarded as a case of exploitation. Since the samples are part of an important contribution, it would suggest that the participants should share the benefits such as financial gain from the research projects. However, this can become complicated in practice as it is unclear what is a fair level of benefit or how benefits can be provided.

Michael: As part of the consent process, participants should be informed about property rights, the potential for financial gain (for the investigator), and if they themselves will share any financial gain. Once the participant is aware before enrolled in the study, the allowing or disallowing of financial gain and property rights should be acceptable. Consent should not be waived in cases if there is expected financial gain for the investigator.

Jack: Unless the research is conducted with the objective of developing a commercial product, proprietary interest in research by the donors can be difficult. It can be hard to put a value on something that has yet to exist especially in cases where there is an assertion of property rights where there is litigation corresponding with perceived value.

References:
Gronowski AM, Moye J, Wendler DS, Caplan AL, Christman M. The use of human tissues in research: what do we owe the research subjects? Clinical Chemistry. 2011; 57 (4): 540-544.

Human Biospecimens: Ethics and Regulations

Overview of Human Biospecimens

The human body and its collection of tissues have been studied since the ancient Greek times. After the Roman Empire fell, anatomical studies slowed down considerably as the use of cadavers became illegal in many places. Researchers were prosecuted for many years if they performed postmortem dissections. In the 15th century, medical schools in Europe allowed their researchers to study the human body and tissues without prosecution. Since then, the study of the human body has advanced significantly. Today, human biospecimens and tissue samples are vital for genetic research. Human biospecimens can be collected from several different sources:

• Prospective tissue collection

• Excess tissue obtained from clinical samples

• Specimens from cadavers

• Tissues with reproductive potential

With the increasing use of human biospecimens in research and clinical trials, issues regarding the ethics and regulations of these specimens needs to continuously be observed.

Governing Treaties, Laws, and Regulations

It is important to understand laws and regulations concerning human biospecimens as it helps researchers with issues of biospecimen ownership and ethical principles about human experimentation. One of the first important efforts of the medical community to regulate this kind of research is the Declaration of Helsinki. While it is not an international legally binding instrument, it has significantly influenced many regulations and national legislations.

Originally adopted in 1964, it has gone through six revisions. In the United States, the Code of Federal Regulations established by the government addresses the protection of the donors. In the Code of Federal regulations is the Common Rule that details the function and role of institutional review boards (IRBs) in the protection of human participants during the research activities. It also outlines the requirements in obtaining informed consent and additional protection for vulnerable groups such as pregnant mothers, neonates, fetuses, children, and prisoners. Some states also have their own laws that govern research using human participants.

Informed Consent

For informed consent, researchers must provide an explanation to potential participants regarding the purposes of the research and expected duration of the study. It should be noted that the descriptions provided should not be general and must be specific to the study. Without being adequately informed about the intended purpose of the research, participants cannot give “informed” consent the key element in the consent process is transparency. Participants should also know all the intended uses of the specimens. If their specimen is required in future research, additional informed consent should be obtained from the donors. However, the IRB can waive the need for informed consent for the use in a secondary project. IRB waiver is more likely if the donor has consented to future research at the time of tissue collection.

The participant also has to be informed regarding the potential risks, benefits, alternatives to participation, and what may be required of them during the study. Additional information required includes compensation and medical treatments that could be available should injury occur to the participants. Participation must be voluntary, and participants should be allowed to withdraw at any time without risk of penalty. Participants should also be provided a contact if they have any questions or concerns regarding the research.

The Common Rule is only applicable to human participants. In some circumstances, it permits research without participant consent. If the research is conducted using anonymous samples without access to the participant’s private information, by definition, informed consent would not be required. The Common Rule also does not apply if the IRB exempts it as the information used does not involve the identification of the donor. Finally, the IRB can waive or change the requirements of the informed consent if:

• The research poses no more than minimal risk to participants

• The welfare and rights of participants are not affected

• The research cannot be conducted without alteration or waiver of informed consent

• The participants are provided with relevant information

Biospecimen Ownership

The ownership of biospecimens has been analyzed in many cases. It has been a question of whether the donor retains ownership rights of their tissue. It has also been debated as an issue of “guardianship” versus “ownership”. In most cases involving excised tissue, courts have concluded that donors do not retain ownership of their excised tissue. However, different rulings have been reached in cases where there has been a previous understanding that the patient would retain their ownership rights. With leftover materials, many are considered to be “abandoned” with patients no longer having any property rights. In tissue obtained postmortem, the Common Rule does not apply as it only applies to living individuals. The Uniform Anatomical Gift Act (UAGA) allows individuals to give their bodies for the study of science. Without the individual’s consent, their spouse or family can also make the gift.

Conclusion

The laws regarding human biospecimens are still evolving. There will be much effort and discussion needed to improve the efficiency of informed consent. With increasing studies using human biospecimens, the frequency of lawsuits may be higher. It is therefore important for new legislatures and regulations as it can help to protect or help both participants and researchers. It is crucial for researchers to strive for transparency and avoid using specimens not outlined in the consent form. The awareness of existing rules is also essential to avoid lawsuits and the destruction of valuable human biospecimens.

Reference:

Allen MJ, Powers MLE, Gronowski KS, Gronowski AM. Human tissue ownership and use in research: what laboratorians and researchers should know. Clinical Chemistry. 2010; 56 (11): 1675-1682.

Preanalytical Variables: Long-Term Storage and Retrieval of Biospecimens

Introduction

The week before last we talked about how pre-analytical variables affect the integrity of human biospecimens, and this week we’ll be following up on this article by discussing the long term storage and retrieval of biospecimens.

The term “storage” comprises of both short and long-term storage of all biospecimens consistent with the study design and planned future use. Depending on the details of their future use, the specimens are either locally or centrally stored. It can also be stored in both locations. The decision will be made depending on the:

• Sample size of biospecimens

• Complexity of collection

• The accrual rate of biospecimens

• Processing procedures

• Logistics

• Cost of storage and retrieval

• Quality issues

• Biorepository governance factors

If the biospecimens are stored for various uses, biorepositories should have duplicates that are close in proximity to the main laboratory. Samples that are to be stored for more than a year should be stored centrally. Duplicates should also be stored on different power supplies or different locations as insurance against natural disasters or equipment failure. Biorepositories are also recommended to have approximately 10 percent of the total mechanical freezers as empty backup freezers to protect against freezer failure. Different storage conditions may be required based on the downstream analyses. Some of the pre-analytical variables that affect long-term storage include:

• The time involved from processing to storage

• Duration of storage

• Temperature

• Facility

• Environmental impact (such as moisture, sunlight, dehydration, humidity, oxidation, evaporation, and desiccation)

• Freeze-thaw cycles

• Some emergencies include: encapsulation of biospecimens in ice after refreezing and microbiological contamination

• Destroyed or no labeling

• Missing or misplaced aliquots

Since biobank material is valuable and hard to replace, the use of systems such as the laboratory information management system (LIMS) should be utilized as it helps allow traceability, confirm chain of custody, and manage biospecimens to improve data reliability and retrieval. Once the integrity of a biospecimen is compromised, it is no longer valuable and becomes useless. It is therefore important to retrieve only those biospecimens that are required. As previously mentioned, duplicate collections of biospecimens are ideal to prevent the destruction of samples.

Blood Sample

The study of the stability of analytes compared to the fresh sample, taking into account the recovery rates, are vital to determine the effects of long-term storage. After long-term storage, the recovery rates may decrease or increase resulting in increased or attenuated risk ratios. It is recommended that hormone, chemistry, and protein analytes are much more stable and stored at -80⁰C for up to 13 months. However, various studies have shown that there are different patterns of stability based on the analyte, time, and temperature of storage. There has been no systematic influence regarding omics analyses observed in samples collected in citrate, heparin, or ethylenediamine triacetic acid (EDTA) if stored at -80⁰C in liquid nitrogen. Long term storage in room temperature and repeated freeze cycles must be avoided. At room, low, and ultra-low temperatures, the extraction of DNA from whole blood samples using bio stabilization technology yielded samples that are pure and that have integrity. Although live cells are stable at room temperature for as long as 48 hours, it should be cryopreserved or cultured in liquid nitrogen to ensure its viability. The recovery of sufficient DNA or those that are of acceptable quality for microarray studies involves the transfer of thawed buffy coat or EDTA whole blood into RNA preservative. Serum or plasma that will be used for miRNA analysis must be extracted immediately or maintained at -80 in RNA free cryotubes.

Urine Sample Protocol

For urine samples, long term storage at temperatures less than -80⁰C without additives is ideal unless it has been specified for certain downstream analyses. Urine samples have been stored at -22⁰C for 12 to 15 years without the use of preservatives while ensuring the stability and measurement validity. Urine used for metabolome and proteome analyses will go through progressive protein degradation if stored at room temperature. While freeze-thaw cycles have minimal impact on the protein profiles, repeated cycles should ideally be avoided.

Saliva Sample Protocol

The protocols for saliva storage are ultimately dependent on the expected downstream analyses. There seems to be minimal impact of protein profile changes despite freeze-thaw cycles. It is recommended that it is stored at -80⁰C. If the saliva samples were divided into aliquots and frozen immediately at -80⁰C, there does not seem to be any differences in cortisol, C-reactive protein, mRNA, or cytokines.

Extracted DNA Sample protocol

The most common method of storage for DNA is still freezing it at -80⁰C. It should be noted that DNA degradation increases with repeated freeze-thaw cycles, higher storage temperature, dilution, and multiple suspensions. Special technologies allow the minimization of storage space and the reduction of shipping and electrical costs. This can be beneficial especially when cryogenic or mechanical equipment is unavailable. It can also be an alternative method for backup storage. Using this technology, there is no degradation or accelerated aging of DNA at room temperature or higher temperatures (50-70⁰C) throughout the 8-month storage duration.

RNA sample protocol

Some of the pre-analytical storage factors that can affect the quality and quantity of analyte or gene expression include the concentration of RNA, temperature, storage time, and repeated thaws. New technology for the dry storage of RNA at room temperature has been developed. This is a technology comparable to RNA that is cryopreserved for up to a year for downstream analyses such as RNA sequencing and real-time polymerase chain reaction.

References:

Ellervik C, Vaught J. Preanalytical variables affecting the integrity of human biospecimens in biobanking. Clinical Chemistry. 2015; 61(7): 913-934. http://clinchem.aaccjnls.org/content/clinchem/61/7/914.full.pdf

Pre-analytical Variables Affecting the Integrity of Human Biospecimens

Introduction

Biorepositories or biobanks function to collect, process, transport, and store biospecimens. The integrity of these biospecimens is crucial for the success of clinical trials and research. There are many factors that can influence the results within research such as:

• Pre-analytical environmental or biological variables

• Pre-analytical technical variables

• Analytical variables

• Post-analytical variables

Pre-analytical variables are defined as factors that can have an impact before the start of the analytical phase. It not only affects the integrity of the tissue samples, but also the results of the analysis. Pre-analytical variables are critical as the analytical integrity of the research can be jeopardized. Seeing as most errors in the laboratory can be attributed to pre-analytical errors this stage is of upmost importance.

Pre-analytical Factors in the Collection of Biospecimens

It is important to adhere to guidelines for general laboratory safety. The collection of biospecimens require a balance of:

• Accrual rate

• Types of biospecimens

• Sample size

• Costs

• Location

• Storage requirements

• Transport logistics

The biospecimens collected can be either invasive or noninvasive. Biospecimens that are collected through non-invasive methods may lead to an increase in sample size due to easiness of collection, reduced costs, and willingness of donor to participate. This method is especially important when dealing with pediatric biobanking. It is important that biological and environmental factors are standardized and documented when interpreting results as it can affect the downstream analysis. It is also vital to take measurements to observe the effects of intervention and the changes over time.

Pre-Analytical Factors That Affect the Collection of Blood Samples and Its Derivatives

The collection of blood samples should be performed by trained staff. Those that are involved in collecting samples from children should specialize in pediatric phlebotomy. The staff needs to be highly trained as this ensures the highest quality of specimens and prevents the donors from experiencing any kind of discomfort. Depending on the research requirements different additives may be required. Different types of additives are coded using different colored collection tubes. Some of the important pre-analytical factors to take note of include:

1. Using the same tube brand and the same lot number throughout the study. This would be ideal as different brands can have different anticoagulants, additives, and may introduce bias.

2. Another important factor is the expiration dates on the tubes as the vacuum in these tubes can decrease with age and negatively impact the blood draw and filling of the tube.

3. Using the same posture such as supine, standing, or supine as these can cause plasma volume changes that may lead to increased analyte levels.

4. Using the recommended needle gauge as a needle that is too thin can lead to hemolysis that distorts the potassium concentrations and hematological cell counts.

5. Using the recommended and same duration of tourniquet use as prolonged use can cause changes in analyte concentrations and hemoconcentration.

6. Avoid inadequate filling as this can result in inaccurate results due to the decrease in blood and additive ratio.

A general rule for common analyses is to use ethylenediaminetetraacetic acid for hematology, DNA, hemoglobin A1C, and a range of proteins. For plasma glucose, it is recommended to use sodium-fluoride tubes while lithium heparin plasma can be used for assays such as kidney function, iron parameters, liver enzymes, thyroid hormones, C-reactive protein, and more.

In remote sites that are resource-poor, capillary dry blood spot (DBS) are easy biospecimens that can be collected. Small volumes of capillary blood from the peripheries can be deposited onto specific paper cards and dried at room temperature for three to four hours. DBS can be used in many analyses. However, some of the pre-analytical variables to note are:

·         Type of collection paper used

·         Type of chemical used in the manufacturing of the paper

·         Thickness of paper

·         The volume of blood deposited

·         Environmental factors such as heat, humidity, sunlight, and moisture

In DNA and RNA collection, there are also biological factors that can affect the biospecimens. These include the donor’s:

·         Gender

·         Age

·         Body mass index

·         Tobacco consumption

Since RNA is more vulnerable to degradation, some of the preanalytical collection factors that can affect the integrity are:

• Tube additive

• Tube type

• Tube sterility

• Type of biospecimen

• The volume of blood collected

• Short-term storage temperature

• Lag time until extraction

In microRNA’s, the pre-analytical variables include:

• Diet

• Age

• Race

• Exercise

• Drugs

• Altitude

• Tobacco use

• Chemicals

• Hemolysis

• Coagulation times

• Temperature

Pre-analytical Factors That Affect the Collection of Urine and Saliva

Urine can be collected in many different ways as it can be used for measurements of many analytes. In urine biospecimens, the preanalytical requirements can be conflicting.  This may result in the requirement of multiple biospecimens. Some of the preanalytical variables for urine collection include:

• Collection method

• Environmental exposure

• Urine dilution

• Dipstick components

• Preservatives or additives used

For saliva, these biospecimens have many advantages as they are easy to collect and can be used in many situations especially if donors are afraid of needles. The preanalytical variables for this biospecimen include:

• The time of collection

• The temperature the specimen is stored

• The collection method

Conclusion

The factors mentioned are pre-analytical variables that affect the biospecimens during the collection phase. However, it is important to note that there are many more pre-analytical variables that can affect the integrity of the biospecimens during the processing, transport, and storage phase.

The Incredible MicroRNA's

What is microRNA?

MicroRNAs (miRNAs) are a group of small non-coding RNAs that are found in tissue samples of animals, plants, and some viruses. Since the discovery of circulating and extracellular miRNAs, there has been a rapid expansion of studies on miRNAs in biofluids like cerebrospinal fluid, plasma, serum, and urine. miRNAs are similar to small interfering RNAs (siRNAs). However, miRNAs originate from RNA transcripts forming short hairpins while siRNAs are from longer parts of double-stranded RNA. miRNAs are plentiful in mammalian cells and seem to target approximately 60% of these genes. miRNAs are thought to have important biological functions as they are evolutionary conserved. A good example is where there is conservation of 90 families of miRNAs as seen in the common ancestor of fish and mammals. The majority of these conserved miRNAs have been shown to play important roles.

Roles of miRNA

miRNA plays a role in RNA silencing and gene expression as post-transcriptional regulators. Within mRNAs, miRNAs function by base-pairing with the complementary molecules causing mRNA molecules to be silenced through cleavage of mRNA strand, destabilization of mRNA, or reduced efficiency of mRNA translation by ribosomes. Despite the low numbers of miRNA, it is estimated that the miRNAs regulate approximately more than 33% of the cellular transcriptome. Therefore, it should be no surprise that the miRNAs have crucial functions in the developmental and cellular processes that have been thought to be involved in many human diseases.

Since miRNAs are relatively stable in biofluids and have a wide range of biological potential, these molecules are suited to be used as non-invasive biomarkers in diagnosis, drug safety, and pre-clinical toxicity. Many studies have proven that secreted miRNAs are involved in conditions such as organ damage, cancers, and coronary heart disease. While the exact role of circulating miRNAs is mostly unknown, they have been observed to be protected from RNAse degradation through the inclusion in membranous particles or protein complexes.

Disease

Since miRNA plays a role in the normal functioning of eukaryotic cells, miRNA dysregulation is therefore linked to disease. For example:

a)       Hereditary Diseases

• It has been found that a mutation in the region of miR-96 results in progressive hearing loss.

• Hereditary keratoconus with anterior polar cataract is seen in mutation in the region of miR-184.

• Growth and skeletal defects can be seen in those with deletion of miR-17 to 92 cluster.

b)      Cancer

• Chronic lymphocytic leukemia was one of the first human diseases that has been associated with miRNA dysregulation. Other miRNAs that have also been linked to cancer are called “oncomirs”. miRNAs are involved in pathways that are crucial in B-cell development such as B-cell receptor signaling, cell-cell interaction, B-cell migration or adhesion, and production or class-switching of immunoglobulins. They also influence the generation of marginal zone, follicular, plasma, B1, and memory B cells.

• Another clinical trial is using miRNA as a screening assay for the detection of colorectal cancer in the early stages.

• miRNAs can also be used to determine prognosis in cancers based on their expression level. For example, a study on non-small-cell lung carcinoma determined that a low level of miR-324a levels is an indication of poor prognosis. In colorectal cancer, a low level of miR-133b and high level of miR-185 is linked with metastasis resulting in poor prognosis.

c)       Heart Disease

• Studies have found that there are specific changes in the expression levels of miRNAs in diseased hearts which points to the involvement of miRNAs in cardiomyopathies.

• miRNAs can also be used to determine the prognosis and risk stratification of cardiovascular diseases.

• In animal models, miRNAs have been associated with the regulation and metabolism of cholesterol.

d)      Nervous System

• miRNAs are thought to be involved in the function and development of the nervous system.

• Neural miRNAs such as miR-124, miR-132 and miR-134 are involved in dendritogenesis.

• Other neural miRNAs are involved in the formation of the synapses. miR-134 and miR-138 are thought to be included in the process of synapse maturation.

• Studies have found that conditions such as bipolar disorder, schizophrenia, anxiety disorders, and major depression have altered miRNA expression.

e)      Obesity

• miRNAs have a vital role in the differentiation of stem cell progenitors into adipocytes. Studies have found that the expression of miRNAs 155, 221, and 222 can inhibit adipogenesis paving a possible genetic treatment for obesity.

• miRNAs of the let-7 family was found to accumulate in tissues as aging occurs. Based on animal models, the excessive expression of let-7 class miRNAs resulted in accelerated aging, insulin resistance, and therefore increases the risk of obesity and diabetes. When let-7 was inhibited, it resulted in an increase in insulin sensitivity and resistance to high-fat-diet-induced obesity. This means the inhibition of let-7 may prevent, reverse, and cure obesity and diabetes.

Conclusion

Although miRNAs have great promise in the screening, diagnosis, treatment, and prevention of various pathological conditions, more research, and clinical trials will be needed in the study of miRNAs to further explore and discover the potential of miRNAs.

References:

1. microRNA. Wikipedia. Accessed 8/22/2018. https://en.wikipedia.org/wiki/MicroRNA#Disease

2. Blondal T, Nielsen SJ, Baker A, et al. Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods. 2013; 59(1): S1-S6.

CRO Services

Introduction

A contract research organization or CRO refers to a company that provides support in the form of research. The research conducted through CRO services can be in one of the following fields:

• Biotechnology

• Pharmaceutical

• Medical device industry

In full a CRO is a company contracted by another organization to help lead and manage their trials, responsibilities, roles, and their function.

Services and Advantages

Some CRO services executed include such things as, but not limited to:

• Biologic assay development

• Biopharmaceutical development

• Commercialization

• Preclinical and clinical research

• Management of clinical trials

• Database design and building

• Data entry and validation

• Medicine and disease coding

• Quality and metric reporting

• Statistical

• Pharmacovigilance (the identification, detection, assessment, observation, and prevention of side effects of pharmaceutical products)

CROs are useful for companies when developing new drugs and medications as they reduce costs. CROs are able to simplify the development of new drugs and entry into drug markets. They also support governmental organizations, foundations, universities, and research institutions. CROs can range from small specialty groups to large international organizations. They aim to provide support for clinical studies and trials. Those that specialize in clinical trials services of a new drug are present from its conception until it is approved by the Food and Drug Administration (FDA) or by the  European Medicines Agency (EMA). Evidently CROs play a crucial role and pharmaceutical companies are continually outsourcing critical functions such as research and manufacturing to CROs.

The number of major corporations that are using CROs in clinical trials and the development of new drugs is increasing. Companies that establish contract with CROs aim to acquire the required expertise without having to hire permanent staff, keeping overhead low. Some CRO trade groups have claimed that contracting with CROs has helped reduce the cost by decreasing the time it takes to conduct a trial. This also means that the company that hires a CRO will not need the required infrastructure, manpower, and office space to conduct these trials. Some CROs can even manage all the aspects in a clinical trial starting from the site and patient selection all the way up until the final regulatory approval.

Regulatory Aspects

The International Council on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) defined CROs as “a person or organization contracted by the sponsor to perform trial-related duties and functions”. Their guidelines highlight the following:

• A sponsor can transfer all their duties and function to a CRO. However, the hiring company will be responsible for the integrity of data acquired from the CRO conducted study. It remains the hiring company’s responsibility to ensure that all the data is factual and backed by science.

• CROs should ensure quality control and quality assurance.

• All duties and functions transferred to a CRO should be in writing. The hiring company should oversee the duties and functions that are carried out on their behalf.

• Duties and functions that are not transferred to a CRO will remain with the sponsor.

Market Size and Growth

The Association of Clinical Research Organizations has estimated that more than half of clinical studies conducted by the pharmaceutical industry have been outsourced to CROs. The most popular field for CROs is therapeutic work including infectious disease, oncology, central nervous system, cardiovascular disease, and metabolic disorders. A further 27% work for within the biotechnology field while the remainder work for governments, foundations, and the medical device industry. The CRO sector is doing extremely well for an industry that just started a decade ago. There is increasing pressure facing medical devices organizations and pharmaceutical companies for the high cost of drugs and they are trying to lower costs without decreasing their profits. One of the best and most common solutions is to outsource clinical trial management as it results in significantly lower overhead costs.

By the year 2013, there were more than 1,100 CROs globally. However, there are many CROs that have gone out of business or that have been acquired. In 2008, it was estimated that the top 10 companies control 56% of the market. A 2007 estimate showed that the market would reach $24 billion in 2010 with a growth rate of 8.5% from 2009 to 2015. In 2016, the research and development spending increased by 15.5% from 2015 to 2020.

Conclusion

CROs are an effective solution as it provides an affordable option for companies to pursue the development and approval of new medication. Before the existence of CROs, this was a hugely expensive endeavor which was only embarked on when the likely hood of regulatory approval was high. With CROs, companies are now able to develop drugs for specific markets.

References:

1. Contract research organization. Wikipedia. Accessed 7/30/2018. https://en.wikipedia.org/wiki/Contract_research_organization

2. Stone K. Contract research organizations (CRO) definition. The Balance. Accessed 7/30/2018. https://www.thebalance.com/contract-research-organizations-cro-2663066

Tissue Procurement Problems

Introduction

Formalin-fixed paraffin embedded (FFPE) tissue blocks are a valuable resource for many significant research programs as researchers tend to select this type of biospecimen as frozen or fresh tissue blocks may be harder to acquire. There is also the challenge that the number of fresh frozen tissue samples may not be able to fulfill the requirements of the research protocol. The FFPE preservation technique demands a fast and rapid fixation after the process of resection of the target biospecimen in a neutral buffered formalin. Once fixed, the specimen is then embedded in paraffin wax. FFPE is now the commonest tissue preparation method used to archive research biospecimens. Currently, almost all surgeries in this day generate FFPE tissue samples. FFPE tissue blocks are crucial as they offer the potential for the discovery of significant information especially in biomedical research programs and drug discovery. Other research applications that utilize FFPE biospecimens include:

• genetic studies

• biomedical identification or authentication

• visualization of tissue structure

This, therefore, makes FFPE tissue blocks ideal for:

• the study of autoimmune diseases – rheumatoid arthritis, systemic lupus erythematosus

• the study of long-term cancers – colon cancer, lung cancer, breast cancer

Challenges

However, there are several challenges that researchers face when it comes to obtaining FFPE tissue samples. Some of the challenges include:

1)     Oversight of the Pathologist

In some cases, the FFPE samples are not obtained or processed appropriately as the certified pathologist is not on site to supervise and ensure that the proper procedures are followed during the procurement of the specimen. Biorepositories should ensure that they hire enough licensed pathologists to ensure that there is no manpower shortage as it could impact the quality of their tissue specimens.

2)     Difficulty Acquiring Samples

There are certain situations where a research team or company requires access to FFPE tissue samples that are hard to come by. For example, FFPE samples from patients with metastatic melanoma might present a challenge to biorepositories. Research teams should partner with a biorepository that has a vast network for tissue procurement as it can help tremendously in the collection of special samples needed in specific research protocols.

3)     Rapid Turnaround

Studies or research that needs a quick procurement of FFPE tissue blocks may pose a challenge to many biorepositories. These research teams that are looking for a rapid turnaround of samples should ask biorepositories about their accelerated procurement method which may then be able to provide the required biospecimens within the time frame.

4)     Transparency

Research teams looking to procure tissue samples need to keep in mind about transparency as studies rely on well-annotated tissue blocks that go through the proper fixation and preservation technique. Before obtaining samples from the biorepository, be sure to ask regarding their specifics and standard of procedure for the fixation and quality assurance protocols. This can impact the result and credibility of the entire study.

5)     Review of FFPE Biospecimens

Some biorepositories obtain their tissue samples from local sources such as the local hospitals. However, not all providers take the time to ensure that the specimens are of the highest quality. Biorepositories are responsible to ensure that a gross and microscopic examination of the FFPE biospecimens to ensure that the samples obtained are of the highest quality.

6)     Sourcing

With the growing number of organizations and biorepositories, research teams that are looking to source biospecimens should consider companies that follow the best practices and have high standards when it comes to their collection and fixation protocols. Unreliable sources may not be able to provide high-quality FFPE tissue samples. Before procuring the required biospecimens, enquire where the organization obtains their samples and the reliability of it.

7)     Patient Information

The patient information from the samples can be valuable and crucial in a research. The more information you can obtain regarding the patient from their data, it can help with some of the results from the research especially in terms of demographics and risk factors of a disease. Patient information also helps you to find the correct patient cohort as some study designs exclude those below or above a certain age. It is therefore important to procure the biospecimens from a biorepository that can provide the necessary data such as the details or a refractory disease, metastatic diseases, or newly diagnosed disease. A new case of cancer or a relapse can also affect the results of a study greatly.

Conclusion

In conclusion, there are many factors to consider when it comes to selecting a new biorepository or organization to partner with to obtain tissue samples. It is important as these specimens ultimately determine the results and credibility of the study. Choosing a credible company that provides the highest quality FFPE tissue samples is one of the most crucial steps in the early stages of a study. Low-quality biospecimens result in wasted hours of study, effort, and decreases the overall morale of the research team.

References:

Doiron L. Typical problems with FFPE tissue samples – and how to solve them. 2014. Folio Conversant. Accessed 7/11/2018. 

https://www.conversantbio.com/blog/bid/387449/Typical-Problems-with-FFPE-Tissue-Samples-And-How-to-Solve-Them

What are FFPE Samples

What is Formalin Fixed Paraffin Embedded Tissue?

Formalin fixed paraffin embedded or FFPE tissues are valuable for both therapeutic applications and research. FFPE is a specific technique used to prepare and preserve tissue specimens utilized in research, examination, diagnostics, and drug development. Tissues are first collected from both diseased and non-diseased donors. The tissue specimen is first preserved through a process called formalin fixing. This step helps to preserve the vital structures and protein within the tissue. It is then embedded into a paraffin wax block and sliced into the required slices, mounted on a microscopic slide, and examined.

The FFPE Process

The process starts by a specimen being selected and then excised from a donor or patient. Samples can also be obtained from other animals such as snakes, mice, or many others. After excision, the tissue is immersed for approximately eighteen to twenty-four hours in a 10% neutral buffered formalin. The tissue is then dehydrated using increasing concentrates of ethanol. Next, the tissue is embedded into paraffin to become FFPE blocks. The methods utilized are dependent on the requirements of the researcher or physician who is requesting the FFPE samples. Specifications about how the issue is cut, size, and purpose of the tissue are all important. Once the procedure is complete a certified pathologist will  evaluate the quality of the specimen.

Storage of FFPE Tissue

FFPE samples can be stored in hospitals, biobanks, and research centers. Storage facilities often keep records of how the tissue was collected, the preservation procedures, and demographic information (such as, but not limited too: the origin, duration, age, ethnicity, gender, and stage of disease) of the donor. The demographic information is an important factor in research and in clinical trials. FFPE samples that are properly preserved are very valuable and can be stored at room temperature for a long period of time.

Applications

FFPE samples are important as they are often used in:

a)       Immunohistochemistry

The sectioned FFPE specimens are mounted on a slide, bathed in a solution containing antibodies, and then stained so that they can be more clearly seen. This method is important for physicians and researchers looking for pathology in the tissue such as Alzheimer’s or cancer.

b)      Oncology

FFPE samples are vital in the field of oncology as tumor tissues have characteristic morphologies allowing researchers to look for certain proteins. These proteins are then used to help in the assessment of treatment and diagnosis.

c)       Hematology

In the study of blood and its disorders, FFPE samples are important in determining the anomalies and discovery of cures. The specimens can be used in studies related to tissue regeneration, genetics, and toxicology.

d)      Immunology

FFPE samples from a donor with autoimmune disease helps in determining the cause and development of therapy for the condition.

Complications or Limitations

One of the possible limitations of the fixation process using formalin is the potential denaturation of the proteins that are present in the tissue making them undetectable to antibodies. To compensate for this issue, antigen retrieval techniques were developed. The antigen retrieval technique specifically recovers DNA, RNA, and proteins from FFPE samples. For this method to work, the quality of FFPE samples are critical. There is also the issue that there is no standard procedure to be used in the preanalytical processing such as fixation and DNA isolation. This means that minor differences such as the different use of instruments, sample handling, and methodology can result in variation that affects the quality of DNA and study results. Some of the factors that have been found to affect study results from FFPE samples are:

1. Inaccurate logging of fixation protocol

2. Variation in fixation time

3. Temperature during fixation

4. Storage conditions of FFPE samples

Quality Control

To ensure the highest quality of FFPE samples, those who collect and store these samples should:

1. Follow ethical and legal standards.

2. Keep a clear and accurate record of donors.

3. Provide information regarding the sampling and collection process

4. Be supervised by a licensed pathologist during the collection of samples

5. Have a complete chain of custody

6. Work only with a carefully selected network of distributors that consistently provide high quality and accurate samples

Fresh Frozen Tissue

Fresh frozen tissues are specimens that are preserved using liquid nitrogen through a method known as “flash freezing”. These specimens are then stored in a freezer that is set at a temperature of less than -80 degrees Celsius. Fresh frozen tissue has different applications than  FFPE samples as they can be used in native morphology studies or molecular analysis as well.

FFPE Samples Vs. Fresh Frozen Tissue

FFPE and fresh frozen tissue have their pros and cons. They are two different types of samples that have different uses dependent on the requirements of the research or clinical study. 

1. FFPE blocks are very hard and can be easily stored at room temperature for decades without the need of special equipment making the type of tissue sample very cost efficient.

2. There is a large archive of FFPE samples available for researchers due to the easiness associated with it storage.

3. FFPE specimens have been used for decades making it incredibly familiar to pathologists.

4. Fresh frozen tissue is much more suitable for the analysis of native proteins, polymerase chain reaction, and next generation DNA sequencing.

5. Fresh frozen tissue ensures the preservation of DNA, RNA, and native proteins.

6. Fresh frozen tissues require specialized equipment for storage. This means mechanical failure, power outages, and carelessness can affect the quality of the samples.

References:

1)      Ward T. The importance of proper formalin fixation of FFPE samples. Personalis. Accessed 6/19/2018. https://www.personalis.com/importance-proper-formalin-fixation-ffpe-specimens/

2)      Doiron L. 5 quality control rules for cancer tissue banks. Folio Conversant. Accessed 6/19/2018. http://www.conversantbio.com/blog/bid/339034/5-Quality-Control-Rules-for-Cancer-Tissue-Banks

3)      FFPE vs frozen tissue samples. BioChain. Accessed 6/19/2018. https://www.biochain.com/general/ffpe-vs-frozen-tissue-samples/

4)      What is FFPE tissue and what are its uses. BioChain. Accessed 6/19/2018. https://www.biochain.com/general/what-is-ffpe-tissue/

Cancer Therapy

Thanks to extended research from human tissue samples we have been able to make major breakthroughs in cancer research. In the twenty-first century, evidence, both epidemiologically and clinically, have supported that the changes in whole-body metabolism can affect oncogenesis, the progression of tumors, and the response of tumor to therapy. It has been observed that metabolic conditions such as hyperglycemia, obesity, hyperlipidemia, and insulin resistance are associated higher with risk of cancer development, accelerated progression of tumors, and poor clinical outcome. Due to these findings, many clinical studies indicate that statins and metformin may help in decreasing cancer-related mortality and morbidity. Phenformin is another drug used to treat diabetics that can help with anticancer effects. However, phenformin was discontinued in the late 1970s due to a high incidence of lactic acidosis. Metformin is the most commonly used antihyperglycemic agent globally. It has an optimal pharmacokinetic profile with:

·         50 – 60% of absolute oral bioavailability

·         Slow absorption

·         Negligible binding to plasma protein

·         Broad tissue distribution

·         No hepatic metabolism

·         Limited drug interactions

·         Rapid urinary interaction

It also has an exceptional safety profile as there is a low number of individuals who have side effects. Statins also have a great safety profile and is currently used by a large population.

Cancer and Cellular Metabolism

The accumulation of evidence has suggested that malignant transformation is linked to changes that affect several factors of metabolism. Metabolic rearrangements associated with cancer have been linked with the inactivation of tumor suppressor genes and activation of proto-oncogenes. However, the accumulation of metabolites such as fumarate, succinate, and 2-hydroxyglutarate (2-HG) drives oncogenesis through the signal transduction cascades. Conclusively, these observations support the notion that signal transduction and intermediate metabolism are associated.

a)       Oncogenes and Metabolism

The signaling pathways from oncogenic drivers are linked to metabolic alterations due to cancer. For example, the expression of the PKM2 (an M2 isoform of pyruvate kinase) encourages the alteration of glycolytic intermediates in the direction of anabolic metabolism while regulating both transcriptional and post-transcriptional program that leads to the addiction of glutamine.

b)      Oncosuppressors and Metabolism

There are some oncosuppressor proteins that can regulate cellular metabolism. The inactivation of tumor suppressor p53 happens in more than 50% of all neoplasms causes a variety of metabolic consequences that could potentially stimulate the Warburg effect. P53 can possibly suppress the transcription of GLUT4 and GLUT1 and stimulate the expression of apoptosis regulator (TIGAR), TP53 induced glycolysis, SCO2, glutaminase 2 (GLS2) and many other pro-autophagic factors. It also interacts physically with glucose-6-phosphate-dehydrogenase (G6PD) with RB1-inducible coiled-coil 1 (RB1CC1).

c)       Oncometabolites and Oncoenzymes

It was found that metabolites can contribute to oncogenesis when mutations such as fumarate hydratase (FH) and succinate dehydrogenase (SDH) was linked to sporadic and familial types of cancer including pheochromocytoma, leiomyoma, renal cell carcinoma, and paraganglioma. once the enzymatic activity of SDH and FH is disrupted, succinate and fumarate accumulate resulting in oncogenesis.

Targeting Cancer Metabolism

The metabolic targets for cancer therapy rewiring of cancer cells is seen as a promising source for new drug targets. Some different approaches have resulted in the identification of agents that can help with targeting glucose metabolism for cancer therapy. However, the low number of metabolic inhibitors reflect the recent rediscovery of the field. There are also some concerns about the uniformity between malignant cells and non-transformed cells that are undergoing proliferation.

a)       Targeting Bioenergetic Metabolism

Some cancer-associated alterations such as the Krebs cycle, glycolysis, glutaminolysis, mitochondrial respiration, and fatty acid oxidation have been studied as potential sites for drug therapy.

b)      Targeting Anabolic Metabolism

The anabolic metabolism in cancer cells increases the output from nucleotide, protein, and protein biosynthesis pathways to help with the generation of new biomass in rapidly proliferating cells (includes both normal and malignant). A high metabolic flux through the pentose phosphate pathway is vital to cancer cells as it generates ribose-5-phosphate and nicotinamide adenine dinucleotide phosphate (NADPH).

c)       Targeting Other Metabolic Pathways

Other pathways involved in the adaptation to metabolic stress may provide drug targets for cancer therapy. This applies to autophagy, hypoxia-inducible factors 1, and nicotinamide adenine dinucleotide metabolism. A competitor of nicotinamide phosphoribosyltransferase (NAMPT) known as FK866 has been observed to have antineoplastic effects in murine tumor models.

Conclusion

The extensive metabolic rewiring in malignant cells provides a large number of possible drug targets. Many agents that target metabolic enzymes are used for decades while others are being developed. Therefore, the use of metabolic modulators that could be complicated by the similarities of highly proliferating normal cells and metabolism of malignant cells, there might be a chance to harness the antineoplastic activity of these drugs clinically. While many efforts were focused on merging metabolic modulators and targeted anticancer drugs, there may be a common view that metabolism and signal transduction are mostly independent if not separate entities. More research is needed to study the extent of how the metabolic functions of oncosuppressive and oncogenic systems contribute to the biological activity.

References:

Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Metabolic targets for cancer therapy. Nature Reviews Drug Discovery. 2013; 12: 829-846.