I participate in an international discussion group relating to individualized tumor response testing (ITRT), otherwise known as cell culture drug response testing (CCDRT). Most recently, the topic of ITRT/CCDRT in non-Hodgkin’s lymphoma (NHL) was raised. One European group utilizes the ATP endpoint. Another European group utilizes a modification of the DISC endpoint (dye exclusion by living cells, in a system capable of selectively identifying the living cells as being either neoplastic or normal). Both ATP and DISC endpoints are based on cell kill/cell death (as opposed to cell proliferation/DNA synthesis, which is the other major ITRT/CCDRT endpoint). The DISC endpoint is capable of discriminating between drug effects of neoplastic versus normal cells, present in the same cell culture, while the ATP is not selective for neoplastic cells versus normal cells.
Generally speaking, the results of cell death endpoints agree with each other pretty well, as long as the cell population being measured is predominately the cell population of interest (in this case, tumor or neoplastic cells). This should not be surprising — after all, a dead cell is a dead cell. The problem comes when there are appreciable numbers of “contaminating” normal/non-neoplastic cells present. In these cases, endpoints which are not specific for tumor cells may produce skewed results.
The table below shows solid tumor correlation coefficients, comparing DISC (“specific for tumor cells”) and MTT (not “specific for tumor cells”) endpoints. These data come from studies in which individual tumors were simultaneously tested in each endpoint, after being cultured in precisely the same conditions, for precisely the same period of time. It can be seen that, in the case of the drug melphalan, when there are greater than 40% tumor cells with solid tumors, the results of DISC and MTT endpoints agree quite well. There were relatively few cases in which specimens with fewer than 40% tumor cells were tested with the non-tumor-selective (MTT) endpoint (because of advance knowledge that, in such specimens, the MTT endpoint was not reliably discriminatory for tumor cells). In the few such cases tested, the MTT/DISC correlations were not very good (r=0.51, r2=0.26).
Comparison between MTT and DISC results for melphalan in solid tumors as a function of percentage of total viable cells which were tumor/neoplastic cells postculture:
TIn contrast, in the case of melphalan in non-Hodgkin’s lymphoma/chronic lymphocytic leukemia, there was progressively serious skewing of MTT results by normal cells, as the post-culture percentage of neoplastic cells dropped below 95%:
The following two scattergrams show similar data graphically (but with solid tumor data limited to ovarian cancer, to simplify the comparisons):
These artifactual results with the metabolic endpoint can seriously comprise the biological validity of the assay results, as shown in the following two graphs.
These data analyses are based on the principle that chemonaive tumors (i.e. from previously-untreated patients) are well known to be more drug sensitive/less drug resistant than are non-chemonaive tumors (i.e. from previously-treated patients):
In the above graph, from the ovarian cancer dataset, it can be seen that, in BOTH DISC and MTT endpoints, specimens from previously-treated patients were significantly more resistant to melphalan than were specimens from chemonaive/previously-untreated patients.
However, there were far different results with the NHL/CLL dataset:
In the case of CLL/NHL, where the effect of normal cells skewing the results of the non-tumor-cell-discriminatory MTT endpoint is much greater, the greater chemosensitivity for specimens from untreated patients was confirmed with the (selective) DISC endpoint, but was not observed with the (non-selective) MTT endpoint.
The problem in NHL/CLL is that the metabolic activity of the neoplastic cells (on a per cell basis) can be low, relative to the metabolic activity of normal cells (e.g. macrophages, cell culture transformed normal T-lymphocytes, etc.). Thus, the normal cells typically present in specimens from lymphatic neoplasms may skew/disort the cell death estimates to a much greater extent than in the case of the typical solid tumors. Thus, results of non-discriminatory endpoints (e.g. MTT, ATP) must be interpreted with caution, in the case of lymphatic neoplasms.
We have similar data in the case of acute non-lymphocytic leukemia (skewing of results by normal cells with non-selective endpoints). We do not have sufficient numbers of datapoints in the case of acute lymphoblastic leukemia to make these comparisons.
It is obvious that non-selective endpoints will have very little utility in neoplasms such as Hodgkin’s Disease and multiple myeloma, where most specimens have very large numbers of “contaminating” normal cells.