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Clinical Validation of Cell Culture Assays

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Functional profiling with cell culture assays for targeted drug therapy

There was a recent study describing correlations between cell culture assay results (cell death in response to Iressa exposure) and survival of 31 patients with non-small cell lung cancer who had received extensive prior chemotherapy. These correlations were based on the actual assay results which had been reported, in real time, prospectively to the doctors who had ordered the assay laboratory tests. There were striking correlations between test results and patient survival (not just response).

By inhibiting anti-apoptosis with Iressa (or even Tarceva), the cells undergo apoptosis and die. And it is detected at the whole cell level in the cell culture assays and reported out - prospectively - that this correlates strikingly with patient survival. Not only is it a very important predictive test, but it is a unique tool for identifying newer, better drugs, testing drug combinations, and serving as a "gold standard" to develop new DNA, RNA, and protein-based tests of drug activity.

EGF-targeted drugs (Iressa, Tarceva, Erbitux) are poorly-predicted by measuring the ostensible target (EGFR), but can be well-predicted by measuring the effect of the drugs on the function of "live" cells. Epidermal Growth Factor (EGF) is a receptor on many normal tissues/cells, and also on many cancer cells. It is a growth hormone, locally secreted by cells. It attaches to a receptor on the cell membrane called Epidermal Growth Factor Receptor (EGFR). It then activates so-called signalling pathways within the cell, a cascade of biochemical events, including phosphorylation of proteins, leading to cell growth/proliferation/division. One type of an enzyme which is involved in the pathway which is involved in protein phosphorylation is called tyrosine kinase.

The assay is the only test that involves direct visualization of the cancer cells at endpoint. This allows for accurate assessment of drug activity, discriminates tumor from non-tumor cells, and provides a permanent archival record, which improves quality, serves as control, and assesses dose response in vitro (includes newly-emergent drug combinations).

Iressa (gefitinib) or Tarceva (erlotinib) induced cell death in short term fresh tumor cultures predicts for long term patient survival in previoulsy-treated non-small cell lung cancer.

Sub-category: Non-Small Cell Lung Cancer

Category: Lung Cancer

Meeting: 2006 ASCO Annual Meeting

Abstract No: 17117

Author (s): L. M. Weisenthal

Abstract: Gefitinib (GEF) may act by inhibiting anti-apoptotic signals transduced by mutant EGFR kinase (Science 305:1163,04). Cell culture assays with cell death endpoints could be informative for GEF activity.

Methods: We tested 568 biopsies of fresh human tumors (TUM) with 2 concentrations of GEF (22 and 11 µg/ml) for 96 hrs, each with 2 separate cell death endpoints (DISC and MTT). Results classified as resistant (RES), intermediate (INT), or sensitive (SEN) based on means and standard deviations of training set data, reported prospectively to 3 different physicians: surgeon, pathologist, and oncologist. Assay evaluability rate > 90%.

Results: Based on overall % control cell death, the following TUM showed (on average) no greater RES or SEN than the universe of 568 assays: NSCLC (n = 72), colon (33), breast (106), ovarian (109), melanoma (23), pancreatic (20), endometrial (12). The following showed (on avg) significantly greater RES: soft tissue sarcomas (n = 24), carcinoid/islet (16), renal (15), and mesothelioma (8). For NSCLC, there was no avg difference between female (32) vs male (35) or untreated (34) vs previously treated (38). For 32 unRxd pts with survival data, there was no significant difference in overall surv for 20 pts with prospectively reported GEF RES (GR) assays vs 12 pts with SEN or INT (GSI) assays. For 31 pts with prior chemoRx (med surv = 155 days), there was significant survival disadvantage for 14 pts with prospectively reported GR vs 17 pts with GSI (median 85 vs 380 days, P2 < 0.0001, HR 3.7; 95% C.I. 2.6-19). For pts with known post-assay Rx, there were 7 pts with GSI subsequently receiving GEF or erlotinib (ERLOT), with med surv = 485 days; 9 pts with GSI not receiving GEF or ERLOT, med surv = 135 days; 10 pts with GR not receiving GEF or ERLOT, med surv = 76 days, and 3 pts with GR receiving GEF or ERLOT, med surv = 75 days. Survival of group of 7 pts was significantly greater than those of groups of 9, 10, and 3 pts (P2 = 0.037, P2 < 0.0001, and P2 = 0.0008, respectively.

Conclusions: GEF-induced cell death in cultures of fresh TUM from prev-treated NSCLC pts may identify pts with favorable prognosis, particularly when treated with GEF or ERLOT.

Functional profiling with cell culture assays for targeted drug therapy
Contact information: 
phone: 714-596-2100
Larry M. Weisenthal, M.D., Ph.D.
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