The Tumor Chemosensitivity Assays (TCAs) and the personalized chemotherapy

at 09.11.2021
A review published on the 19th of June 2021 by the journal “Medicina” of the Lithuanian University of Health Sciences presents the latest data in the field of Tumor Chemosensitivity Assays (TCAs) and Pharmacogenetics in cancer treatment. The paper presents the latest results of chemoresistance and chemosensitivity research, proposing personalized oncology, based on the evaluation of the tumor chemosensitivity of each patient.
According to the study [1], chemotherapy is still an important option for the treatment of various cancer types and it is unavoidable especially in the metastatic stage. In the last 50 years, many chemotherapeutic drugs have been introduced, but there is still no satisfactory improvement in the clinical outcome of cancer patients. Cancer is a heterogeneous disease and the responses to the same chemotherapeutic may vary from one patient to another one, even if cancer cells are originated from the same organ (the histologically same phenotype).
To this end, the research is moving towards two directions: it uses, on the one hand, tumor chemosensitivity tests based on the functional biology of the tumor to determine the chemical sensitivity of each patient to different chemotherapeutics; on the other hand, pharmacogenetic tests based on genome-technologies. Pharmacogenetics is a new field studying how certain variants (polymorphisms) of genes affect the body's response to certain drugs. At present, the combination of these tests seems to be the best solution in oncology practice.

TCAs have a satisfactory negative predictive value (showing the possibility of drug resistance), ranging from roughly 66% to 95%, and a moderate positive predictive value (showing the possibility of drug sensitivity), ranging from roughly 50% to 85%. On the other hand, pharmacogenetics is, to some extent, able to give some clues for the possibility of the side effects of the drugs although it is still not in clinical use. For this reason, the combination of TCAs and pharmacogenetics could provide oncologists with useful data to manage the disease. For example, while TCAs could allow the elimination of ineffective drugs (due to the high negative predictive value), pharmacogenetics could help the oncologists to choose the best possible drug options amongst the tested drugs by the TCA test.

TCAs are designed to select the most appropriate chemotherapy option for individual cancer patients by indicating resistance or sensitivity to drugs. Over the years, TCAs have made satisfactory progress, evolving from some technologically simple assays (e.g., clonogenic assay) to technologically advanced assays (e.g., luminescence-based assays like ATP-TCA or organoids). Thanks to technological achievements, even microfluidic systems have been started to be used for TCAs. As an example, the results of a study performed with a microfluidic system that has been used for real-time screening of drugs in a 3D environment indicated that the microfluidic system detects the resistance and sensitivity of cancer cells to chemotherapeutics in less than 12 h. Moreover, a patient-specific mathematical model has been created to be used to perform TCA and allows one to predict the clinical response to up to 31 drugs within 5 days after bone marrow biopsy.
TCAs are able to guide oncologists about which anti-cancer drugs are more likely to work well enough (or not) on a given patient. Some reports have shown that TCAs could help oncologists to predict the response rate, recurrence, platinum-based drug resistance, progression-free survival as well as the overall survival rate of cancer patients. TCAs have been successfully used to select the better treatment regimen in patients with colorectal liver metastasis and it was shown that the assay-based treatment led to better response rates to drugs. Some reports in the literature clearly show that there is a correlation between the results of TCAs and patient outcomes. Moreover, it was reported that there is a good correlation between the genes predicted to be involved in mechanisms of drug sensitivity/resistance and in vitro chemosensitivity. This is of particular importance in the definition of predictive signatures to guide individualized chemotherapy. Taking the above into account, TCAs keep gaining attention as is proven by the increasing number of TCA-based publications over the years.

TCA Methods
1.Human Tumor Clonogenic Assay (HTCA) 
Clonal growth of mammalian cells was first achieved in the 1950s and based on the clonal growth capability of tumor cells, the human tumor clonogenic assay (HTCA) was firstly tested by Hamburger and Salmon in 1977 [2]. HTCA is a soft agar system designed for growing tumor tissues in cell culture conditions and thus allows one to have a prediction about the responses to chemotherapy in cancer patients. The HTCA might be a useful tool for the evaluation of the anti-tumor effects of drugs in vitro. However, as is mentioned above, this method was used to determine drug response (sensitive/resistance), especially in the 1980s, and it seems to be losing attention. The main reason for this is that it is time-consuming, subjective, and not suitable for automation. For these reasons, this type of assay seems not to be helpful anymore in terms of the prediction of response to chemotherapy in patients, but it has still had a great value in stem cell-based research activities.

2.MTT Based Chemosensitivity Assays
The MTT assay is a simple and rapid colorimetric cell viability assay for setting the metabolic activity of cells. The principle of the assay is the reduction of MTT tetrazolium salt to a blue/purple formazan crystal by living cells but not by dead cells. It can also be used to measure the chemosensitivity of tumor cells. MTT-based chemosensitivity assays were evaluated in several types of cancer such as childhood leukemia, brain, colorectal, acute myeloid leukemia, lung, and ovarian cancer.
However, the MTT assay has some disadvantages. For example, damaged or inactivated mitochondria are able to produce formazan crystals, some chemicals can interact with MTT salt resulting in false results in viability, or tested agents may interfere with mitochondrial dehydrogenase activity, resulting in activation or inhibition of mitochondrial dehydrogenases and thus over/underestimation of the MTT assay results. Therefore, any tumor chemosensitivity/chemoresistance data in the literature obtained from the MTT assay should be interpreted with great caution to avoid false-positive/negative results.

3.The ATP bioluminescence Assay 
Adenosine triphosphate (ATP) molecules act as energy sources with biological systems and can be found in all metabolically active cells [3]. After cell death, the synthesis state of the ATP cell disappears, and endogenous ATPs achieve rapid degradation easily. For this reason, intracellular ATP content has been described as the main indicator of cell viability and is becoming one of the methods for determining viability. The ATP bioluminescence test has been defined as the fastest, most sensitive, and simplest method of viability testing [4].

The assay was first developed by Lundin and colleagues as a somatic cell viability assay. The ATP-TCA is a standardized system that can be adapted to a variety of uses with both cell lines and primary cell cultures. Generally, tumor chemosensitivity/chemoresistance assays use the culture of tumor cells in vitro, and this method may also cause stromal cell contamination in the tested sample. The response of stromal/epithelial cells to chemotherapy may greatly differ and thus contamination of stromal cells may cause unsuccessful treatment because of misinterpreted results. Therefore, serum-free culture medium and polypropylene plates are used in this method to prevent the growth of non-neoplastic cells over a 6-day incubation period. Then, intracellular ATP is extracted by a detergent-based lysis solution and relative ATP levels are measured as bioluminescence light by a luciferin–luciferase reaction. 

ATP-based chemosensitivity assays have been evaluated on many tumor types such as pancreatic cancer, ovarian cancer, colorectal, breast, non-small cell lung cancer. Many studies have shown the reliability of ATP as a sensitive measure of cell viability for various cell lines. ATP-TCA seems to be useful particularly in ovarian cancer. In a study [5] performed with ovarian carcinoma cells, it was reported that ATP-TCA measured cisplatin resistance with >90% accuracy. The authors suggested that the ATP-based chemosensitivity/chemoresistance assay has a high sensitivity, linearity, and precision for measuring the activity of single agents and drug combinations.

In another study [6], a significant correlation was reported between drug sensitivity/resistance and clinical response (p = 0.007). In the same study, the assay demonstrated a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 95%, 44%, 66%, and 89%, respectively. The NPV in this study is especially notable because it gives an appreciable idea of the patients who will show recurrence (due to drug resistance) within 12 months of the post-chemotherapy period. In a recent study [7], the sensitivity, specificity, positive predictive value, and negative predictive value for the clinical chemotherapy sensitivity were found to be as 88.6%, 77.8%, 83%, and 84.8% in ovarian cancer patients, respectively. These values should be considered acceptable and promising for the future of ATP-TCA. The other study [8] conducted on patients with unresectable non-small cell lung cancer showed that there was a good correlation between ATP-TCA results and clinical outcomes of patients. Another study [9] analyzed the correlations between the clinical outcomes of patients and tumor chemosensitivity assay in stage III lung cancer patients. The results indicated that the disease-free survival rates were detected longer in patients with higher drug sensitivity compared to patients who display drug resistance (18 vs. 8.5 months, p < 0.05).
ATP-TCA has been further studied in a more advanced fashion in deadly skin disease, malignant melanoma. In a study performed by Ugurel și collab. [10] the sensitivity index values of dacarbazine-treated patients were compared with classical chemotherapy (cisplatin, paclitaxel, treosulfan, gemcitabine) agent-treated patients. They found that there was no significant difference between these two different therapy protocols in terms of the superiority of any protocol over the other. However, classical chemotherapy-given patients developed more severe side effects compared to the dacarbazine protocol.
In conclusion, ATP-TCA seems to provide promising retrospective ex vivo/in vivo correlations with acceptable positive and negative predictive values. Also, ATP-TCA provides a rational approach for the preclinical evaluation of novel active combination regimens for diverse malignant diseases. More importantly, ATP-TCA-directed chemotherapy can produce impressive response rates although the long-term results (progression-free and overall survival) may suffer from the lack of satisfactory results. Also, ATP-TCA-directed chemotherapy appears to be of particular value for platinum-refractory patients. However, more clinical studies are still needed for firm conclusions, although the correlations between the assay results and response to treatment are highly promising. ATP-TCA is a well-accepted and promising methodology as it provides reliable data in hematological cancers as well. In a study [11], it has recently been reported that ATP-TCA demonstrated a significant correlation with the complete response for chemotherapy and could be a useful tool to optimize personalized treatments for patients with acute myeloid leukemias.

ATP-TCA is able to predict not only the best chemotherapy regimen for any given patient but also the survival rate. Accordingly, in a recent study, it was shown that patients with higher drug sensitivity tended to have longer disease-free survival (18 vs. 8.5 months) than patients displaying drug resistance. Taken altogether, ATP-TCA might be considered as a helpful methodology in terms of obtaining better therapy outcomes in cancer patients.

In addition to the mentioned methods, some other techniques are also used to test drug resistance and/or sensitivity such as extreme drug resistance (EDR-Test)/chemotherapy resistance assay (CTR-Test) [12], tissue explant assay [13], differential staining cytotoxicity assay [14], fluorescent cytoprint assay (FCA) [15], collagen-gel droplet embedded culture drug sensitivity test [16].

At the moment, numerous clinical trials examining the relationship between in vitro tumor response and clinical outcomes are available. These data suggest that in vitro drug response assays can accurately predict drug resistance and can identify patients who are more or less likely to benefit from a given agent. These highly promising results make it possible to design tailored (patient-specific) regimens for each different patient.

In conclusion, the paper asserts that the personalization of chemotherapy in cancer is inevitable due to the vast majority of newly-approved chemotherapeutic drugs in recent years. However, oncologists eagerly demand to know which drug would be suitable for which patient. Therefore, TCAs are supposed to gain more importance to provide oncologists with data to be used for this specific task.
[4] Lomakina, G.Y., Modestova, Y.A. & Ugarova, N.N. Bioluminescence assay for cell viability. Biochemistry Moscow 80, 701–713 (2015). Riss J, Khanna C, Koo S, Chandramouli GV, Yang HH, Hu Y, Kleiner DE, Rosenwald A, Schaefer CF, Ben-Sasson SA, Yang L, Powell J, Kane DW, Star RA, Aprelikova O, Bauer K, Vasselli JR, Maranchie JK, Kohn KW, Buetow KH, Linehan WM, Weinstein JN, Lee MP, Klausner RD, Barrett JC. Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma. Cancer Res. 2006 Jul 15;66(14):7216-24. doi: 10.1158/0008-5472.CAN-06-0040. PMID: 16849569.
[5] Andreotti, P.; Cree, I.; Kurbacher, C.M.; Hartmann, D.M.; Linder, D.; Harel, G.; Gleiberman, I.; Caruso, P.; Ricks, S.H.; Untch, M. Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: Clinical correlation for cisplatin resistance of ovarian carcinoma. Cancer Res. 1995, 55, 5276–5282.
[6] Konecny, G.; Crohns, C.; Pegram, M.; Felber, M.; Lude, S.; Kurbacher, C.; Cree, I.A.; Hepp, H.; Untch, M. Correlation of drug response with the ATP tumorchemosensitivity assay in primary FIGO stage III ovarian cancer. Gynecol. Oncol. 2000, 77, 258–263.
[7] Zhang, J.; Li, H. Heterogeneity of tumor chemosensitivity in ovarian epithelial cancer revealed using the adenosine triphosphatetumor chemosensitivity assay. Oncol. Lett. 2015, 9, 2374–2380.
[8] Moon, Y.W.; Choi, S.H.; Kim, Y.T.; Sohn, J.H.; Chang, J.; Kim, S.K.; Park,M.S.; Chung, K.Y.; Lee, H.J.; Kim, J.H. Adenosine triphosphatebased chemotherapy response assay (ATP-CRA)-guided platinum-based 2-drug chemotherapy for unresectable nonsmall-cell lung cancer. Cancer 2007, 109, 1829–1835.]
[9] Chen, Z.; Zhang, S.; Ma, S.; Li, C.; Xu, C.; Shen, Y.; Zhao, J.; Miao, L. Evaluation of the in vitro Chemosensitivity and Correlation with Clinical Outcomes in Lung Cancer using the ATP-TCA. Anti Cancer Agents Med. Chem. 2018, 18, 139–145.]
[10] Ugurel, S.; Loquai, C.; Terheyden, P.; Schadendorf, D.; Richtig, E.; Utikal, J.; Gutzmer, R.; Rass, K.; Sunderkötter, C.; Stein, A.; et al. Chemosensitivity-directed therapy compared to dacarbazine in chemo-naive advanced metastatic melanoma: A multicenter randomized phase-3 DeCOG trial. Oncotarget 2017, 8, 76029–76043. 
[11] Xia, F.; Ma, S.; Bian, Y.; Yu, D.; Ma, W.; Miao, M.; Huang, C.; Miao, L. A retrospective study of the correlation of in vitro chemosensitivity using ATP-TCA with patient clinical outcomes in acute myeloid leukemia. Cancer Chemother. Pharmacol. 2019, 85, 509–515. 
[12] Eltabbakh, G.H. Extreme drug resistance assay and response to chemotherapy in patients with primary peritoneal carcinoma. J. Surg. Oncol. 2000, 73, 148–152. Eltabbakh, G.H.; Piver, M.S.; Hempling, R.E.; Recio, F.O.; Lele, S.B.; Marchetti, D.L.; Baker, T.R.; Blumenson, L.E. Correlation between extreme drug resistance assay and response to primary paclitaxel and cisplatin in patients with epithelial ovarian cancer. Gynecol. Oncol. 1998, 70, 392–397. Holloway, R.W.; Mehta, R.S.; Finkler, N.J.; Li, K.T.; McLaren, C.E.; Parker, R.J.; Fruehauf, J.P. Association between in vitro platinum resistance in the EDR assay and clinical outcomes for ovarian cancer patients. Gynecol. Oncol. 2002, 87, 8–16. 
[13] Brower, S.L.; Fensterer, J.E.; Bush, J.E. The ChemoFx assay: An ex vivo chemosensitivity and resistance assay for predicting patient response to cancer chemotherapy. Methods Mol. Biol. 2008, 414, 57–78. [PubMed], Suchy, S.L.; Landreneau, R.J.; Schuchert, M.J.;Wang, D.; Ervin, P.R., Jr.; Brower, S.L. Adaptation of a chemosensitivity assay to accurately assess pemetrexed in ex vivo cultures of lung cancer. Cancer Biol. Ther. 2013, 14, 39–44.
[14] Alley, M.C.; Scudiero, D.A.; Monks, P.A.; Hursey, M.L.; Czerwinski, M.J.; Fine, D.L.; Abbott, B.J.; Mayo, J.G.; Shoemaker, R.H.B.; Boyd, M.R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1998, 48, 589–601. Weisenthal, L.M. Differential staining cytotoxicity assay: A review. Cancer Cell Cult. 2011, 731, 259–283.Bird, M.C.; Bosanquet, A.G.; Forskitt, S.; Gilby, E.D. Semi-micro adaptation of a 4-day differential staining cytotoxicity (DiSC) assay for determining the in-vitro chemosensitivity of haematological malignancies. Leuk. Res. 1986, 10, 445–449.
[15] Meitner, P.A. The fluorescent cytoprint assay: A new approach to in vitro chemosensitivity testing. Oncology 1991, 5, 75–81. Leone, L.A.; Meitner, P.A.; Myers, T.J.; Grace,W.R.; Gajewski,W.H.; Fingert, H.J.; Rotman, B. Predictive value of the fluorescent cytoprint assay (FCA): A retrospective correlation study of in vitro chemosensitivity and individual responses to chemotherapy. Cancer Investig. 1991, 9, 491–503.
[16] Goto, H.; Kitagawa, N.; Sekiguchi, H.; Miyagi, Y.; Keino, D.; Sugiyama, M.; Sarashina, T.; Miyagawa, N.; Yokosuka, T.; Hamanoue, S.; et al. The Collagen Gel Droplet–embedded Culture Drug Sensitivity Test in Relapsed Hepatoblastoma. J. Pediatr. Hematol. 2017, 39, 395–401. Kobayashi, H.; Tanisaka, K.; Doi, O.; Kodama, K.; Higashiyama, M.; Nakagawa, H.; Miyake, M.; Taki, T.; Hara, S.; Yasutomi, M.; et al. An in vitro chemosensitivity test for solid human tumors using collagen gel droplet embedded cultures. Int. J. Oncol. 1997, 11, 449–455. Takamura, Y.; Kobayashi, H.; Taguchi, T.; Motomura, K.; Inaji, H.; Noguchi, S. Prediction of chemotherapeutic response by collagen gel droplet embedded culture-drug sensitivity test in human breast cancers. Int. J. Cancer 2002, 98, 450–455. 

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