EFFICACY-TO-EFFECTIVENESS CLINICAL TRIALS.

Efficacy trials, which assess treatments in optimally selected patients under advantageous conditions for relatively short time periods, are necessary to gain regulatory approval for marketing. In contrast, effectiveness trials, which test treatments across a spectrum of patients in real-world conditions with follow-up periods that match typical treatment regimens, provide critical information on drug effects in those patients who may ultimately receive the treatment. We previously proposed a study design that integrates efficacy and effectiveness trials into a 2-component "efficacy-to-effectiveness (E2E) trial," in which if the initial efficacy trial component is positive, then the trial immediately and seamlessly transitions to the effectiveness trial component. However, we believe that total study duration could be even further shortened by simultaneously addressing efficacy and effectiveness too (EE2). An EE2 trial rigorously demonstrates efficacy, but uses broad inclusion characteristics of effectiveness trials. An example of a study using EE2 design, the IMMEDIATE (Immediate Myocardial Metabolic enhancement During Initial Assessment and Treatment in Emergency Care) trial, is provided.

Do radiation-induced bystander effects correlate to the intrinsic radiosensitivity of individuals and have clinical significance?

It is well known that patients can vary in their normal tissue response to radiotherapy, and this can be problematic. As a result, radiobiologists have been using in vitro models to assess variation in response and elucidate the genetic determinants of this variation. However, the clinical relevance of these models is currently unknown. In this study, blood samples from healthy controls (n = 20) and colorectal carcinoma patients (n = 60) were cultured in vitro to assess two radiobiological end points in parallel: intrinsic radiosensitivity assayed by chromosomal aberrations (G(2) scores) and radiation-induced bystander effects assayed by viability testing. Increased intrinsic radiosensitivity was observed in colorectal carcinoma donors (55%) compared to the healthy donors (5%) (P < 0.005). Similarly, more pronounced radiation-induced bystander effects were observed in the colorectal carcinoma donors compared to the healthy donors after 24 h exposure but not after 96 h exposure to donor irradiated cell conditioned medium (ICCM) (P < 0.05). All scores were tested for correlation with the age, sex and clinical stage of the colorectal carcinoma patients. The only statistically significant correlation was found in samples from severe Dukes D patients (P < 0.005), which had low/radioresistant G(2) scores. No correlation was found between radiation-induced intrinsic sensitivity and bystander effects, which suggests that they may have separate underlying molecular mechanisms, but they both show clinical relevance in individual patient samples.

Radiation and chemotherapy bystander effects induce early genomic instability events: telomere shortening and bridge formation coupled with mitochondrial dysfunction.

The bridge breakage fusion cycle is a chromosomal instability mechanism responsible for genomic changes. Radiation bystander effects induce genomic instability; however, the mechanism driving this instability is unknown. We examined if radiation and chemotherapy bystander effects induce early genomic instability events such as telomere shortening and bridge formation using a human colon cancer explant model. We assessed telomere lengths, bridge formations, mitochondrial membrane potential and levels of reactive oxygen species in bystander cells exposed to medium from irradiated and chemotherapy-treated explant tissues. Bystander cells exposed to media from 2Gy, 5Gy, FOLFOX treated tumor and matching normal tissue showed a significant reduction in telomere lengths (all p values <0.018) and an increase in bridge formations (all p values <0.017) compared to bystander cells treated with media from unirradiated tissue (0Gy) at 24h. There was no significant difference between 2Gy and 5Gy treatments, or between effects elicited by tumor versus matched normal tissue. Bystander cells exposed to media from 2Gy irradiated tumor tissue showed significant depolarisation of the mitochondrial membrane potential (p=0.012) and an increase in reactive oxygen species levels. We also used bystander cells overexpressing a mitochondrial antioxidant manganese superoxide dismutase (MnSOD) to examine if this antioxidant could rescue the mitochondrial changes and subsequently influence nuclear instability events. In MnSOD cells, ROS levels were reduced (p=0.02) and mitochondrial membrane potential increased (p=0.04). These events were coupled with a decrease in percentage of cells with anaphase bridges and a decrease in the number of cells undergoing telomere length shortening (p values 0.01 and 0.028 respectively). We demonstrate that radiation and chemotherapy bystander responses induce early genomic instability coupled with defects in mitochondrial function. Restoring mitochondrial function through overexpression of MnSOD significantly rescues nuclear instability events; anaphase bridges and telomere length shortening.

An example of medical device-based projection of clinical trial enrollment: Use of electrocardiographic data to identify candidates for a trial in acute coronary syndromes.

BACKGROUND: To identify potential participants for clinical trials, electronic health records (EHRs) are searched at potential sites. As an alternative, we investigated using medical devices used for real-time diagnostic decisions for trial enrollment. METHODS: To project cohorts for a trial in acute coronary syndromes (ACS), we used electrocardiograph-based algorithms that identify ACS or ST elevation myocardial infarction (STEMI) that prompt clinicians to offer patients trial enrollment. We searched six hospitals' electrocardiograph systems for electrocardiograms (ECGs) meeting the planned trial's enrollment criterion: ECGs with STEMI or > 75% probability of ACS by the acute cardiac ischemia time-insensitive predictive instrument (ACI-TIPI). We revised the ACI-TIPI regression to require only data directly from the electrocardiograph, the e-ACI-TIPI using the same data used for the original ACI-TIPI (development set n = 3,453; test set n = 2,315). We also tested both on data from emergency department electrocardiographs from across the US (n = 8,556). We then used ACI-TIPI and e-ACI-TIPI to identify potential cohorts for the ACS trial and compared performance to cohorts from EHR data at the hospitals. RESULTS: Receiver-operating characteristic (ROC) curve areas on the test set were excellent, 0.89 for ACI-TIPI and 0.84 for the e-ACI-TIPI, as was calibration. On the national electrocardiographic database, ROC areas were 0.78 and 0.69, respectively, and with very good calibration. When tested for detection of patients with > 75% ACS probability, both electrocardiograph-based methods identified eligible patients well, and better than did EHRs. CONCLUSION: Using data from medical devices such as electrocardiographs may provide accurate projections of available cohorts for clinical trials.

Clusterin and chemotherapy sensitivity under normoxic and graded hypoxic conditions in colorectal cancer.

BACKGROUND AND AIMS: In vitro studies have shown that clusterin modulates treatment sensitivity in a number of human cancers; however, the interaction between clusterin expression and hypoxia in controlling treatment response in CRC has not previously been examined. The aim of this study was to assess the effect of clusterin overexpression in CRC cells on sensitivity to 5-fluorouracil (5-FU), oxaliplatin and FOLFOX treatment under normoxic and graded hypoxic conditions. METHODS: SW480 colon cancer cells were transfected with full length Clusterin cDNA to generate a clusterin overexpressing cell line. Overexpression was confirmed by western blot analysis. The response of parental and clusterin overexpressing cells to 5-FU, oxaliplatin and FOLFOX was examined using a crystal violet-based proliferation assay under normoxic conditions, 3% and 1% hypoxic conditions. The levels of apoptosis and G2/M arrest in FOLFOX-treated cells were assessed by flow cytometry. RESULTS: Under normoxic conditions, clusterin overexpressing cells were more sensitive to FOLFOX treatment (p = 0.01); under 3% and 1% hypoxic conditions, overexpressing clusterin cells were more sensitive to 5-FU, oxaliplatin and FOLFOX, p values <0.05 for all conditions. Under normoxic conditions, overexpressing clusterin cells showed significantly higher levels of apoptosis when treated with FOLFOX compared to untransfected cells; levels of G2M cells were not significantly different. Under both 3% and 1% hypoxia, the percentage of cells undergoing apoptosis following FOLFOX treatment was significantly higher in overexpressing clusterin cells. CONCLUSION: These in vitro findings suggest that tumours expressing high levels of clusterin, particularly if hypoxic in nature, may benefit from treatments such as FOLFOX.

Mitochondrial mutagenesis induced by tumor-specific radiation bystander effects.

The radiation bystander effect is a cellular process whereby cells not directly exposed to radiation display cellular alterations similar to directly irradiated cells. Cellular targets including mitochondria have been postulated to play a significant role in this process. In this study, we utilized the Random Mutation Capture assay to quantify the levels of random mutations and deletions in the mitochondrial genome of bystander cells. A significant increase in the frequency of random mitochondrial mutations was found at 24 h in bystander cells exposed to conditioned media from irradiated tumor explants (p = 0.018). CG:TA mutations were the most abundant lesion induced. A transient increase in the frequency of random mitochondrial deletions was also detected in bystander cells exposed to conditioned media from tumor but not normal tissue at 24 h (p = 0.028). The increase in both point mutations and deletions was transient and not detected at 72 h. To further investigate mitochondrial dysfunction, mitochondrial membrane potential and reactive oxygen species were assessed in these bystander cells. There was a significant reduction in mitochondrial membrane potential and this was positively associated with the frequency of random point mutation and deletions in bystander cells treated with conditioned media from tumor tissue (r = 0.71, p = 0.02). This study has shown that mitochondrial genome alterations are an acute consequence of the radiation bystander effect secondary to mitochondrial dysfunction and suggests that this cannot be solely attributable to changes in ROS levels alone.

Increased topoisomerase IIalpha expression in colorectal cancer is associated with advanced disease and chemotherapeutic resistance via inhibition of apoptosis.

Topoisomerase IIalpha is a nuclear enzyme that regulates the tertiary structure of DNA. The influence of topoisomerase IIalpha gene (TOP2A) or protein alterations on disease progression and treatment response in colorectal cancer (CRC) is unknown. The study investigated the clinical relevance of topoisomerase IIalpha in CRC using in vivo and in vitro models. Differentially expressed genes in early and late-stage CRC were identified by array comparative genomic hybridization (CGH). Cellular location of gene amplifications was determined by fluorescence in situ hybridization (FISH). Topoisomerase IIalpha levels, proliferation index, and HER2 expression were examined in 228 colorectal tumors by immunohistochemistry. Overexpression of topoisomerase IIalpha in vitro was achieved by liposome-based transfection. Cell growth inhibition and apoptosis were quantified using the crystal violet assay and flow cytometry, respectively, in response to drug treatment. Amplification of TOP2A was identified in 3 (7.7%) tumors using array CGH and confirmed using FISH. At the protein level, topoisomerase IIalpha staining was observed in 157 (69%) tumors, and both staining and intensity levels were associated with an aggressive tumor phenotype (p values 0.04 and 0.005, respectively). Using logistic regression analysis, topoisomerase IIalpha remained significantly associated with advanced tumor stage when corrected for tumor proliferation (p=0.007) and differentiation (p=0.001). No association was identified between topoisomerase IIalpha and HER2. In vitro, overexpression of topoisomerase IIalpha was associated with resistance to irinotecan (p=0.001) and etoposide chemotherapy (p=0.03), an effect mediated by inhibition of apoptosis. Topoisomerase IIalpha overexpression is significantly associated with alterations in tumor behavior and response to drug treatment in CRC. Our results suggest that gene amplification may represent an important mechanism underlying these changes.