Johnathan M. Lancaster

Oncogenic pathway signatures in human cancers as a guide to targeted therapies

The development of an oncogenic state is a complex process involving the accumulation of multiple independent mutations that lead to deregulation of cell signalling pathways central to the control of cell growth and cell fate. The ability to define cancer subtypes, recurrence of disease and response to specific therapies using DNA microarray-based gene expression signatures has been demonstrated in multiple studies. Various studies have also demonstrated the potential for using gene expression profiles for the analysis of oncogenic pathways. Here we show that gene expression signatures can be identified that reflect the activation status of several oncogenic pathways. When evaluated in several large collections of human cancers, these gene expression signatures identify patterns of pathway deregulation in tumours and clinically relevant associations with disease outcomes. Combining signature-based predictions across several pathways identifies coordinated patterns of pathway deregulation that distinguish between specific cancers and tumour subtypes. Clustering tumours based on pathway signatures further defines prognosis in respective patient subsets, demonstrating that patterns of oncogenic pathway deregulation underlie the development of the oncogenic phenotype and reflect the biology and outcome of specific cancers. Predictions of pathway deregulation in cancer cell lines are also shown to predict the sensitivity to therapeutic agents that target components of the pathway. Linking pathway deregulation with sensitivity to therapeutics that target components of the pathway provides an opportunity to make use of these oncogenic pathway signatures to guide the use of targeted therapeutics.

Genomic signatures to guide the use of chemotherapeutics

Using in vitro drug sensitivity data coupled with Affymetrix microarray data, we developed gene expression signatures that predict sensitivity to individual chemotherapeutic drugs. Each signature was validated with response data from an independent set of cell line studies. We further show that many of these signatures can accurately predict clinical response in individuals treated with these drugs. Notably, signatures developed to predict response to individual agents, when combined, could also predict response to multidrug regimens. Finally, we integrated the chemotherapy response signatures with signatures of oncogenic pathway deregulation to identify new therapeutic strategies that make use of all available drugs. The development of gene expression profiles that can predict response to commonly used cytotoxic agents provides opportunities to better use these drugs, including using them in combination with existing targeted therapies.NOTE: In the version of this article initially published online, the affiliations of some authors were incorrectly listed. R.S. and J.C. should be affiliation 4, and the correct address for this affiliation should be Division of Gynecologic Surgical Oncology, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, Florida 33612, USA. Also, Center for Applied Genomics and Technology should be omitted from affiliation 1. The error has been corrected for all versions of the article.Using in vitro drug sensitivity data coupled with Affymetrix microarray data, we developed gene expression signatures that predict sensitivity to individual chemotherapeutic drugs. Each signature was validated with response data from an independent set of cell line studies. We further show that many of these signatures can accurately predict clinical response in individuals treated with these drugs. Notably, signatures developed to predict response to individual agents, when combined, could also predict response to multidrug regimens. Finally, we integrated the chemotherapy response signatures with signatures of oncogenic pathway deregulation to identify new therapeutic strategies that make use of all available drugs. The development of gene expression profiles that can predict response to commonly used cytotoxic agents provides opportunities to better use these drugs, including using them in combination with existing targeted therapies.NOTE: In the version of this article initially published online, the affiliations of some authors were incorrectly listed. R.S. and J.C. should be affiliation 4, and the correct address for this affiliation should be Division of Gynecologic Surgical Oncology, H. Lee Moffitt Cancer Center &Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, Florida 33612, USA. Also, Center for Applied Genomics and Technology should be omitted from affiliation 1. The error has been corrected for all versions of the article.

Physician Referral for Fertility Preservation in Oncology Patients: A National Study of Practice Behaviors

PURPOSEnCancer survival rates are improving, and the focus is moving toward quality survival. Fertility is a key aspect of quality of life for cancer patients of childbearing age. Although cancer treatment may impair fertility, some patients may benefit from referral to a specialist before treatment. However, the majority of studies examining patient recall of discussion and referral for fertility preservation (FP) show that less than half receive this information. This study examined the referral practices of oncologists in the United States.nnnMETHODSnThis study examined oncologists referral practice patterns for FP among US physicians using the American Medical Association Physician Masterfile database. A 53-item survey was administered via mail and Internet to a stratified random sample of US physicians.nnnRESULTSnForty-seven percent of respondents routinely refer cancer patients of childbearing age to a reproductive endocrinologist. Referrals were more likely among female physicians (P = .004), those with favorable attitudes (P = .043), and those whose patients routinely ask about FP (odds ratio = 2.09; 95% CI, 1.31 to 3.33).nnnCONCLUSIONnLess than half of US physicians are following the guidelines from the American Society of Clinical Oncology, which suggest that all patients of childbearing age should be informed about FP.

An Integrated Genomic-Based Approach to Individualized Treatment of Patients With Advanced-Stage Ovarian Cancer

PURPOSEnThe purpose of this study was to develop an integrated genomic-based approach to personalized treatment of patients with advanced-stage ovarian cancer. We have used gene expression profiles to identify patients likely to be resistant to primary platinum-based chemotherapy and also to identify alternate targeted therapeutic options for patients with de novo platinum-resistant disease.nnnPATIENTS AND METHODSnA gene expression model that predicts response to platinum-based therapy was developed using a training set of 83 advanced-stage serous ovarian cancers and tested on a 36-sample external validation set. In parallel, expression signatures that define the status of oncogenic signaling pathways were evaluated in 119 primary ovarian cancers and 12 ovarian cancer cell lines. In an effort to increase chemotherapy sensitivity, pathways shown to be activated in platinum-resistant cancers were subject to targeted therapy in ovarian cancer cell lines.nnnRESULTSnGene expression profiles identified patients with ovarian cancer likely to be resistant to primary platinum-based chemotherapy with greater than 80% accuracy. In patients with platinum-resistant disease, we identified expression signatures consistent with activation of Src and Rb/E2F pathways, components of which were successfully targeted to increase response in ovarian cancer cell lines.nnnCONCLUSIONnWe have defined a strategy for treatment of patients with advanced-stage ovarian cancer that uses therapeutic stratification based on predictions of response to chemotherapy, coupled with prediction of oncogenic pathway deregulation, as a method to direct the use of targeted agents.

Patterns of Gene Expression That Characterize Long-term Survival in Advanced Stage Serous Ovarian Cancers

Purpose: A better understanding of the underlying biology of invasive serous ovarian cancer is critical for the development of early detection strategies and new therapeutics. The objective of this study was to define gene expression patterns associated with favorable survival. Experimental Design: RNA from 65 serous ovarian cancers was analyzed using Affymetrix U133A microarrays. This included 54 stage III/IV cases (30 short-term survivors who lived <3 years and 24 long-term survivors who lived >7 years) and 11 stage I/II cases. Genes were screened on the basis of their level of and variability in expression, leaving 7,821 for use in developing a predictive model for survival. A composite predictive model was developed that combines Bayesian classification tree and multivariate discriminant models. Leave-one-out cross-validation was used to select and evaluate models. Results: Patterns of genes were identified that distinguish short-term and long-term ovarian cancer survivors. The expression model developed for advanced stage disease classified all 11 early-stage ovarian cancers as long-term survivors. The MAL gene, which has been shown to confer resistance to cancer therapy, was most highly overexpressed in short-term survivors (3-fold compared with long-term survivors, and 29-fold compared with early-stage cases). These results suggest that gene expression patterns underlie differences in outcome, and an examination of the genes that provide this discrimination reveals that many are implicated in processes that define the malignant phenotype. Conclusions: Differences in survival of advanced ovarian cancers are reflected by distinct patterns of gene expression. This biological distinction is further emphasized by the finding that early-stage cancers share expression patterns with the advanced stage long-term survivors, suggesting a shared favorable biology.

Microsomal epoxide hydrolase polymorphism as a risk factor for ovarian cancer

Microsomal epoxide hydrolase (EPHX) is one of many enzymes involved in the metabolism of endogenous and exogenous toxicants. Polymorphic forms of the human EPHX gene have been described that vary in enzymatic activity, and one, Tyr113His, has been associated with hepatocellular carcinoma susceptibility. We demonstrated that EPHX was highly expressed in the human ovary, and investigated whether specific EPHX genotypes are associated with ovarian cancer susceptibility. Seventy‐three Caucasian patients with ovarian cancer and 75 Caucasian‐female controls without cancer were genotyped for the Tyr113His polymorphism by a polymerase chain reaction‐restriction fragment length polymorphism assay. The frequency of the homozygous high‐activity genotype was 41% in the control population and 64% in the ovarian cancer patients. The odds ratio for ovarian cancer with this genotype was 2.6 (95% confidence interval 1.3, 5.0; P < 0.01). The increased ovarian cancer risk associated with the high‐activity genotype could reflect differences in metabolic activation of endogenous or exogenous carcinogens.

Pharmacogenomic Strategies Provide a Rational Approach to the Treatment of Cisplatin-Resistant Patients With Advanced Cancer

PURPOSEnStandard treatment for advanced non-small-cell lung cancer (NSCLC) includes the use of a platinum-based chemotherapy regimen. However, response rates are highly variable. Newer agents, such as pemetrexed, have shown significant activity as second-line therapy and are currently being evaluated in the front-line setting. We utilized a genomic strategy to develop signatures predictive of chemotherapeutic response to both cisplatin and pemetrexed to provide a rational approach to effective individualized medicine.nnnMETHODSnUsing in vitro drug sensitivity data, coupled with microarray data, we developed gene expression signatures predicting sensitivity to cisplatin and pemetrexed. Signatures were validated with response data from 32 independent ovarian and lung cancer cell lines as well as 59 samples from patients previously treated with cisplatin.nnnRESULTSnGenomic-derived signatures of cisplatin and pemetrexed sensitivity were shown to accurately predict sensitivity in vitro and, in the case of cisplatin, to predict treatment response in patients treated with cisplatin. The accuracy of the cisplatin predictor, based on available clinical data, was 83.1% (sensitivity, 100%; specificity 57%; positive predictive value, 78%; negative predictive value, 100%). Interestingly, an inverse correlation was seen between in vitro cisplatin and pemetrexed sensitivity, and importantly, between the likelihood of cisplatin and pemetrexed response in patients.nnnCONCLUSIONnThe use of genomic predictors of response to cisplatin and pemetrexed can be incorporated into strategies to optimize therapy for advanced solid tumors.

Pulmonary embolism after major abdominal surgery in gynecologic oncology

OBJECTIVE: To estimate the incidence and prognostic significance of postoperative pulmonary embolism after gynecologic oncology surgery. METHODS: All patients who underwent gynecologic oncology surgery from June 2001 to June 2003 and received venous thromboembolism prophylaxis with only intermittent pneumatic compression and early ambulation were identified from our database. Patients were grouped by procedure (major/minor abdominal or nonabdominal surgery), diagnosis (malignant/nonmalignant), and cancer subtype. Groups were compared by &khgr;2 analysis and logistic regression. Survival was studied with the Kaplan-Meier method and Mantel-Byar test. RESULTS: A total of 1,373 surgical patients were identified over the 2-year period, including 839 major abdominal surgery cases and 534 minor abdominal surgery or nonabdominal surgery cases. Of the 839 patients, 507 had a diagnosis of cancer, and 332 were benign. The incidence of pulmonary embolism among cancer patients undergoing major abdominal surgery was 4.1% (21/507) compared with 0.3% (1/332) among patients undergoing major abdominal surgery with benign findings (P < .001, odds ratio [OR] 13.8, 95% confidence interval [CI] 1.9–102.1). The incidence of pulmonary embolism among patients undergoing minor/nonabdominal surgery was 0.4% (2/536). Cancer diagnosis and age more than 60 years were identified as risk factors for pulmonary embolism (P = .009, OR 0.31, 95% CI 0.13–0.74). One-year survival for patients with and those without pulmonary embolism were 48.0% ± 12% and 77.0% ± 2%, respectively. CONCLUSION: Patients with cancer undergoing major abdominal surgery and using pneumatic compression for thromboembolic prophylaxis had a 14-fold greater odds of developing a pulmonary embolism compared with patients with benign disease. Randomized studies are needed to determine whether additional prophylactic measures may benefit this high-risk group of patients. LEVEL OF EVIDENCE: II-3

LIN28B Polymorphisms Influence Susceptibility to Epithelial Ovarian Cancer

Defective microRNA (miRNA) biogenesis contributes to the development and progression of epithelial ovarian cancer (EOC). In this study, we examined the hypothesis that single nucleotide polymorphisms (SNP) in miRNA biogenesis genes may influence EOC risk. In an initial investigation, 318 SNPs in 18 genes were evaluated among 1,815 EOC cases and 1,900 controls, followed up by a replicative joint meta-analysis of data from an additional 2,172 cases and 3,052 controls. Of 23 SNPs from 9 genes associated with risk (empirical P < 0.05) in the initial investigation, the meta-analysis replicated 6 SNPs from the DROSHA, FMR1, LIN28, and LIN28B genes, including rs12194974 (G>A), an SNP in a putative transcription factor binding site in the LIN28B promoter region (summary OR = 0.90, 95% CI: 0.82-0.98; P = 0.015) which has been recently implicated in age of menarche and other phenotypes. Consistent with reports that LIN28B overexpression in EOC contributes to tumorigenesis by repressing tumor suppressor let-7 expression, we provide data from luciferase reporter assays and quantitative RT-PCR to suggest that the inverse association among rs12194974 A allele carriers may be because of reduced LIN28B expression. Our findings suggest that variants in LIN28B and possibly other miRNA biogenesis genes may influence EOC susceptibility.

Cell proliferation and apoptosis in human uterine leiomyomas and myometria

Abstract. To determine the role of cell proliferation and apoptosis in uterine leiomyoma growth, we studied protein expression of two major regulatory proteins of apoptosis – Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) – and two endogenous markers of cell replication – proliferating cell nuclear antigen (PCNA) and Ki-67 – in tumors and matched myometrium from premenopausal women. Conventional mitotic indices also were determined, and all proliferation data were correlated to tumor size. In situ end-labeling of fragmented DNA and routine histology were used to assess apoptosis. Our results showed that the apoptosis-regulating proteins (Bcl-2 and Bax) were expressed in the cytoplasm of the leiomyoma and myometrial smooth muscle cells throughout the menstrual cycle. Bax expression differed from Bcl-2 in that it also was found in the cytoplasm of vascular smooth muscle cells of the myometria and tumors. Both tumors and myometrial samples expressed 26-kDa and 21-kDa proteins that reacted with antibodies directed towards Bcl-2 and Bax, respectively. Apoptosis was not a prominent feature of uterine leiomyomas or myometrium. PCNA- and Ki-67-labeling and mitotic counts were significantly (P<0.05) higher in leiomyomas than in matched myometrial samples. Proliferative activity was variable for individual tumors of the same patient and independent of tumor size. Our results suggest that altered apoptosis by overexpression of Bcl-2 or by decreased expression of Bax does not appear to be a major factor in uterine leiomyoma growth. We conclude that increased cell proliferation is the most significant contributor to growth and that the proliferative state is autonomous for each tumor in a given patient and is independent of tumor size.