Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

CYTOGENETIC AND DNA CONTENT ANALYSIS IN MULTIPLE MYELOMA

In this dissertation, the cytogenetic characteristics of bone marrow cells from 41 multiple myeloma patients were investigated. These cytogenetic data were correlated with the total DNA content as measured by flow cytometry. Both the cytogenetic information and DNA content were then correlated with clinical data to determine if diagnosis and prognosis of multiple myeloma could be improved.^ One hundred percent of the patients demonstrated abnormal chromosome numbers per metaphase. The average chromosome number per metaphase ranged from 42 to 49.9, with a mean of 44.99. The percent hypodiploidy ranged from 0-100% and the percent hyperdiploidy from 0-53%. Detailed cytogenetic analyses were very difficult to perform because of the paucity of mitotic figures and the poor chromosome morphology. Thus, detailed chromosome banding analysis on these patients was impossible.^ Thirty seven percent of the patients had normal total DNA content, whereas 63% had abnormal amounts of DNA (one patient with less than normal amounts and 25 patients with greater than normal amounts of DNA).^ Several clinical parameters were used in the statistical analyses: tumor burden, patient status at biopsy, patient response status, past therapy, type of treatment and percent plasma cells. Only among these clinical parameters were any statistically significant correlations found: pretreatment tumor burden versus patient response, patient biopsy status versus patient response and past therapy versus patient response.^ No correlations were found between percent hypodiploid, diploid, hyperdiploid or DNA content, and the patient response status, nor were any found between those patients with: (a) normal plasma cells, low pretreatment tumor mass burden and more than 50% of the analyzed metaphases with 46 chromosomes; (b) normal amounts of DNA, low pretreatment tumor mass burden and more than 50% of the metaphases with 46 chromosomes; (c) normal amounts of DNA and normal quantities of plasma cells; (d) abnormal amounts of DNA, abnormal amounts of plasma cells, high pretreatment tumor mass burden and less than 50% of the metaphases with 46 chromosomes.^ Technical drawbacks of both cytogenetic and DNA content analysis in these multiple myeloma patients are discussed along with the lack of correlations between DNA content and chromosome number. Refined chromosome banding analysis awaits technical improvements before we can understand which chromosome material (if any) makes up the "extra" amounts of DNA in these patients. None of the correlations tested can be used as diagnostic or prognostic aids for multiple myeloma. ^

Bayesian and survival model for tumor relapse status and disease-specific survival, with applications to breast cancer

Breast cancer is the most common non-skin cancer and the second leading cause of cancer-related death in women in the United States. Studies on ipsilateral breast tumor relapse (IBTR) status and disease-specific survival will help guide clinic treatment and predict patient prognosis.^ After breast conservation therapy, patients with breast cancer may experience breast tumor relapse. This relapse is classified into two distinct types: true local recurrence (TR) and new ipsilateral primary tumor (NP). However, the methods used to classify the relapse types are imperfect and are prone to misclassification. In addition, some observed survival data (e.g., time to relapse and time from relapse to death)are strongly correlated with relapse types. The first part of this dissertation presents a Bayesian approach to (1) modeling the potentially misclassified relapse status and the correlated survival information, (2) estimating the sensitivity and specificity of the diagnostic methods, and (3) quantify the covariate effects on event probabilities. A shared frailty was used to account for the within-subject correlation between survival times. The inference was conducted using a Bayesian framework via Markov Chain Monte Carlo simulation implemented in softwareWinBUGS. Simulation was used to validate the Bayesian method and assess its frequentist properties. The new model has two important innovations: (1) it utilizes the additional survival times correlated with the relapse status to improve the parameter estimation, and (2) it provides tools to address the correlation between the two diagnostic methods conditional to the true relapse types.^ Prediction of patients at highest risk for IBTR after local excision of ductal carcinoma in situ (DCIS) remains a clinical concern. The goals of the second part of this dissertation were to evaluate a published nomogram from Memorial Sloan-Kettering Cancer Center, to determine the risk of IBTR in patients with DCIS treated with local excision, and to determine whether there is a subset of patients at low risk of IBTR. Patients who had undergone local excision from 1990 through 2007 at MD Anderson Cancer Center with a final diagnosis of DCIS (n=794) were included in this part. Clinicopathologic factors and the performance of the Memorial Sloan-Kettering Cancer Center nomogram for prediction of IBTR were assessed for 734 patients with complete data. Nomogram for prediction of 5- and 10-year IBTR probabilities were found to demonstrate imperfect calibration and discrimination, with an area under the receiver operating characteristic curve of .63 and a concordance index of .63. In conclusion, predictive models for IBTR in DCIS patients treated with local excision are imperfect. Our current ability to accurately predict recurrence based on clinical parameters is limited.^ The American Joint Committee on Cancer (AJCC) staging of breast cancer is widely used to determine prognosis, yet survival within each AJCC stage shows wide variation and remains unpredictable. For the third part of this dissertation, biologic markers were hypothesized to be responsible for some of this variation, and the addition of biologic markers to current AJCC staging were examined for possibly provide improved prognostication. The initial cohort included patients treated with surgery as first intervention at MDACC from 1997 to 2006. Cox proportional hazards models were used to create prognostic scoring systems. AJCC pathologic staging parameters and biologic tumor markers were investigated to devise the scoring systems. Surveillance Epidemiology and End Results (SEER) data was used as the external cohort to validate the scoring systems. Binary indicators for pathologic stage (PS), estrogen receptor status (E), and tumor grade (G) were summed to create PS+EG scoring systems devised to predict 5-year patient outcomes. These scoring systems facilitated separation of the study population into more refined subgroups than the current AJCC staging system. The ability of the PS+EG score to stratify outcomes was confirmed in both internal and external validation cohorts. The current study proposes and validates a new staging system by incorporating tumor grade and ER status into current AJCC staging. We recommend that biologic markers be incorporating into revised versions of the AJCC staging system for patients receiving surgery as the first intervention.^ Chapter 1 focuses on developing a Bayesian method to solve misclassified relapse status and application to breast cancer data. Chapter 2 focuses on evaluation of a breast cancer nomogram for predicting risk of IBTR in patients with DCIS after local excision gives the statement of the problem in the clinical research. Chapter 3 focuses on validation of a novel staging system for disease-specific survival in patients with breast cancer treated with surgery as the first intervention. ^

COMPARISON OF MEASUREMENTS OF ABSORBED DOSE TO WATER USING A WATER CALORIMETER AND IONIZATION CHAMBERS FOR CLINICAL RADIOTHERAPY PHOTON AND ELECTRON BEAMS

With the development of the water calorimeter direct measurement of absorbed dose in water becomes possible. This could lead to the establishment of an absorbed dose rather than an exposure related standard for ionization chambers for high energy electrons and photons. In changing to an absorbed dose standard it is necessary to investigate the effect of different parameters, among which are the energy dependence, the air volume, wall thickness and material of the chamber. The effect of these parameters is experimentally studied and presented for several commercially available chambers and one experimental chamber, for photons up to 25 MV and electrons up to 20 MeV, using a water calorimeter as the absorbed dose standard and the most recent formalism to calculate the absorbed dose with ion chambers.^ For electron beams, the dose measured with the calorimeter was 1% lower than the dose calculated with the chambers, independent of beam energy and chamber.^ For photon beams, the absorbed dose measured with the calorimeter was 3.8% higher than the absorbed dose calculated from the chamber readings. Such differences were found to be chamber and energy independent.^ The results for the photons were found to be statistically different from the results with the electron beams. Such difference could not be attributed to a difference in the calorimeter response. ^

Comparison of Bayesian and frequentist approaches in clinical outcomes between hospitals

Many public health agencies and researchers are interested in comparing hospital outcomes, for example, morbidity, mortality, and hospitalization across areas and hospitals. However, since there is variation of rates in clinical trials among hospitals because of several biases, we are interested in controlling for the bias and assessing real differences in clinical practices. In this study, we compared the variations between hospitals in rates of severe Intraventricular Haemorrhage (IVH) infant using Frequentist statistical approach vs. Bayesian hierarchical model through simulation study. The template data set for simulation study was included the number of severe IVH infants of 24 intensive care units in Australian and New Zealand Neonatal Network from 1995 to 1997 in severe IVH rate in preterm babies. We evaluated the rates of severe IVH for 24 hospitals with two hierarchical models in Bayesian approach comparing their performances with the shrunken rates in Frequentist method. Gamma-Poisson (BGP) and Beta-Binomial (BBB) were introduced into Bayesian model and the shrunken estimator of Gamma-Poisson (FGP) hierarchical model using maximum likelihood method were calculated as Frequentist approach. To simulate data, the total number of infants in each hospital was kept and we analyzed the simulated data for both Bayesian and Frequentist models with two true parameters for severe IVH rate. One was the observed rate and the other was the expected severe IVH rate by adjusting for five predictors variables for the template data. The bias in the rate of severe IVH infant estimated by both models showed that Bayesian models gave less variable estimates than Frequentist model. We also discussed and compared the results from three models to examine the variation in rate of severe IVH by 20th centile rates and avoidable number of severe IVH cases. ^

Verification of the clinical implementation of the respiratory gated beam delivery technique with synchrotron-based proton irradiation

The clinical advantage for protons over conventional high-energy x-rays stems from their unique depth-dose distribution, which delivers essentially no dose beyond the end of range. In order to achieve it, accurate localization of the tumor volume relative to the proton beam is necessary. For cases where the tumor moves with respiration, the resultant dose distribution is sensitive to such motion. One way to reduce uncertainty caused by respiratory motion is to use gated beam delivery. The main goal of this dissertation is to evaluate the respiratory gating technique in both passive scattering and scanning delivery mode. Our hypothesis for the study was that optimization of the parameters of synchrotron operation and respiratory gating can lead to greater efficiency and accuracy of respiratory gating for all modes of synchrotron-based proton treatment delivery. The hypothesis is tested in two specific aims. The specific aim #1 is to assess the efficiency of respiratory-gated proton beam delivery and optimize the synchrotron operations for the gated proton therapy. A simulation study was performed and introduced an efficient synchrotron operation pattern, called variable Tcyc. In addition, the simulation study estimated the efficiency in the respiratory gated scanning beam delivery mode as well. The specific aim #2 is to assess the accuracy of beam delivery in respiratory-gated proton therapy. The simulation study was extended to the passive scattering mode to estimate the quality of pulsed beam delivery to the residual motion for several synchrotron operation patterns with the gating technique. The results showed that variable Tcyc operation can offer good reproducible beam delivery to the residual motion at a certain phase of the motion. For respiratory gated scanning beam delivery, the impact of motion on the dose distributions by scanned beams was investigated by measurement. The results showed the threshold for motion for a variety of scan patterns and the proper number of paintings for normal and respiratory gated beam deliveries. The results of specific aims 1 and 2 provided supporting data for implementation of the respiratory gating beam delivery technique into both passive and scanning modes and the validation of the hypothesis.

A simulation study of the standard design, the rolling six design, the CRM, and the modified CRM in Phase I clinical trials

The Phase I clinical trial is considered the "first in human" study in medical research to examine the toxicity of a new agent. It determines the maximum tolerable dose (MTD) of a new agent, i.e., the highest dose in which toxicity is still acceptable. Several phase I clinical trial designs have been proposed in the past 30 years. The well known standard method, so called the 3+3 design, is widely accepted by clinicians since it is the easiest to implement and it does not need a statistical calculation. Continual reassessment method (CRM), a design uses Bayesian method, has been rising in popularity in the last two decades. Several variants of the CRM design have also been suggested in numerous statistical literatures. Rolling six is a new method introduced in pediatric oncology in 2008, which claims to shorten the trial duration as compared to the 3+3 design. The goal of the present research was to simulate clinical trials and compare these phase I clinical trial designs. Patient population was created by discrete event simulation (DES) method. The characteristics of the patients were generated by several distributions with the parameters derived from a historical phase I clinical trial data review. Patients were then selected and enrolled in clinical trials, each of which uses the 3+3 design, the rolling six, or the CRM design. Five scenarios of dose-toxicity relationship were used to compare the performance of the phase I clinical trial designs. One thousand trials were simulated per phase I clinical trial design per dose-toxicity scenario. The results showed the rolling six design was not superior to the 3+3 design in terms of trial duration. The time to trial completion was comparable between the rolling six and the 3+3 design. However, they both shorten the duration as compared to the two CRM designs. Both CRMs were superior to the 3+3 design and the rolling six in accuracy of MTD estimation. The 3+3 design and rolling six tended to assign more patients to undesired lower dose levels. The toxicities were slightly greater in the CRMs.^

Evaluating the Utility of Clinical Criteria for the Identification of Lynch Syndrome among Endometrial Cancer Patients

Background: Lynch Syndrome (LS) is a familial cancer syndrome with a high prevalence of colorectal and endometrial carcinomas among affected family members. Clinical criteria, developed from information obtained from familial colorectal cancer registries, have been generated to identify individuals at elevated risk for having LS. In 2007, the Society of Gynecologic Oncology (SGO) codified criteria to assist in identifying women presenting with gynecologic cancers at elevated risk for having LS. These criteria have not been validated in a population-based setting. Materials and Methods: We retrospectively identified 412, unselected endometrial cancer cases. Clinical and pathologic information were obtained from the electronic medical record, and all tumors were tested for expression of the DNA mismatch repair proteins through immunohistochemistry. Tumors exhibiting loss of MSH2, MSH6 and PMS2 were designated as probable Lynch Syndrome (PLS). For tumors exhibiting immunohistochemical loss of MLH1, we used the PCR-based MLH1 methylation assay to delineate PLS tumors from sporadic tumors. Samples lacking methylation of the MLH1 promoter were also designated as PLS. The sensitivity and specificity for SGO criteria for detecting PLS tumors was calculated. We compared clinical and pathologic features of sporadic tumors and PLS tumors. A simplified cost-effectiveness analysis was also performed comparing the direct costs of utilizing SGO criteria vs. universal tumor testing. Results: In our cohort, 43/408 (10.5%) of endometrial carcinomas were designated as PLS. The sensitivity and specificity of SGO criteria to identify PLS cases were 32.7 and 77%, respectively. Multivariate analysis of clinical and pathologic parameters failed to identify statistically significant differences between sporadic and PLS tumors with the exception of tumors arising from the lower uterine segment. These tumors were more likely to occur in PLS tumors. Cost-effectiveness analysis showed clinical criteria and universal testing strategies cost $6,235.27/PLS case identified and $5,970.38/PLS case identified, respectively. Conclusions: SGO 5-10% criteria successfully identify PLS cases among women who are young or have significant family history of LS related tumors. However, a larger proportion of PLS cases occurring at older ages with less significant family history are not detected by this screening strategy. Compared to SGO clinical criteria, universal tumor testing is a cost effective strategy to identify women presenting with endometrial cancer who are at elevated risk for having LS.