In Vitro Drug Testing
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Mosaic Blue Assay™
Ki-67 and phospho-histone H3 (pHH3)
For over 20 years, clinical investigators have been using chemosensitivity testing to determine sensitivity and resistance to chemotherapeutic agents. Chemosensitivity testing is a laboratory test that is performed by disaggregating fresh live tumor tissue into single cells or small spheroids, exposing these cells to chemotherapeutic agents, and evaluating the agents’ effectiveness in vitro. These assays provide the most meaningful information about drug resistance. Studies have demonstrated that if a patient’s cancer cells are resistant to a suprapharmacologic exposure of chemotherapeutic drug in vitro, then the patient has less than a 1% chance of responding to that drug.
There are several types of commercially available chemotherapeutic assays. While each assay may differ in technique, they all have similar methods: 1) fresh cancer is procures and shipped to a lab using methods to maintain cell viability; 2) cells are isolated and plated under conditions that suppress the growth of normal cells but allow cancer cells to grow; 3) cancer cells are exposed to drugs; and 4) the inhibitory or cytotoxic effect of drugs is characterized.
The results of the chemosensitivity assay can be applied to patient care. By identifying individual agents to which a patient’s cancer is highly resistant, oncologists improve patient care by switching to a more effective agent. This spares the patient exposure to ineffective therapy and subsequent side effects.
The soft agar colony forming assay is one of the most commonly used chemosensitivity assays for solid tumors. However, this assay is limited by being time consuming, labor intensive, material depleting, and having subjective analyses.
e rationale for using the alamar blue soft agar assay in a 96-well plate includes:
- Soft agar suppresses normal and fibroblast growth
- The assay can be performed with several replicates and drug concentrations, both as single agents and in combination
- The assay uses fewer tumor cells, allowing use of limited patient material as well as additional in vitro testing
- The dye is non-toxic and allows for post-treatment evaluation of cells
- The assay is progressive, allowing for greater evaluability
Mosaic Spot Assay™
The Mosaic Spot Assay™, which is based on the principals of the DiSC Assay, is a highly accurate, validated, reproducible, 4-day assay for detecting drug resistance in tumor cells. Living cancer cells are differentially stained with Wright Giemsa in hematologic tumors exposed to chemotherapeutic agents. The Mosaic Spot Assay™ was developed for use in hematological malignancies such as leukemia and lymphoma. Based on 510 published clinical correlations, this assay identifies patients that will not respond to a cancer therapeutic with 92% accuracy. The Mosaic Spot Assay™ is performed using non-dividing cells and has the advantage of having a morphological endpoint. It can also be used to assess activity of new agents and profile drug cross resistance patterns.
The Mosaic Spot Assay™ results predict both clinical response and survival outcome, suggesting that this assay is useful as a surrogate in the clinic for selecting chemotherapeutic agents and indicating prognosis for patients with hematological malignancies. Bird et al. reported a correlation between in vitro assay results and clinical response in patients with chronic lymphocytic leukemia (CLL) or Non-Hodgkin’s Lymphoma (NHL) upon evaluation of a median of 5 drugs tested per patient, including 4-hydroperoxycyclophosphamide (4HC).
A landmark publication demonstrated that fludarabine test-resistant CLL patients, as compared with fludarabine test-sensitive CLL patients, had significantly lower response rates (7% versus 69%) and decreased survival (median 7.9 months versus 41.7 months; relative risk = 14.8; p< 0.0001). These results suggest that Mosaic Spot Assay directed therapy may enhance selection of preferred treatment options, and improve survival rates.
Polycythemia vera (PV) is a rare myeloproliferative disorder characterized by an EPO – independent overproduction of red blood cells. Diagnostic features include enlarged spleen, reddened appearance of the skin, elevated hemoglobin, and decreased serum levels of EPO.
Recently, the in vitro growth of erythroid colonies in the absence of EPO, known as endogenous erythroid colonies (EEC), has found clinical utility in the diagnosis and prognosis of PV. This assay utilizes hematopoietic stem cells present in bone marrow or peripheral blood to quantify erythrocyte cell lineages when cultured in a semi-solid matrix, typically methylcellulose supplemented with nutrients and cytokines. Cytokines include recombinant human stem cell factor (rh SCF), interleukin-3 (rh IL-3) and granulocyte/macrophage colony-stimulating factor (rh GM-CSF). The combination of these growth factors stimulates the proliferation of erythroid colonies during long term cultures.
The individual erythroid progenitors proliferate to form clusters or colonies and are enumerated based on their morphology using light microscopy. Colony forming unit-erythroid (CFU-E) refers to the smallest and most rapidly maturing erythroid colonies, usually comprised of 8-200 erythroblasts in 1-2 clusters. Burst forming unit-erythroid (BFU-E) are distinguishable by their proliferative capacity giving rise to larger, multi-clustered colonies, usually comprised of 3 or more clusters of erythroblasts. Erythroblasts are recognizable by their distinctive reddish-orange hue, which is further emphasized by the use of a blue filter during microscopic visualization.
At Mosaic Laboratories, the EEC assay is used to evaluate drugs for activity vs. polycythemia vera by determining the ability of compounds to inhibit the formation of BFU-E and CFU-E’s.