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February 1, 2020
Careful Monitoring Before and After Infusion Is Critical to Successful Treatment
Careful monitoring of patients throughout the duration of chimeric antigen receptor (CAR)-T cell therapy, including their laboratory profiles, helps guide and refine clinical management—from preparing and considering potential candidates to monitoring the long-term recovery of those who receive this powerful new therapy. Two CAR-T cell products, tisagenlecleucel and axicabtagene ciloleucel, have been approved by the Food and Drug Administration (FDA) and are quickly becoming mainstays in hematologic and oncologic treatment strategies. This minireview considers CAR-T cell therapy from the perspective of clinical laboratories, with the goal of empowering clinical laboratorians with information so that patients receive optimal care regardless of clinical setting.
THE ROLE OF LABORATORIES BEFORE CAR-T CELL INFUSION
Collecting source material is critical to the CAR-T cell manufacturing process. Since CAR-T cells are autologous, i.e. the donor is also the recipient, patients must be evaluated carefully before they become candidates for CAR-T cell therapy and cellular collection by apheresis. In the case of tisagenlecleucel, patients must stop certain treatments, including allogeneic therapies and immunosuppressants, for up to 12 weeks before apheresis collection. Monitoring hematologic parameters or immunosuppressant levels in the blood may be necessary to ensure patients are adequately prepared leading up to cellular collection and continued during treatment to adjust their therapy as needed to maintain their health status.
Although most cellular collections are successful, certain laboratory parameters are crucial to determine whether a patient can proceed with collection. There must be enough T cells in the peripheral blood to be collected, as measured by absolute lymphocyte count and/or peripheral blood CD3 counts. After collection, cells must be prepared and transported for manufacturing, either in-house where the cells were collected and the patient will be infused, or at an external central manufacturing facility, the typical model for most sites.
Many important considerations for product preparation and transport center on the safety of both patient and product. Labs must use special handling for any product that requires cryopreservation before the product gets packaged and shipped. This includes performing cell counts on a hematology analyzer, characterizing the cellular composition by flow cytometry, adding cryoprotectant and proper labeling, and checking both patient and product identification multiple times.
During manufacturing, which can take 2 weeks, patients need clinical monitoring to ensure that they remain stable until the CAR-T cells can be infused. The manufacturing process is typically monitored by release testing, which helps ensure product safety, purity, and potency. Laboratory assessment typically includes product composition by flow cytometric characterization of CAR-T cells; product sterility by microbial culture and testing for endotoxin and mycoplasma contamination; and functional assays to assess in vitro cytotoxic function and activation (9).
Once manufacturing is complete and testing has confirmed that the product can be released for use, the product is then frozen, shipped back to the infusion site, and stored in a cellular therapy laboratory until the patient is ready for product infusion.
AFTER CAR-T CELL INFUSION
After a patient has been infused with CAR-T cell product, the T cells ideally recognize their target antigen, activate, and begin to proliferate and exert anti-tumor effects. Response to therapy can be monitored by detecting malignant cells. The clinical lab examines peripheral blood or other potentially affected tissues by standard means such as examining peripheral blood smears for morphology and/or using flow cytometry to detect malignant cells by protein expression.
Detecting CAR-T cells themselves is not as straightforward. CAR-T cells can exhibit atypical morphologic features, and methods to detect the CAR protein itself are not commercially available. In addition, the potential customization of CAR proteins prohibits at this time development of any single assay to detect CAR-T cells.
Patients can exhibit expected and unexpected effects from CAR-T cell therapy infusion. A well-characterized and potentially serious adverse effect is cytokine release syndrome (CRS). Characterized by high fever, organ dysfunction, hypotension, and increasing oxygen requirements, CRS ranges from mild to life-threatening and is attributed to extremely high levels of cytokines, specifically interleukin 6 (IL-6).
The American Society for Transplantation and Cellular Therapy (ASTCT) recently published guidelines for defining and grading CRS. While certain aspects of CRS are almost certainly determined by laboratory values, such as transaminitis and other signs of organ dysfunction, laboratory parameters are not included as factors to determine the presence or severity of CRS. Some tests, such as serum IL-6 quantitation, are not widely available, which translates into a long turnaround time since they must be sent out. The nonspecific nature of other lab parameters that might be altered during the course of CRS—such as ferritin and C-reactive protein—as well as the potential lag time between symptoms and altered lab values, support treating patients empirically based off their clinical presentation.
Neurotoxicity, also called immune effector cell-associated neurotoxicity syndrome, is another adverse effect of CAR-T cell infusion and can present with neurological symptoms such as encephalopathy, agitation, delirium, and seizures. Laboratory evaluations currently do not aid in characterizing neurotoxicity. The ASTCT encouraged continued exploration of potentially useful laboratory characterization of patients who demonstrate clinical signs and symptoms of adverse events from CAR-T cell therapy. Additional laboratory characterization of patients who experience adverse effects from CAR-T cells could help inform the larger community about laboratory testing that has diagnostic, therapeutic, or prognostic potential for CRS and neurotoxicity.
Notably, CAR-T cells can effectively eliminate normal cells if they express the target antigen; this phenomenon is known as on-target/off-tumor toxicity. In the context of the FDA-approved therapies that target CD19, B cell aplasia is an anticipated effect, since normal B cells also express CD19 and are therefore subject to elimination by anti-CD19 CAR-T cells. This requires monitoring patients who receive CAR-T cells directed against CD19 to assess whether they develop B cell aplasia, mainly by measuring serum gammaglobulins. Hypoglobulinemia can be managed clinically with immunoglobulin replacement therapy.
Clinical laboratorians should be aware of the potential laboratory abnormalities that accompany treatment with CAR-T cells. For example, patients might experience cytopenias in the period following infusion. In this instance, they might resemble a patient who has undergone a hematopoietic progenitor cell transplant, although they received entirely different cells. Prolonged B cell aplasia and hypogammaglobulinemia associated with CAR-T cells directed to CD19 could predispose them to develop infections. These patients might then be similar to other patients who are predisposed to infections, and appropriate microbial testing is warranted to ensure prompt therapeutic intervention if necessary.
Serologic monitoring of antibody titers to previous vaccinations can also be prudent. Interestingly, although B cell aplasia can be profound, plasma cells have been shown to persist in patients after they’ve been treated with CAR-T cells. Plasma cells, which do not express CD19, are major antibody-producing cells of the immune system and help form memory to vaccines and antibody-mediated immune responses; plasma cell persistence could be protective for a patient. In addition, the lentiviral vector used to deliver the CAR genetic material into T cells in tisagenlecleucel can, depending on the assay, result in false-positive HIV test results.
Laboratory testing and investigation reflects the variety of clinical scenarios of patients who receive CAR-T cells and requires an individualized approach. For instance, while B cell aplasia and hypogammaglobulinemia is specific to anti-CD19 CAR-T cell therapy, the potential for false-positive HIV test results might not be specific to the CAR-T cell target, but rather the viral vector used to introduce genetic material. Other side effects such as cytopenias and CRS might be generalized to treatment with CAR-T cell therapies regardless of a therapy’s target.
Patients also must be monitored over time for development of unanticipated adverse effects of CAR-T cells, such as relapse of the primary malignancy, secondary malignancies, and mutagenic potential of the CAR-T cells themselves. Long-term monitoring involves careful consideration of the previous CAR-T cell therapy to ensure any potential effects of the treatment are identified. Awareness of the anticipated, unanticipated, specific, and general side effects can help guide clinical laboratories to investigate how testing can be improved upon as more becomes known about the different CAR-T cells—both those now in clinical use and the ones to come.
NEW THERAPIES ON THE HORIZON
CARs can differ in clinical targets, genetic structure, and even the cell in which the CAR resides. Even as the two FDA-approved CAR-T cell therapies disseminate in practice, researchers are continuing to develop other CAR-T cells and other genetically modified cellular therapies.
CAR-T cells are very attractive as a potential therapy because they are fairly simple to construct, customizable, and have already shown the potential to alter the clinical course of diseases with otherwise very poor survival. The CAR construct can be modified to recognize different targets, such as B cell maturation antigen on multiple myeloma cells or mesothelin on certain solid tumors. Both these therapies are under clinical development and have undergone testing in humans.
Development of CAR-T cells expressing different target antigens requires thoroughly evaluating their side effect profiles in preclinical and clinical trials to identify any on-target/off-tumor effects, their potential clinical impact, and strategies to monitor and treat patients.
Laboratory evaluation of patients throughout the process of CAR-T cell therapy is critical to identify information that might be useful to monitor success or mitigate side effects of this therapy. Furthermore, knowledge and familiarity with the potential clinical and laboratory presentations of patients who undergo CAR-T cell therapy will encourage all providers to anticipate and adapt quickly to the changing CAR-T cell clinical landscape and will help to optimize care for patients who receive currently used CAR-T cells as well as those to come.