Theodore S. Lawrence

MicroRNA miR-34 Inhibits Human Pancreatic Cancer Tumor-Initiating Cells

Background MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells. Methodology/Principal Findings We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines MiaPaCa2 and BxPC3, and the potential link to pancreatic cancer stem cells. Restoration of miR-34 expression in the pancreatic cancer cells by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cells to chemotherapy and radiation. We identified that CD44+/CD133+ MiaPaCa2 cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo. Conclusions/Significance Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer cells. Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination. Restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53–miR34, potentially via inhibiting pancreatic cancer stem cells.

Uncertainties in CT-based radiation therapy treatment planning associated with patient breathing

PURPOSE To evaluate uncertainties associated with treatment-planning computed tomography (CT) data obtained with the patient breathing freely. METHODS AND MATERIALS Patients with thoracic or abdominal tumors underwent a standard treatment-planning CT study while breathing quietly and freely, followed by CT scans while holding their breath at normal inhalation and normal exhalation. Identical treatment plans on all three CT data sets for each patient pointed out differences in: (a) radiation path lengths; (b) positions of the organs; (c) physical volumes of the lung, liver, and kidneys; (d) the interpretation of plan evaluation tools such as dose-volume histograms and normal tissue complication probability (NTCP) models; and (e) how well the planning CT data set represented the average of the inhalation and exhalation studies. RESULTS Inhalation and exhalation data differ in terms of radiation path length (nearly one quarter of the cases had path-length differences > 1 cm), although the free breathing and average path lengths do not exhibit large differences (0-9 mm). Liver and kidney movements averaged 2 cm, whereas differences between the free breathing and average positions averaged 0.6 cm. The physical volume of the liver between the free breathing and static studies varied by as much as 12%. The NTCP calculations on exhale and inhale studies varied from 3 to 43% for doses that resulted in a 15% NTCP on the free-breathing studies. CONCLUSION Free-breathing CT studies may improperly estimate the position and volume of critical structures, and thus may mislead evaluation of plans based on such volume dependent criteria such as dose-volume histograms and NTCP calculations.

Estimation of tumor control probability model parameters from 3-D dose distributions of non-small cell lung cancer patients

Tumor control probability (TCP) model calculations may be used in a relative manner to evaluate and optimize three-dimensional (3-D) treatment plans. Using a mathematical model which makes a number of simplistic assumptions, TCPs can be estimated from a 3-D dose distribution of the tumor given the dose required for a 50% probability of tumor control (D50) and the normalized slope (gamma) of the sigmoid-shaped dose-response curve at D50. The purpose of this work was to derive D50 and gamma from our clinical experience using 3-D treatment planning to treat non-small cell lung cancer (NSCLC) patients. Our results suggest that for NSCLC patients, the dose to achieve significant probability of tumor control may be large (on the order of 84 Gy) for longer (> 30 months) local progression-free survival.

Phase I Trial of Radiation Dose Escalation With Concurrent Weekly Full-Dose Gemcitabine in Patients With Advanced Pancreatic Cancer

PURPOSE The primary objective of this phase I trial was to determine the maximum-tolerated dose of radiation that could be delivered to the primary tumor concurrent with full-dose gemcitabine in patients with advanced pancreatic cancer. PATIENTS AND METHODS Thirty seven patients with unresectable (n = 34) or incompletely resected pancreatic cancer (n = 3) were treated. Gemcitabine was administered as a 30-minute intravenous infusion at a dose of 1,000 mg/m(2) on days 1, 8, and 15 of a 28-day cycle. Radiation therapy was initiated on day 1 and directed at the primary tumor alone, without prophylactic nodal coverage. The starting radiation dose was 24 Gy in 1.6-Gy fractions. Escalation was achieved by increasing the fraction size in increments of 0.2 Gy, keeping the duration of radiation constant at 3 weeks. A second cycle of gemcitabine alone was intended after a 1-week rest. RESULTS Two of six assessable patients experienced dose-limiting toxicity at the final planned dose level of the trial (42 Gy in 2.8-Gy fractions), one with grade 4 vomiting and one with gastric/duodenal ulceration. Two additional patients at this dose level experienced late gastrointestinal toxicity that required surgical management. CONCLUSION The final dose investigated (42 Gy) is not recommended for further study considering the occurrence of both acute and late toxicity. However, a phase II trial of this novel gemcitabine-based chemoradiotherapy approach, at a radiation dose of 36 Gy in 2.4-Gy fractions, is recommended on the basis of tolerance, patterns of failure, and survival data.

The reproducibility of organ position using active breathing control (ABC) during liver radiotherapy

PURPOSE To evaluate the intrafraction and interfraction reproducibility of liver immobilization using active breathing control (ABC). METHODS AND MATERIALS Patients with unresectable intrahepatic tumors who could comfortably hold their breath for at least 20 s were treated with focal liver radiation using ABC for liver immobilization. Fluoroscopy was used to measure any potential motion during ABC breath holds. Preceding each radiotherapy fraction, with the patient setup in the nominal treatment position using ABC, orthogonal radiographs were taken using room-mounted diagnostic X-ray tubes and a digital imager. The radiographs were compared to reference images using a 2D alignment tool. The treatment table was moved to produce acceptable setup, and repeat orthogonal verification images were obtained. The positions of the diaphragm and the liver (assessed by localization of implanted radiopaque intra-arterial microcoils) relative to the skeleton were subsequently analyzed. The intrafraction reproducibility (from repeat radiographs obtained within the time period of one fraction before treatment) and interfraction reproducibility (from comparisons of the first radiograph for each treatment with a reference radiograph) of the diaphragm and the hepatic microcoil positions relative to the skeleton with repeat breath holds using ABC were then measured. Caudal-cranial (CC), anterior-posterior (AP), and medial-lateral (ML) reproducibility of the hepatic microcoils relative to the skeleton were also determined from three-dimensional alignment of repeat CT scans obtained in the treatment position. RESULTS A total of 262 fractions of radiation were delivered using ABC breath holds in 8 patients. No motion of the diaphragm or hepatic microcoils was observed on fluoroscopy during ABC breath holds. From analyses of 158 sets of positioning radiographs, the average intrafraction CC reproducibility (sigma) of the diaphragm and hepatic microcoil position relative to the skeleton using ABC repeat breath holds was 2.5 mm (range 1.8-3.7 mm) and 2.3 mm (range 1.2-3.7 mm) respectively. However, based on 262 sets of positioning radiographs, the average interfraction CC reproducibility (sigma) of the diaphragm and hepatic microcoils was 4.4 mm (range 3.0-6.1 mm) and 4.3 mm (range 3.1-5.7 mm), indicating a change of diaphragm and microcoil position relative to the skeleton over the course of treatment with repeat breath holds at the same phase of the respiratory cycle. The average population absolute intrafraction CC offset in diaphragm and microcoil position relative to skeleton was 2.4 mm and 2.1 mm respectively; the average absolute interfraction CC offset was 5.2 mm. Analyses of repeat CT scans demonstrated that the average intrafraction excursion of the hepatic microcoils relative to the skeleton in the CC, AP, and ML directions was 1.9 mm, 0.6 mm, and 0.6 mm respectively and the average interfraction CC, AP, and ML excursion of the hepatic microcoils was 6.6 mm, 3.2 mm, and 3.3 mm respectively. CONCLUSION Radiotherapy using ABC for patients with intrahepatic cancer is feasible, with good intrafraction reproducibility of liver position using ABC. However, the interfraction reproducibility of organ position with ABC suggests the need for daily on-line imaging and repositioning if treatment margins smaller than those required for free breathing are a goal.

Phase II Trial of High-Dose Conformal Radiation Therapy With Concurrent Hepatic Artery Floxuridine for Unresectable Intrahepatic Malignancies

PURPOSE A phase II trial was conducted to determine if high-dose radiation with concurrent hepatic arterial floxuridine would improve survival in patients with unresectable intrahepatic malignancies. PATIENTS AND METHODS Three-dimensional conformal high-dose radiation therapy was delivered concurrently with hepatic arterial floxuridine in 128 patients. The radiation dose was based on a normal-tissue complication probability model and subjected the patient to an estimated maximum risk of radiation-induced liver disease of 10% to 15%. The study design provided more than 80% power to detect a two-fold increase in median survival compared with historical controls at a 5% significance level. RESULTS The median radiation dose delivered was 60.75 Gy (1.5-Gy fractions bid). At a median follow-up time of 16 months (26 months in patients who were alive) the median survival was 15.8 months (95% CI, 12.6 to 18.3 months), significantly longer than in the historical control. The actuarial 3-year survival was 17%. The total dose was the only significant predictor of survival. Primary hepatobiliary tumors had a significantly greater tendency to remain confined to the liver than did colorectal cancer metastases. Overall toxicity was acceptable, with 27 patients (21%) and 11 patients (9%) developing grade 3 and 4 toxicity, respectively, and one treatment-related death. CONCLUSION The results suggest that, compared with historical controls, high-dose focal liver irradiation with hepatic artery floxuridine prolongs survival in patients with unresectable chemotherapy-refractory metastatic colorectal cancer and primary hepatobiliary tumors. This provides a rationale for intensification of local therapy for unresectable hepatobiliary cancers and integration of this regimen with newer systemic therapy for patients with colorectal cancer.

Integration of EGFR inhibitors with radiochemotherapy

Laboratory studies that led to the development of epidermal growth factor receptor (EGFR) inhibitors indicated that such inhibitors would be effective when given to patients with tumours that are driven by activated EGFR. However, initial clinical studies have shown modest responses to EGFR inhibitors when used alone, and it has not yet been possible to clearly identify which tumours will respond to this therapy. As a result, EGFR inhibitors are now used in combination with radiation therapy, chemotherapy and, more recently, with concurrent radiochemotherapy. In general, these clinical trials have been designed without much preclinical data. What do we need to know to make these combinations successful in the clinic?

Functional diffusion map as an early imaging biomarker for high-grade glioma: correlation with conventional radiologic response and overall survival.

PURPOSE Assessment of radiologic response (RR) for brain tumors utilizes the Macdonald criteria 8 to 10 weeks from the start of treatment. Diffusion magnetic resonance imaging (MRI) using a functional diffusion map (fDM) may provide an earlier measure to predict patient survival. PATIENTS AND METHODS Sixty patients with high-grade glioma were enrolled onto a study of intratreatment MRI at 1, 3, and 10 weeks. Receiver operating characteristic curve analysis was used to evaluate imaging parameters as a function of patient survival at 1 year. Both log-rank and Cox proportional hazards models were utilized to assess overall survival. RESULTS Greater increases in diffusion in response to therapy over time were observed in those patients alive at 1 year compared with those who died as a result of disease. The volume of tumor with increased diffusion by fDM at 3 weeks was the strongest predictor of patient survival at 1 year, with larger fDM predicting longer median survival (52.6 v 10.9 months; log-rank, P < .003; hazard ratio [HR] = 2.7; 95% CI, 1.5 to 5.9). Radiologic response at 10 weeks had similar prognostic value (median survival, 31.6 v 10.9 months; log-rank P < .0007; HR = 2.9; 95% CI, 1.7 to 7.2). Radiologic response and fDM differed in 25% of cases. A composite index of response including fDM and RR provided a robust predictor of patient survival and may identify patients in whom RR does not correlate with clinical outcome. CONCLUSION Compared with conventional neuroimaging, fDM provided an earlier assessment of equal predictive value, and the combination of fDM and RR provided a more accurate prediction of patient survival than either metric alone.

Analysis of clinical complication data for radiation hepatitis using a parallel architecture model

PURPOSE The detailed knowledge of dose volume distributions available from the three-dimensional (3D) conformal radiation treatment of tumors in the liver (reported elsewhere) offers new opportunities to quantify the effect of volume on the probability of producing radiation hepatitis. We aim to test a new parallel architecture model of normal tissue complication probability (NTCP) with these data. METHODS AND MATERIALS Complication data and dose volume histograms from a total of 93 patients with normal liver function, treated on a prospective protocol with 3D conformal radiation therapy and intraarterial hepatic fluorodeoxyuridine, were analyzed with a new parallel architecture model. Patient treatment fell into six categories differing in doses delivered and volumes irradiated. By modeling the radiosensitivity of liver subunits, we are able to use dose volume histograms to calculate the fraction of the liver damaged in each patient. A complication results if this fraction exceeds the patients functional reserve. To determine the patient distribution of functional reserves and the subunit radiosensitivity, the maximum likelihood method was used to fit the observed complication data. RESULTS The parallel model fit the complication data well, although uncertainties on the functional reserve distribution and subunit radiosensitivity are highly correlated. CONCLUSION The observed radiation hepatitis complications show a threshold effect that can be described well with a parallel architecture model. However, additional independent studies are required to better determine the parameters defining the functional reserve distribution and subunit radiosensitivity.

Mechanism of Radiosensitization by the Chk1/2 Inhibitor AZD7762 Involves Abrogation of the G2 Checkpoint and Inhibition of Homologous Recombinational DNA Repair

The median survival for patients with locally advanced pancreatic cancer treated with gemcitabine and radiation is approximately 1 year. To develop improved treatment, we have combined a Chk1/2-targeted agent, AZD7762, currently in phase I clinical trials, with gemcitabine and ionizing radiation in preclinical pancreatic tumor models. We found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitized MiaPaCa-2 cells to radiation. AZD7762 inhibited Chk1 autophosphorylation (S296 Chk1), stabilized Cdc25A, and increased ATR/ATM-mediated Chk1 phosphorylation (S345 Chk1). Radiosensitization by AZD7762 was associated with abrogation of the G(2) checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent gamma-H2AX expression. AZD7762 was also a radiation sensitizer in multiple tumor xenograft models. In both MiaPaCa-2- and patient-derived xenografts, AZD7762 significantly prolonged the median time required for tumor volume doubling in response to gemcitabine and radiation. Together, our findings suggest that G(2) checkpoint abrogation and homologous recombination repair inhibition both contribute to sensitization by Chk1 inhibition. Furthermore, they support the clinical use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer.