Question: What is the clinical context which will be informed by this biomarker and what is the unmet need for the development of new biomarkers?

The first step to initiating a study protocol on blood-based biomarkers is identifying the significance of the proposed biomarker of interest and assessing if there is prior research to support its relevance. In order to determine the answer to the above questions, it is imperative to do a thorough review of literature using database search engines such as PubMed, EMBASE, and Cochrane Library. This will allow investigators to discover areas where a knowledge gap exists and provide opportunity to fill those gaps with the proposed study.

Example: PD-L1 is a commonly studied biomarker for multiple different types of cancer and is the only validated predictive biomarker in immunotherapy cancer treatment. However, despite FDA approval of its use, this tissue-based biomarker is not perfect.19 Several biomarkers, including the use of tumor mutation burden, have been proposed in conjunction with PD-L1 to build a better predictive model.20 To determine the knowledge gaps present, investigators can use Medical Subject Headings terms such as ‘immunotherapy’, ‘cancer’, and ‘biomarker’ to search through medical databases. The investigator must justify the study of their proposed biomarker in the context of the current literature.

Question: How is it different from other studies of the same biomarker in the same population? Does this represent a methodologic or analytic advancement?

Once the significance of the proposed biomarker is determined, the next step is assessing the level of innovation of the study. It is important to clearly state what specific aspects of the suggested study are novel compared with the existing body of scientific knowledge. This offers protocol readers perspective on how the study will benefit the scientific community and further advance predictive biomarkers for ICI efficacy.

Example: A one-time assessment of tumor PD-L1 is not an accurate representation of the patient’s tumor (tumor heterogeneity) at baseline or through the course of therapy. Innovative methods to assess PD-L1 may include measuring expression of PD-L1 in circulating tumor cells in the blood over the course of therapy as a way of obtaining repeated measurements.21

Question: What are the steps involved and is there justification for these steps? What are the study goals? Will this be an integral or integrated biomarker study? What next steps will the results inform?

This section of the study protocol outlines the logistical plan for conducting the study. It involves first stating the primary and secondary goals of the study and then describing the steps required to achieve these goals. The steps listed should include approach for pre-analytic, analytic, and post-analytic factors, along with justification for why the approach was picked. In clinical trials, biomarker studies can be conducted either as an integral or integrated study. During integral studies, the biomarker being assessed is imperative for the continuation of the trial and the primary study objective is designed around the biomarker.22 While in integrated studies, tests are conducted to identify or validate biomarkers that can be used in future trials and a hypothesis is tested as a secondary study objective.22 This portion of the protocol should conclude with how the results obtained will inform future investigations.

Example: Determining if repeated measurements of PD-L1 via circulating tumor cells throughout immunotherapy treatment may better predict durable benefit from ICI. Pre-analytic factors to consider include: blood sample collection, processing (how circulating tumor cells are obtained from a blood sample), and storage. Analytic factors involve the actual testing of the sample (ie, which assay will be used to test for PD-L1). Post-analytic factors include how the data will be recorded and interpreted. The study is classified as an integrated biomarker study as it will be measured in all participants and will not determine study eligibility or treatment received. The results from this study will shed light on how repeated measurements of PD-L1 during therapy will influence its use as predictive biomarker throughout treatment.

Question: Are the principal investigator and the team adequately qualified?

The roles of each team member are described in the Statement of work for study coordination section of this paper. In addition to this, there should be an explanation regarding the study principal investigator (PI) qualifications for undertaking the project. The investigators must have technical and clinical expertise to perform the study. There are often institutional requirements as well in regard to training for handling patient information, working with human specimens, and remaining compliant with the Healthcare Insurance Portability and Accountability Act (HIPAA).

Example: Experience in the circulating tumor cell field is desirable if the blood-based biomarker involves serial measurement of PD-L1 in circulating tumor cells (CTCs) over time. Investigators involved in the study must undergo research, ethics, and compliance training and obtain certificates of successful completion in order to participate.

Question: Are there adequate resources to carry out the study to completion?

In order to successfully complete blood-based biomarker studies, a wide variety of research personnel are required—research coordinators, data managers, and regulatory staff. Further, sufficient laboratory staff is needed for data acquisition and analysis. Additionally, access to equipment or necessary supplies should be assessed as well. In this section, inclusion of a budget may be useful and is further expanded on in the Budget requirements section.

Example: It is strongly recommended to process fresh blood in a timely fashion for circulating tumor cell isolation. Proper coordination between research labs and personnel is needed to make sure essential equipment is ready and sample is properly dropped off. In the instance of storing processed blood components, appropriate refrigeration and freezing conditions are critical.

Question: How will the data be recorded? Who will maintain the codes for the patient data? What type of output will be generated?

After data is collected, it must be stored securely and specific members of the research team should be identified to maintain a separate data sheet associating each patient to a unique code. Database management can be done in a number of ways and is further addressed in the Database management section. Options include storing the data on a secure institutional network drive, controlling access with encryption, or password-protected cloud storage. It is crucial to have the data backed up as well. Determining what additional data to collect, such as demographic and clinical information, is reviewed in the Standardized documentation of demographics and clinical information section.

Example: If evaluating the role of PD-L1 on CTCs as a novel biomarker, each study participant will be assigned a unique patient identifier, which will be maintained on a password-protected database. Clinical, pathologic, and biomarker data collected from the study will be inputted into a separate password-protected database which is linked only through the use of the unique patient identifier.

Question: How will the statistical analysis be designed, analyzed, and validated?

Considerable thought must be given as to how many replicates are needed to reduce the measurement error (eg, reducing batch effect and minimize assay-to-assay variability). To associate the biomarker to the clinical outcome, the type of the outcome must be selected. These include association of the biomarker of interest with ICI response or time-to-event variable, such as PFS. A benefit of using continuous data analyses (with appropriate data transformation, such as log) is that all information is retained. Based on this endpoint, sample size calculations are needed to ensure the study has adequate power to test the hypothesis.

Example: In order to determine relationship between PD-L1 in CTC as a predictive biomarker of PFS, ensure that a multivariate analysis is performed, missing data are minimized and adjusted for, and validation studies are planned.

Statistical design

Statistical analysis

• Data cleaning and normalization.

• Understanding and addressing missing data via methods such as multiple imputation or doubly robust methods.

• Determining the appropriate statistical approach, identifying and adjusting confounding factors via statistical analyses. For a binary response outcome, we can consider a logistic regression or a Cox regression for a time-to-event outcome, such as PFS. The goodness-of-fit of the model should be checked and the appropriate transformation should be used on the biomarker data (such as log) to ensure a good fit of the model.

• Evaluating sampling or selection bias in the observational study and developing propensity score models to correct for such potential bias.

• Appropriately addressing the multiple comparison issue if multiple biomarkers are tested simultaneously. For example, the Bonferroni procedure and Tukey’s procedure can be used to control for the family-wise type I error.

• Avoid dichotomizing the continuous biomarker measurement for analysis to retain statistical power unless there is a clinical meaningful threshold commonly used for the biomarker. Repeated or arbitrary methods that create many possible thresholds for a cut-off to test for predictive ability or the lowest p value are discouraged and have been criticized for their ad-hoc nature, potential for overfitting, and lack of correction for multiple comparison.24 25

Statistical validation

To evaluate the predictive ability of the biomarker, the area under the receiver operating characteristic (ROC) curve can be computed to measure the ability of the model to correctly classify responders and non-responders.26 For the survival outcome, such as PFS, the c-index and time-dependent ROC curve27 28 can be used for evaluation of the discriminative ability of the model, and Brier score29 for evaluation of the predictive accuracy of the model. All the calculations should be based on a 5-fold or 10-fold cross-validation. Moreover, the added predictive ability of a new biomarker can be assessed using method described by Pencina et al.30 If the sample size allows, the biomarker should be validated in a separate cohort.

Total blood

At least 5 mL of blood needs to be collected in a 13×75 mm vacutainer tube with K2EDTA. The blood should be mixed via inverting the tube 10 times at 90° in aliquots containing 1.5 mL low-binding protein Eppendorf tubes or cryovials with O-ring sealed screw leads. The sample must be stored frozen at −80°C until data collection. Figure 2 notes common liquid biopsy markers that are obtained from each blood component.

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Figure 2

Common liquid biopsy markers origin with respect to blood components. NK, natural killer.

Plasma

At least 5 mL of blood needs to be collected in a vacutainer tube with K2EDTA. It is important to ensure it is properly filled as the additives are calibrated for optimum blood to additive ratio.40 The blood should be well mixed via inverting the tube four to six times at 90°. The samples are centrifuged for 10 min at 1300× g at 4°C. The plasma is removed from the top layer and aliquoted in 1.5 mL low-binding Eppendorf or cryovial tubes with O-ring sealed screw leads. The buffy coats (white layer) can be transferred to support aliquot tubes with the addition of 1.2 mL of ‘RNA Later’. Plasma should be stored frozen at −80°C until it is assayed. Refer to online supplement section 4 for sample standard operating procedure (SOP) for plasma processing.

Serum

At least 5 mL of blood needs to be collected in a vacutainer tube containing silica clot activator, polymer gel, silicone-coated interior, or equivalent. Allow sample to sit upright at room temperature for 30–60 min in order for clot formation to occur, may need to sit longer if patient was taking anticoagulant.40 It must be processed immediately or stored at 4°C (only up to 4 hours). In order to process the sample, it is centrifuged for 10 min at 1300× g at 4°C. The serum is removed from the top layer and aliquot 300 µL in 1.5 mL low-binding protein Eppendorf tubes or cryovials (decrease molecule of interest binding to tube surface) with O-ring sealed screw leads (prevent evaporation). Prior to assay, the serum is stored frozen at −80°C. Refer to online supplement section 4 for sample serum SOP.

White blood cells

Obtain 10 mL of blood in vacutainer tubes that are EDTA coated. The blood should be mixed well via inverting the tube 10 times at 90°. Using two 15 mL falcon tubes (or one 50 mL tube), layer 5 mL of blood on 5 mL of density gradient medium in each tube (ie, Ficoll). These tubes should be centrifuged for 40 min at 400× g at room temperature. Remove the WBC layer in the middle of the tube with a long pipette tip and place in a different 15 mL conical tube that contains 10 mL of phosphate buffered saline (PBS), pH 7.4. Then centrifuge the tubes again for 10 min at 400× g at room temperature. The WBC pellet is then washed by removing the supernatant and resuspending the pellet in 10 mL of PBS. This is again centrifuged for 10 min at 400× g at room temperature and the same washing process is repeated. Using a hemocytometer, the cells are counted and then added to freezing media in order to store samples at 1×10⁷ cells/mL. Aliquot into 1.5 mL cryovials. Prior to assay, the samples should be kept frozen at −80°C. If commercially available, complete cell preparation tubes containing anticoagulant, liquid density medium, and inert gel barrier are used, then processing of the samples can start with centrifuge with subsequent sample washing and freezing media.

Red blood cells

Obtain 5 mL of blood in a 13×75 mm vacutainer tube with K2EDTA. The blood should be mixed well via inverting the tube 10 times at 90°. Then for 10 min, the sample is centrifuged at 1000× g at 4°C. The top layer (plasma) is removed using a pipette. Next, the white buffy layer containing leukocytes is removed. The RBCs are then lysed in 4 volume of ice cold high-performance liquid chromatography grade water and centrifuged for 15 min at 10 000× g at 4°C. The erythrocyte lysate (supernatant) is collected and placed in 1.5 mL low-binding Eppendorf tubes or cryovials. Prior to assay, the samples should be stored at −80°C.

Platelets

Obtain 5 mL of blood in a 13×75 mm vacutainer tube with K2EDTA. The blood should be mixed well via inverting the tube 10 times at 90°. The sample should then be centrifuged for 15 min at 160–200× g at room temperature. The supernatant-containing plasma rich in platelets is then collected and placed into a new tube. This supernatant is then centrifuged at 700× g for 15 min at room temperature. Remove the supernatant and resuspend the pellet with the addition of PBS. Complete the rinse two more times and then resuspend the pelleted platelets in PBS or media with EDTA at 2 mM. Using a hemocytometer, count the platelets and adjust concentration to 1–3×10⁸ platelets/mL in PBS or media. Prior to assay, the samples can be stored at −80°C in low-binding protein Eppendorf tubes or cryovials.

DNA

Obtain 5 mL of blood in a 13×75 mm vacutainer tube with K2EDTA. The blood should be well mixed via inverting the tube 10 times at 90°. Aliquot 1 mL of blood into 1.5 mL low-binding protein Eppendorf tubes or cryovials with O-ring sealed screw leads. Prior to assay, the sample should be stored at −20°C or −80°C.

RNA

Obtain 2.5 mL of blood in a 16×100 mm RNA-stabilizer blood RNA tube. The blood should be mixed well via inverting the tube 10 times at 90°. Prior to assay, the sample should be stored at −20°C (<6 months) or −80°C (>6 months to years). In order to collect blood for RNA analysis, a blood collector set should be used, not a syringe. When collecting other specimens at the same time, ensure that RNA is the last specimen collected. If only RNA is being collected, then a small amount of blood is drawn into a ‘discard tube’.

Circulating cell free DNA

Cell free DNAs are usually isolated from plasma or serum instead of whole blood. Minimal time delay between blood draw and sample processing is preferred, as DNA concentration increases with more blood cell lysis.41 K2EDTA tubes are fine for short interval blood processing. If longer storage time is required between blood draw and processing, preservative blood collection tubes are recommended (ie, Streck Cell Free DNA Blood Collection Tubes).

Circulating tumor cells

Sample requirements vary among the numerous isolation techniques for CTCs. Generally, at least 5 mL of blood is needed in vacutainer tube with spray coated, or equivalent K2EDTA (tube size 13×75 mm). For studies involving CTCs, to avoid the potential skin/epithelial cell contamination by needle aspiration during the blood draw, the first few millimeters of blood needs to be discarded. It is strongly recommended that the sample is processed freshly, within 4 hours after blood draw to minimize cell loss. If shipping or storing is needed, obtain samples in blood preservative collection tubes such as CellSave tubes (Veridex) and BCT tubes (Streck). Studies have shown tubes with preservatives were able to retrieve comparable results from CTCs up to 96 hours at room temperature.42 The blood should be mixed well via inverting the tube 10 times at 90° prior to processing.

EVs and exosomes

EVs are usually isolated from plasma or serum instead of whole blood, in order to minimize vesicle secretion after blood draw. Samples should be stored in −20°C (short term) or −80°C (long term) prior to isolation. One of the most commonly used isolation methods is differential ultracentrifugation. Specifically, centrifuge and collect supernatant at 12,000× g for 20 min. Then, dilute and balance samples using sterile PBS pH 7.4, centrifuge at 100,000× g for 90 min.43 Replace the supernatant with sterile PBS and repeat centrifuge at 100,000× g for 90 min. Finally, EVs should then be suspended in 100 µL PBS pH 7.4 or Radioimmunoprecipitation assay buffer (RIPA buffer) with protease inhibitor cocktail, depending on downstream applications. Notably, the process for exosome collection is identical to EV collection and historically researchers have referred to lipid bilayer delimited particles released from a cell as either exosome or EV.44 Figure 3 notes how blood samples are processed for each biomarker being tested.

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Figure 3

Blood sample processing. CTC, circulating tumor cell; EVs, extracellular vesicles; PBS, phosphate buffered saline.

Peripheral blood mononuclear cells

Add 5 mL of Lymphocyte Separation Medium to the bottom of a 15 mL centrifuge tube and transfer 10 mL of blood using a pipette into the tube. Place tubes in the centrifuge at 1600 rpm with brake off for 30 min. Once completed and separation has occurred, remove the interface containing the lymphocytes to a different 15 mL centrifuge tube, fill it with PBS, and mix by inverting tube at 90° angle. Place back into centrifuge for 10 min at 1350 rpm with brake on low. Remove tubes and decant supernatant into waste container and resuspend the cells. Fill with PBS and spin down at 1250 rpm with brake on high. Following this, wash and perform hypotonic lysis by decanting and resuspending the cells again, add 2 mL sterile water and gently mix then add 10 mL of PBS within 30 s. Centrifuge at 1250 rpm for 10 min with brake on high. Resuspend the cells and add 10 mL of PBS. Mix by inverting the tube at 90° angle a few times. Remove 50 µL and place into a 96 well plate and add 50 µL trypan blue to count. Final count should be 0.2×10⁶. To store, add freezing medium (90% AB serum plus 10% dimethylsulfoxide) to the cells and place into cryovials. Store at −80°C. Refer to online supplement section 5 for PBMC processing SOP example.

Storage of samples

All patient samples should be stored at a secure facility that includes fridges and freezers that can be locked and are only accessible by approved research personnel. There must be the capability to store samples at −20°C and −80°C, as well as liquid nitrogen tanks. In case of power issues, there should be a backup generator or power plan in place, as well as alarms that indicate any issues with maintaining temperature or power. All of the fridges and freezers themselves should have careful temperature records, a CO₂ backup system to maintain temperature as low as −70°C in case of a power outage, and a dedicated computer to store information. Investigators handling specimens should monitor and record storage conditions, number of freeze thaw cycles, and number of equipment failures (ie, temperature changes).45 For uniform temperature, validation of freezer units (ie, temperature mapping of freezer interior) and regular defrosting is recommended. An inventory system should be in place denoting location of each aliquot with documentation of specific box, shelf, and freezer number.

Shipment of samples

If researchers are working with other institutions or even international groups, then oftentimes samples must be shipped to other locations. When this is the case, researchers should keep in mind the shipping temperature, time, distance, weather, and transportation method. Ideally, samples should be sent overnight during the weekday, in order to decrease sample loss. In order to maintain frozen temperature, ensure that dry ice or liquid nitrogen is available. If using dry ice, ensure that all personnel handling have appropriate training per federal regulations and certification is on file. Due to temperature sensitivity of samples, a courier who is able to refill the dry ice should be used, especially if there are shipping delays. The package should also contain a device to constantly monitor temperature. Put filled cardboard boxes with samples in a plastic Ziploc bag along with absorbent pad and seal it. Place bagged boxes in the bottom of shipping box, these boxes must have Styrofoam insert surrounded by cardboard outer box. Fill shipping box with as much dry ice as possible (pelletized dry ice is ideal). Place Human Specimens Exempt label on shipping container. Ensure shipment is sent overnight on Mondays–Wednesdays only. Prior to shipping the samples, the sending institution should contact the receiving institution and ensure research personnel will be available to accept the package. Further paperwork should be included in the package that lists all samples by their identification number and description of all specimens. Once arrived at its destination, it must be promptly stored in appropriate conditions. Notably, the thawing method used is important in maintaining sample integrity. Using additives like human serum albumin, dextran, and fetal bovine serum was better than human AB serum; using medium pre-warmed to 25°C–37°C yielded better results than chilled medium.36

Institutional start-up

If collaborating with a federal agency, an Intergovernmental Personnel Act agreement may be necessary. This allows for temporary assignment of employees between federal agencies and state and local governments, colleges, and universities. Additionally, there may be consideration of a Cooperative Research and Development Agreement, which allows federal labs to participate in collaborative work with non-federal institutions, such as universities. In these instances, data use agreements will be required in order to share data between collaborating agencies.

Project start-up

The initial pre-site selection meeting (determine if investigators and clinical site are adequately equipped to complete the study) and start-up meetings (occurs after clinical site meets all regulatory requirements and obtains IRB approval) are all arranged by the research coordinators. The project proposal is submitted by the coordinator to IRB and Research Data Center for local and regulatory approval. At this step, a draft regarding plan for sample collection is created.

Patient prescreening

Coordinators create a patient tracking system and proceed to screen patients presenting for clinic appointments for eligibility of enrolling into the study. IRB-approved recruitment materials including letters and brochures can be used. Once patients are identified, the coordinator communicates with the PI to confirm that the patient meets all eligibility criteria. At this time, the coordinator will introduce the idea of consenting to the study to patients and continue communication with follow-up telephone contact.

Patient screening and enrollment

Once patient is determined to be eligible, the study coordinator will meet the patient and further explain the project. The coordinator then proceeds ahead with obtaining informed consent from the patient and HIPAA authorization. The coordinator should log screen failures and successful enrollments. Once consented, the coordinator will help arrange and schedule all necessary tests and lab orders. They will also help with obtaining required demographic and survey data and drug reconciliation.

Sample collection and processing

The coordinator assists in the collection of patient blood samples and helps route them to the appropriate locations. They help label samples, maintain the sample database, and shipping log.

Central lab sample and shipping

If samples are being shipped to central labs or other collaborating institutions then coordinators help with preparing the shipping label, packaging (ie, dry ice), adhering to exempt human specimen standards, and following up with the receiving locations regarding sample arrival and results obtained.

Data entry and maintenance

Coordinators enter visit data into the patient database within a specified time frame. Prior to inputting data, they de-identify any patient imaging and protected health information. They also aid in monitoring and entering any adverse events (condition which appears or worsens after participant is enrolled) or serious adverse events (results in death or life-threatening problem leading to inpatient hospitalization) experienced by patients. During this time, coordinators also answer any questions that patients have.

Regulatory updates

Maintenance of accurate training logs and delegation is completed by the coordinator. The regulatory binder contains documents such as investigator curriculum vitae, delegation of authority/duty logs, IRB approvals, Human Subjects Protections training/certifications, and HIPAA training certification. Coordinators will also be in charge of completing the annual continuing review for safety and IRB approval, as well as provide any updates or amendments.

Patient long-term follow-up

As patients continue in the study, coordinators will assess patient survival, long-term follow-up clinic visits, and updating clinical data.