Pharmacogenetic analysis of paclitaxel transport and metabolism genes in breast cancer.

Paclitaxel is commonly used in the treatment of breast cancer. Variability in paclitaxel clearance may contribute to the unpredictability of clinical outcomes. We assessed genomic DNA from the plasma of 93 patients with high-risk primary or stage IV breast cancer, who received dose-intense paclitaxel, doxorubicin and cyclophosphamide. Eight polymorphisms in six genes associated with metabolism and transport of paclitaxel were analyzed using Pyrosequencing. We found no association between ABCB1, ABCG2, CYP1B1, CYP3A4, CYP3A5 and CYP2C8 genotypes and paclitaxel clearance. However, patients homozygous for the CYP1B1*3 allele had a significantly longer progression-free survival than patients with at least one Valine allele (P=0.037). This finding could reflect altered paclitaxel metabolism, however, the finding was independent of paclitaxel clearance. Alternatively, the role of CYP1B1 in estrogen metabolism may influence the risk of invasive or paclitaxel resistant breast cancer in patients carrying the CYP1B1*3 allele.

Expression of drug pathway proteins is independent of tumour type.

Current clinicopathological staging systems have the advantage of standardized criteria for assessing tumour stage, and a relationship between advancing tumour stage and poor prognosis has been established for most cancers. However, these tools have not led to clear criteria for therapy selection in individual patients. Indeed, the concept of therapy based on anatomical location seems quaint. Therefore, a representative drug pathway (irinotecan) was evaluated across common tumour types to test the hypothesis that pharmacological proteins are expressed independent of anatomical location. Many enzymes are involved in controlling the disposition of irinotecan, including the cellular target (TOP1), metabolism enzymes (CES2, UGT1A1, CYP3A4, CYP3A5), and cellular transporters of the anti-cancer agent (ABCB1, ABCC1, ABCC2, ABCC3, ABCC5, ABCG2). These 11 proteins were evaluated in tissue microarrays containing colon, breast, prostate, ovary, and lung cancers; brain tumours; melanoma; lymphoma; and selected normal tissues. A total of 255 tumours and 37 normal tissue samples were evaluable for all proteins. Linear discriminant analysis designed to predict the tissue type from the protein expression levels revealed a 49.6% misclassification rate, indicating that protein expression of this drug pathway is not associated with tissue type. Cluster analysis identified a variety of tumours with the same pharmacological profile. The anatomy independence of drug pathways stimulates efforts to move away from our traditional approaches to the selection of cancer therapy.

Unfavourable expression of pharmacologic markers in mucinous colorectal cancer.

Patients with mucinous colorectal cancer generally have worse prognoses than those with the nonmucinous variety. The reason for this disparity is unclear, but may result from a differential response to adjuvant chemotherapy. We examined known molecular markers for response to common chemotherapy in these two histological subtypes. In all, 21 patients with mucinous and 30 with nonmucinous Dukes C colorectal cancer were reviewed for demographic data and outcome. Total RNA from the tumours and adjacent normal mucosa was isolated and reverse transcribed. Quantitative expression levels of drug pathway genes were determined using TaqMan RT-PCR (5-fluorouracil (5-FU): TYMS, DPYD, ECGF1; oxaliplatin: GSTP1 (glutathione S-transferase pi), ERCC1 and 2; irinotecan: ABCB1, ABCG2, CYP3A4, UGT1A1, CES2, TOP1). Mucinous tumours significantly overexpressed both TYMS and GSTP1 relative to nonmucinous tumours and patient-matched normal mucosa. No significant differences in expression of the remaining markers were found. Mean follow-up was 20 months; 17 patients had recurrent disease. Among patients receiving 5-FU, those with mucinous tumours experienced shorter disease-free survival (DFS) than those with nonmucinous tumours (median DFS 13.8 vs 46.5 months, P=0.053). Mucinous colorectal cancer overexpresses markers of resistance to 5-FU and oxaliplatin. Likewise, DFS may be decreased in patients with mucinous tumours who receive 5-FU. The presence of mucin should be carefully evaluated in developmental trials of new agents for treating colorectal cancer.

RNA profiling of cyclooxygenases 1 and 2 in colorectal cancer.

Cyclooxygenases (particularily Cox-2) are involved in carcinogenesis and metastatic cancer progression. The expression profiles of the cyclooxygenases and the roles they play in established tumours of similar stage remains unclear. We report that Cox-1 and Cox-2 expression is highly variable in Dukes' C tumours, and changes in Cox-1 expression may be of importance.

Relationship of cytomegalovirus viral load in blood to pneumonitis in lung transplant recipients.

We developed a multiplex, quantitative, real-time, polymerase chain reaction assay for cytomegalovirus (CMV) and used it to measure the CMV viral load in weekly blood specimens from 43 lung transplant recipients. The median viral load in blood samples immediately preceding bronchoscopy was 1150 copies/microg human DNA for 12 subjects with pneumonitis compared to 91 copies for 31 subjects without (P=0.02, Mann-Whitney U test). Each log10 increase in CMV viral load resulted in an increase of 1.92 in the odds ratio for CMV pneumonitis (95% confidence interval 1.03-3.56). CMV viral load was elevated (>100 copies/microg human DNA) for a median of 21 days before bronchoscopy in those subjects with pneumonitis versus 0 days in those without (P=0.004). We conclude that the risk of CMV pneumonitis after lung transplantation is related to the level of CMV DNA in blood. Quantitative PCR should be evaluated prospectively for the preemptive management of CMV in lung transplant recipients.

Tree-based recursive partitioning methods for subdividing sibpairs into relatively more homogeneous subgroups.

We propose a new splitting rule for recursively partitioning sibpair data into relatively more homogeneous subgroups. This strategy is designed to identify subgroups of sibpairs such that within-subgroup analyses result in increased power to detect linkage using Haseman-Elston regression. We assume that the subgroups can be defined by patterns of non-genetic binary covariates measured on each sibpair. The data we consider consists of the squared difference of a quantitative trait measurement on each sibpair, estimates of identity-by-descent (IBD) values at each genetic marker, and binary covariate data describing characteristics of the sibpair (e.g., race, sex, family history of disease). To test the efficacy of this method in linkage analysis, we performed two simulation experiments. In the first, we simulated a mixture consisting of 66.6% of the sibpairs with no linkage and 33.3% of the sibpairs with genetic linkage to one marker. The two groups were distinguished by the value of a single binary covariate. We also simulated one unlinked marker and one random covariate to include as noise in the data. In the second experiment, we simulated a mixture consisting of 55% of the sibpairs with no genetic linkage, 22.5% of the sibpairs with genetic linkage to one marker, and 22.5% of the sibpairs with linkage to a different marker. Each subgroup was defined by a distinct pattern of two binary covariates. We also simulated one unlinked marker and two random covariates to include as noise in the data. Our simulation studies found that we can significantly increase the overall power to detect linkage by fitting Haseman-Elston regression models to homogeneous subgroups with only a small increase in the false-positive rate. Second, the splitting rule can correctly identify important covariates and linked markers. Third, recursive partitioning of sibpair data using this splitting rule can correctly identify sibpair subgroups. These results indicate that partitioning sibpairs into homogeneous subgroups is feasible and significantly increases the power to detect linkage, thus demonstrating the practical utility and potential this new methodology holds.

Injury in the era of genomics.

The traditional approach to the study of biology employs small-scale experimentation that results in the description of a molecular sequence of known function or relevance. In the era of the genome the reverse is true, as large-scale cloning and gene sequencing come first, followed by the use of computational methods to systematically determine gene function and regulation. The overarching goal of this new approach is to translate the knowledge learned from a systematic, global analysis of genomic data into a complete understanding of biology. For investigators who study shock, the specific goal is to increase understanding of the adaptive response to injury at the level of the entire genome. This review describes our initial experience using DNA microarrays to profile stress-induced changes in gene expression. We conclude that efforts to apply genomics to the study of injury are best coordinated by multi-disciplinary groups, because of the extensive expertise required.

Evaluation of a reduced MR imaging sequencing protocol in adult patients with stroke.

PURPOSE: To compare a reduced (three-sequence) magnetic resonance (MR) imaging protocol with a full (eight- to 10-sequence) MR imaging protocol in adults suspected of having stroke. MATERIALS AND METHODS: Six neuroradiologists interpreted a consecutive sample of 265 MR images in patients suspected of having stroke. Each read reduced-protocol images in a discrete series of 40 patients (one read images in only 15) and corresponding full-protocol images 1 month later (reduced/full protocol). Five of the readers each read images in 10 additional cases, five each as full/full and reduced/reduced protocol controls. kappa values between full and reduced protocols, reader assessment of protocol adequacy, confidence level, and need for additional sequences or examinations were evaluated. RESULTS: In the reduced/full protocol, the kappa value for detecting ischemia was 0.797; and that for detecting any clinically important abnormality, 0.635. Statistically similar kappa values were found with the full/full control design (kappa = 0.802 and 0.715, respectively). The full protocol was judged more adequate than the reduced protocol (2.0 of 5.0 points vs 1.6, P <.001) and generated greater diagnostic confidence (8.6 of 10.0 points vs 8.9, P =.01), less need for additional sequences (2.7 of 6.0 points vs 1.5, P <.001), and more requests for additional examinations (28.4% vs 36.3%). CONCLUSION: Disagreement between interpretations of reduced- and full-protocol images might be attributable to baseline-level intraobserver inconsistency, as demonstrated in control designs. A greater number of sequences did not lead to greater consistency.

Gene expression profiling with oligonucleotide microarrays distinguishes World Health Organization grade of oligodendrogliomas.

Oligodendrogliomas are the second most common type of glial neoplasm with distinct prognostic and therapeutic implications. Although refinements have led to improved clinical stratification, current grading schemes are still limited by subjective histopathological criteria. In this report, we have used oligonucleotide array technology to perform expression profiling in morphologically classic oligodendrogliomas. Expression information from approximately 1100 genes divided tumors into two molecularly distinct groups that corresponded exactly to their previously assigned histological grades. Subsequent gene clustering identified a subset of 196 transcripts showing a common, differential expression pattern between tumor grades. A number of these genes have been associated with the maintenance of cytoarchitecture, cellular differentiation and maturation, immunogenicity, and chemotherapeutic resistance. These results demonstrate the utility of gene expression profiling as an objective, ancillary tool for grading oligodendrogliomas and a potential approach for classifying diffuse gliomas where histological assessment may be difficult or ambiguous.

Classification methods for confronting heterogeneity.

Recursive partitioning/tree models are discussed as a method of dissecting the complex nature of traits with different causal mechanisms operating in different subsets of the data (e.g., different genes operating in different subsets of families). In addition to the straightforward application of classification and regression trees to define more homogeneous subsets of the data on which to conduct further analysis, developments incorporating linkage analysis into the definition of the regression trees (Shannon et al., 2000) are discussed. The pros and cons of recursive partitioning vs. the related approach of context-dependent analysis (Turner et al., 1999) are also reviewed as two promising analysis strategies that may be useful for genetic dissection of complex traits.

The impact of ganciclovir-resistant cytomegalovirus infection after lung transplantation.

BACKGROUND: Cytomegalovirus (CMV) resistance to ganciclovir has become increasingly common in acquired immunodeficiency syndrome patients but has only rarely been reported in recipients of solid organ transplants. METHODS: A retrospective study of ganciclovir susceptibility testing of CMV isolates recovered from lung transplant recipients was performed. Patients with CMV isolates having partial (1 or =3 microg/ml) to ganciclovir determined by plaque reduction assay were included in a case-control study to identify risk factors for ganciclovir resistance. RESULTS: Between 2/91 and 5/98, 18 patients (5.2% of patients transplanted) were found to have CMV infections with some degree of ganciclovir resistance (4 partially, 14 fully resistant). More positive viral blood cultures (3.2+/-2.5 vs. 1.6+/-1.4 CMV positive cultures, P=0.02) and more episodes of CMV pneumonitis (0.24+/-0.23 vs. 0.10+/-0.17 episodes/bronchoscopy, P=0.02) occurring before the detection of resistance were seen among resistant patients than controls. Ganciclovir-resistant patients received more antithymocyte globulin during induction (70+/-44 vs. 45+/-39 mg/kg, P=0.03) and received ganciclovir for a greater number of days (79+/-52 vs. 64+/-53 days, P=0.005) before the detection of resistance than controls. Ganciclovir-resistant patients had a shorter survival and an earlier onset of bronchiolitis obliterans syndrome compared with patients in the transplant database at Washington University. CONCLUSIONS: Ganciclovir-resistant CMV infection is a serious complication of solid organ transplantation associated with more episodes of viremia, more frequent disease, earlier onset of bronchiolitis obliterans and shorter survival. The use of antithymocyte globulin and prolonged exposure to ganciclovir are risk factors for the development of ganciclovir resistance.

Combining classification trees using MLE.

We propose a probability distribution for an equivalence class of classification trees (that is, those that ignore the value of the cutpoints but retain tree structure). This distribution is parameterized by a central tree structure representing the true model, and a precision or concentration coefficient representing the variability around the central tree. We use this distribution to model an observed set of classification trees exhibiting variability in tree structure. We propose the maximum likelihood estimate of the central tree as the best tree to represent the set. This MLE retains the interpretability of a single tree model and has excellent generalizability. We implement an ascent search for the MLE tree structure using a data set of 13 classification trees that predict the presence or absence of cancer based on immune system parameters.

Meta-analysis of randomized controlled trials comparing laparoscopic and open appendectomy.

We performed a meta-analysis to determine whether laparoscopic or open appendectomy gives better outcomes for patients with suspected acute appendicitis. Studies were selected from the MEDLINE database, personal files, and meeting abstracts. Eleven of 21 randomized controlled trials were included in the meta-analysis. Pooled effect size estimates were calculated using a random effects model. Laparoscopic appendectomy reduced time to full functioning by 5.48 days (95% confidence interval [CI] 3.70 to 7.26; p < 0.001), improved postoperative pain at 24 hours measured by a visual analog scale from 0 to 10 by 1.19 points (95% CI -2.14 to -0.24 points; p=0.014), and decreased the absolute risk for wound infection by 3.2% (95% CI -5.6% to -0. 8%; p=0.009). Operating time was increased by 17.12 min (95% CI 14.19 to 20.03; p < 0.0001). There was no difference between the two surgeries for length of hospital stay, readmission rate, and intra-abdominal abscess formation. Laparoscopic appendectomy improves patient outcomes.

True and false positive peaks in genomewide scans: applications of length-biased sampling to linkage mapping.

Disease-susceptibility loci are now being mapped via genomewide scans in which a linkage statistic is computed at each of a large number of markers. Such disease-susceptibility loci may be identified via a peak in the test statistic when the latter is plotted against the genetic map. In this paper we establish, by appealing to renewal theory, that true positive peaks are expected to be longer than false positive peaks. These results are verified by a realistic simulation of a genomewide linkage study based on the affected-sib-pair design. Since longer peaks are more likely to contain a gene of interest than are shorter peaks, these differences may aid in linkage mapping, justifying assignment of lower priority to shorter peaks. However, since these differences are generally small, statistics based on both peak length and height may not be much more powerful than those based on height alone. The results presented here also provide a theoretical framework for methods that use the length of shared haplotypes in populations to map disease genes.

An application of decision theory to patient screening for an autologous tumour vaccine trial.

Patients are eligible for accrual onto a phase I autologous tumour vaccine clinical trial if their resected and dissociated tumour achieves a minimum viable cell count. Because tumour pre-processing and cell count determination are expensive, there has been developed a screening procedure based on tumour mass to screen out those tumours unlikely to yield sufficient viable cells. If theta is the ratio of the expected benefit of an accrual onto the study to the cost of tumour pre-processing and cell counting, then we maximize long-run benefit by pre-processing and counting only those tumours whose masses exceed a cutoff mc, such that Pr(sufficient tumour cells masses = mc) = 1/theta. We derive algorithms for estimating mc and evaluate them under a variety of assumptions concerning the cell count/mass relationship. These include explicit equations for mc under parametric assumptions as well as more general algorithms based on non-parametric smoothing techniques. We show that when theta deviates substantially from 2, these methods outperform simple inverse interpolation.

The effect of graft function on FK506 plasma levels, dosages, and renal function, with particular reference to the liver.

Plasma FK506 was studied in 49 liver, 13 heart, 3 double-lung or heart-lung, and 21 kidney recipients. The levels were correlated with the drug doses used, kidney function, and liver function. In all varieties of recipients, there was an early rise in the FK506 plasma levels that occurred at the time of intravenous administration of the drug. At the same time or shortly after, there were increases in serum creatinine that were transitory except in liver recipients with continuing suboptimal graft function. The quality of hepatic function dominated all aspects of FK506 management in the liver recipients. Those who received well-functioning grafts could be given about the same drug doses as recipients of kidneys and the thoracic organs. Liver recipients with defective grafts had astronomical rises in plasma FK506, a high incidence of renal failure, and probably increased neurotoxicity. In kidney transplant recipients, the FK506 plasma levels and doses were essentially the same in patients with prompt versus delayed renal function. These studies have highlighted the necessity, first, of close pharmacologic monitoring of patients who are given FK506 in the presence of abnormal liver function, and second, of using smaller intravenous induction doses than in past practice.