ABSTRACT
The phosphoinositide 3-kinase (PI3K) signaling pathway is significantly altered in a wide variety of human cancers, driving cancer cell growth and survival. Consequently, a large number of PI3K inhibitors are now in clinical development. To begin to improve the selection of patients for treatment with PI3K inhibitors and to identify de novo determinants of patient response, we sought to identify and characterize candidate genomic and phosphoproteomic biomarkers predictive of response to the selective PI3K inhibitor, GDC-0941, using the NCI-60 human tumor cell line collection. In this study, sixty diverse tumor cell lines were exposed to GDC-0941 and classified by GI(50) value as sensitive or resistant. The most sensitive and resistant cell lines were analyzed for their baseline levels of gene expression and phosphorylation of key signaling nodes. Phosphorylation or activation status of both the PI3K-Akt signaling axis and PARP were correlated with in vitro response to GDC-0941. A gene expression signature associated with in vitro sensitivity to GDC-0941 was also identified. Furthermore, in vitro siRNA-mediated silencing of two genes in this signature, OGT and DDN, validated their role in modulating sensitivity to GDC-0941 in numerous cell lines and begins to provide biological insights into their role as chemosensitizers. These candidate biomarkers will offer useful tools to begin a more thorough understanding of determinants of patient response to PI3K inhibitors and merit exploration in human cancer patients treated with PI3K inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Indazoles/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Sulfonamides/pharmacology , Transcriptome/drug effects , Acetylglucosamine/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 2 , Cluster Analysis , Gene Knockdown Techniques , Humans , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Receptor, ErbB-3/metabolism , Signal Transduction , Statistics, NonparametricABSTRACT
Oxaliplatin is widely used to treat colorectal cancer, as both adjuvant therapy for resected disease and palliative treatment of metastatic disease. However, a significant number of patients experience serious side effects, including prolonged neurotoxicity, from oxaliplatin treatment creating an urgent need for biomarkers of oxaliplatin response or resistance to direct therapy to those most likely to benefit. As a first step to improve selection of patients for oxaliplatin-based chemotherapy, we have conducted an in vitro cell-based small interfering RNA (siRNA) screen of 500 genes aimed at identifying genes whose loss of expression alters tumor cell response to oxaliplatin. The siRNA screen identified twenty-seven genes, which when silenced, significantly altered colon tumor cell line sensitivity to oxaliplatin. Silencing of a group of putative resistance genes increased the extent of oxaliplatin-mediated DNA damage and inhibited cell-cycle progression in oxaliplatin-treated cells. The activity of several signaling nodes, including AKT1 and MEK1, was also altered. We used cDNA transfection to overexpress two genes (LTBR and TMEM30A) that were identified in the siRNA screen as mediators of oxaliplatin sensitivity. In both instances, overexpression conferred resistance to oxaliplatin. In summary, this study identified numerous putative predictive biomarkers of response to oxaliplatin that should be studied further in patient specimens for potential clinical application. Diverse gene networks seem to influence tumor survival in response to DNA damage by oxaliplatin. Finally, those genes whose loss of expression (or function) is related to oxaliplatin sensitivity may be promising therapeutic targets to increase patient response to oxaliplatin.
Subject(s)
Biological Phenomena/genetics , Genomics/methods , Neoplasms/genetics , Neoplasms/pathology , Organoplatinum Compounds/pharmacology , Biological Phenomena/drug effects , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , DNA Damage/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Screening Assays, Antitumor , Genes, Neoplasm/genetics , Humans , Models, Biological , Oxaliplatin , RNA, Small Interfering/metabolism , Reproducibility of Results , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.
Subject(s)
Artifacts , Breast Neoplasms/genetics , Gene Expression Profiling , RNA, Neoplasm/analysis , Reverse Transcriptase Polymerase Chain Reaction , Breast Neoplasms/metabolism , Gene Expression Profiling/methods , Humans , Immunohistochemistry , Paraffin Embedding , Reproducibility of Results , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Time FactorsABSTRACT
A review of 122 cases of symptomatic coarctation of the aorta in neonates and infants confirmed the need for early corrective surgery in the majority of cases, in relation to the usual coexistence of associated cardiovascular malformations. The operative risk in the newborn up to one month of age was very high (42 %) but fell very sharply thereafter (5%). Restenosis was the principal late complication (36 %), persistent hypertension being rare in this age group. The risk of restenosis (not a significant vital risk during secondary surgery) should not weigh in the balance against life saving surgery where clinical deterioration is observed despite medical therapy or due to associated malformations.
Subject(s)
Aortic Coarctation/surgery , Age Factors , Aortic Coarctation/complications , Humans , Infant , Infant, Newborn , Postoperative Complications/etiology , Prognosis , Recurrence , Time FactorsABSTRACT
The technique of left subclavian-main left coronary artery is described as the treatment of anomalous origin of the left coronary artery from the pulmonary artery, without the aid of cardiopulmonary bypass, which, however, remains on standby. Through a left postero-lateral thoracotomy, the left main coronary artery is detached from the main pulmonary artery with a cuff of pulmonary wall after lateral clamping of the pulmonary artery. Tapes are encercling the pulmonary artery and the descending thoracic aorta, making them ready for an eventual connection to the standby bypass, in case the coronary clamping is not well tolerated. The anastomosis between the left subclavian artery dissected free and the prepared left main coronary artery is then possible and easy even in a small infant. This technique has been used in three infants aged three to thirty months without any mortality nor particular morbidity.
Subject(s)
Coronary Vessel Anomalies/surgery , Coronary Vessels/surgery , Subclavian Artery/surgery , Child, Preschool , Extracorporeal Circulation , Humans , Infant , Methods , Pulmonary Artery/abnormalitiesABSTRACT
The authors report four cases of metabolic cardiomyopathy with lipid infiltration diagnosed by skeletal muscle and myocardial biopsy in children with no clinical signs of muscular dystrophy. Normal or increased serum and urinary carnitine levels excluded a primary carnitine deficiency. A deficiency in muscular-palmityl-carnitine-transferase was demonstrated. This pathogenic mechanism may be an indication for treatment with carnitine, but the results are less spectacular than in primary carnitine deficiency states.