Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma.

Through exomic sequencing of ten hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCC) and subsequent evaluation of additional affected individuals, we discovered novel inactivating mutations of ARID2 in four major subtypes of HCC (HCV-associated HCC, hepatitis B virus (HBV)-associated HCC, alcohol-associated HCC and HCC with no known etiology). Notably, 18.2% of individuals with HCV-associated HCC in the United States and Europe harbored ARID2 inactivation mutations, suggesting that ARID2 is a tumor suppressor gene that is relatively commonly mutated in this tumor subtype.

Detection and quantification of rare mutations with massively parallel sequencing.

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

Mutant proteins as cancer-specific biomarkers.

Cancer biomarkers are currently the subject of intense research because of their potential utility for diagnosis, prognosis, and targeted therapy. In theory, the gene products resulting from somatic mutations are the ultimate protein biomarkers, being not simply associated with tumors but actually responsible for tumorigenesis. We show here that the altered protein products resulting from somatic mutations can be identified directly and quantified by mass spectrometry. The peptides expressed from normal and mutant alleles were detected by selected reaction monitoring (SRM) of their product ions using a triple-quadrupole mass spectrometer. As a prototypical example of this approach, we demonstrated that it is possible to quantify the number and fraction of mutant Ras protein present in cancer cell lines. There were an average of 1.3 million molecules of Ras protein per cell, and the ratio of mutant to normal Ras proteins ranged from 0.49 to 5.6. Similarly, we found that mutant Ras proteins could be detected and quantified in clinical specimens such as colorectal and pancreatic tumor tissues as well as in premalignant pancreatic cyst fluids. In addition to answering basic questions about the relative levels of genetically abnormal proteins in tumors, this approach could prove useful for diagnostic applications.

The genetic landscape of the childhood cancer medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Integrated analysis of homozygous deletions, focal amplifications, and sequence alterations in breast and colorectal cancers.

We have performed a genome-wide analysis of copy number changes in breast and colorectal tumors using approaches that can reliably detect homozygous deletions and amplifications. We found that the number of genes altered by major copy number changes, deletion of all copies or amplification to at least 12 copies per cell, averaged 17 per tumor. We have integrated these data with previous mutation analyses of the Reference Sequence genes in these same tumor types and have identified genes and cellular pathways affected by both copy number changes and point alterations. Pathways enriched for genetic alterations included those controlling cell adhesion, intracellular signaling, DNA topological change, and cell cycle control. These analyses provide an integrated view of copy number and sequencing alterations on a genome-wide scale and identify genes and pathways that could prove useful for cancer diagnosis and therapy.

An integrated genomic analysis of human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.

Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells.

Angiopoietin (Ang)-2, a context-dependent agonist/antagonist for the vascular-specific Tie2 receptor, is highly expressed by endothelial cells at sites of normal and pathologic angiogenesis. One prevailing model suggests that in these settings, Ang-2 acts as an autocrine Tie2 blocker, inhibiting the stabilizing influence of the Tie2 activator Ang-1, thereby promoting vascular remodeling. However, the effects of endogenous Ang-2 on cells that are actively producing it have not been studied in detail. Here, we demonstrate that Ang-2 expression is rapidly induced in endothelial cells by the transcription factor FOXO1 after inhibition of the phosphatidylinositol 3-kinase/Akt pathway. We employ RNAi and blocking antibodies to show that in this setting, Ang-2 unexpectedly functions as a Tie2 agonist, bolstering Akt activity so as to provide negative feedback on FOXO1-regulated transcription and apoptosis. In addition, we show that Ang-2, like Ang-1, activates Tie2/Akt signaling in vivo, thereby inhibiting the expression of FOXO1 target genes. Consistent with a role for Ang-2 as a Tie2 activator, we demonstrate that Ang-2 inhibits vascular leak. Our data suggests a model in which Ang-2 expression is induced in stressed endothelial cells, where it acts as an autocrine Tie2 agonist and protective factor.

Non-contact holmium:YAG laser thermal keratoplasty for hyperopia: two-year follow-up.

PURPOSE: We evaluated the safety and efficacy of the non-contact holmium:YAG laser thermal keratoplasty (LTK) for the treatment of mild to moderate hyperopia without astigmatism. METHODS: A prospective, non-comparative case series included 50 eyes of 28 patients (aged > or =40 years) who had stable refraction and an astigmatic component < +0.50 diopters (D). We applied the non-contact pulsed holmium:YAG laser to treat the hyperopic spherical component using the Hyperion LTK System. All patients had minimum 12-month follow-up and 64% (18 patients) had 24-month follow-up. RESULTS: The mean age of patients was 48.4 +/- 8.23 years (range: 40 to 62 years). The preoperative hyperopic mean spherical equivalent refraction was +2.32 +/- 0.975 D (range: +1.00 to +4.75 D). Postoperatively, the subjective manifest refraction decreased from the preoperative mean value of +2.32 D to a mean -0.09 D at 1 month after surgery and regressed to +0.315 D at the last follow-up examination, resulting in a mean correction of +2.005 +/- 0.81 D at 24 months after surgery. Preoperatively, mean uncorrected visual acuity in LogMAR units was 0.798 +/- 0.353 and at 12 months after surgery, it was a mean 0.108 +/- 0.136. Keratometric power increased from 42.595 +/- 1.949 D before surgery to 44.605 +/- 1.626 D at 24 months after surgery. CONCLUSIONS: Holmium:YAG LTK was an acceptable alternative for the correction of mild to moderate hyperopia in this middle-aged population.

Functional and anatomic results of macular hole surgery complicated by massive indocyanine green subretinal migration.

PURPOSE: To report the functional and anatomic results of macular hole (MH) surgery complicated by massive subretinal migration of indocyanine green (ICG) dye. DESIGN: Interventional case report. METHODS: We performed standard pars plana vitrectomy surgery for a stage 3, senile idiopathic MH. After posterior vitreous detachment and vitreous removal, we instilled 2 ml of ICG (0.5%, 270 mOsm); the surgery was complicated by diffuse subretinal migration of the ICG dye but peeling of the internal limiting membrane (ILM) was performed (despite the obvious difficulties from the low contrast between the green-stained ILM overlying a green-stained subretinal space) and the rest of the procedure was completed with a final injection of 16% C3F8. RESULTS: Post-surgical optical coherence tomography confirmed the anatomic closure of the MH. Digital photography with the excitation and barrier filters for ICG showed a striking autofluorescence along the inferior vascular arcade, which remained intense 7 months after surgery. Despite the massive subretinal migration of ICG, visual acuity (VA) improved to 20/30. CONCLUSIONS: This is the first report of VA recovery despite massive subretinal migration of ICG dye during MH surgery. Subretinal migration of ICG dye may be a potential complication during MH surgery; this should alert the surgeon to limit its use, despite the possible absence of clinically apparent toxic effects.

Posterior capsule rupture and vitreous loss during phacoemulsification with or without the use of an anterior chamber maintainer.

PURPOSE: To compare the incidence of posterior capsule rupture and vitreous loss during phacoemulsification with and without the use of an anterior chamber maintainer (ACM) SETTING: Aristotle University Eye Clinic, Thessaloniki, Greece. METHODS: The surgical records of patients who had phacoemulsification by experienced surgeons over a 2-year period were reviewed. The cases were divided into 2 groups: surgery performed without the use of an ACM (no-ACM group) and surgery performed with the use of an ACM (ACM group). RESULTS: The no-ACM group comprised 231 cases and the ACM group, 312 cases. Posterior capsule rupture occurred in 19 cases (8.22%) and 22 cases (7.05%), respectively. Of the patients with posterior capsule rupture, 6 (31.57%) in the no-ACM group and 16 (72.72%) in the ACM group had vitreous loss and required an anterior vitrectomy; the difference between the 2 groups was statistically significant (P =.03). CONCLUSIONS: The use of an ACM in phacoemulsification provided a consistently deep anterior chamber and approximately the same rate of complications as with a standard technique in the hands of experienced surgeons. However, when posterior capsule rupture occurred, the incidence of vitreous loss was significantly higher in eyes with an ACM.

A-scan quantitative echography of senile cataracts and correlation with phacoemulsification parameters.

PURPOSE: To study the echographic appearance of various types of senile cataracts using a-scan quantitative echography and correlate these findings with phacoemulsification parameters indicating surgical lens hardness. METHODS: The study involved 71 eyes with senile cataracts and 20 additional eyes with normal human lenses. Cataract and clear lenses were studied using A-scan quantitative echography and with a tissue sensitivity gain setting of 74 db. We assessed the lens internal reflectivity in the A-scan echograms within a range of 0% to 100%, and calculated the mean of all spikes. All eyes with cataract underwent clear corneal phacoemulsification. At the end of the operation we recorded the phacoemulsification time and mean percent power. We determined the correlations of echographic and phacoemulsification data. RESULTS: Quantitative echography in the 71 studied eyes with cataract revealed acoustic reflections having various configurations according to the biomicroscopic type of cataract, i.e. cortical, nuclear, corticonuclear, brunescent, white intumescent. The mean of all echographic spikes within the cataract lenses varied from 3% (in some brunescent very sclerotic lenses) to 53%. Echographic reflections were not observed in the A-scan echograms of 20 additional eyes with normal human lenses. The mean of internal lens spikes correlated negatively with phacoemulsification time (p < 0.001; r = -0.703) and mean percent phacoemulsification power (p < 0.001; r = -0.57). CONCLUSIONS: The results indicate that A-scan quantitative echography may be used as an adjunctive tool in the preoperative evaluation of cataracts, particularly in cases with compromized visualization of the lens (small pupil, corneal opacities).

Perfluorocarbon liquid utilization in primary vitrectomy repair of retinal detachment with multiple breaks.

PURPOSE: To evaluate the efficacy of pars plana vitrectomy in conjunction with intraoperative perfluoro-n-octane (PFO) use as initial treatment of retinal detachment (RD) with multiple breaks located at various distances from the ora serrata. METHODS: Twenty-two consecutive eyes (15 phakic, 2 aphakic, and 5 pseudophakic) presenting with RD with multiple breaks and tears underwent primary pars plana vitrectomy, PFO retinal reattachment, transcleral cryopexy or endolaser treatment of breaks, PFO/air exchange, and final injection of 18% perfluoropropane (C3F8). Scleral buckles were not used. The mean follow-up period was 29 months. RESULTS: Temporary PFO utilization attached the posterior retina and facilitated the safe removal of vitreous at its base and around the retinal tears. Intraoperative complications included new breaks (3 eyes), enlargement of breaks (2 eyes), and a small bubble of subretinal PFO (1 eye). Postoperatively, the retina remained attached during follow-up in 19 eyes. Cataract developed or progressed in 13 phakic eyes. CONCLUSIONS: Pars plana vitrectomy in conjunction with intraoperative PFO utilization is effective as initial treatment of RDs with multiple breaks. The main limitation of this technique is the postoperative progressive cataract formation in phakic eyes.

Angiopoietins have distinct modular domains essential for receptor binding, dimerization and superclustering.

Angiopoietins are a recently discovered family of angiogenic factors that interact with the endothelial receptor tyrosine kinase Tie2, either as agonists (angiopoietin-1) or as context-dependent agonists/antagonists (angiopoietin-2). Here we show that angiopoietin-1 has a modular structure unlike any previously characterized growth factor. This modular structure consists of a receptor-binding domain, a dimerization motif and a superclustering motif that forms variable-sized multimers. Genetic engineering of precise multimers of the receptor-binding domain of angiopoietin-1, using surrogate multimerization motifs, reveals that tetramers are the minimal size required for activating endothelial Tie2 receptors. In contrast, engineered dimers can antagonize endothelial Tie2 receptors. Surprisingly, angiopoietin-2 has a modular structure and multimerization state similar to that of angiopoietin-1, and its antagonist activity seems to be a subtle property encoded in its receptor-binding domain.

Retinal complications of phakic intraocular lenses.

High myopia has always been a challenge for refractive correction. Current laser surgical techniques, however, fall short of correcting high refractive errors due to lack of predictability, regression, corneal ectasia, and introduction of high order optical aberration. Phakic intraocular lenses (IOL) have been proposed as an effective refractive surgical procedure for the correction of severe myopia, but, despite recent advances in implant material technology and design, their concept is still under clinical investigation. Most of the concern regarding the complications of phakic IOLs focuses on the anterior segment of the eye. This review examines the posterior segment complications associated with phakic IOL implantation, evaluates possible pathogenetic mechanisms and discusses posterior segment complications, prevention and management.

Sulcus implantation of a 3-piece, 6.0 mm optic, hydrophobic foldable acrylic intraocular lens in phacoemulsification complicated by posterior capsule rupture.

To evaluate the safety of implanting a 3-piece, 6.0 mm optic, foldable acrylic intraocular lens (IOL) in cases of posterior capsule rupture during phacoemulsification. Department of Ophthalmology, Ahepa Hospital, Aristotle University Medical School, and Interbalkan Medical Center, Thessaloniki, Greece. This prospective noncomparative case series included 28 eyes (28 patients) having phacoemulsification complicated by extensive posterior capsule rupture with or without vitreous loss. In all eyes, a 3-piece, 6.0 mm optic, foldable acrylic IOL (AcrySof MA60BM, Alcon) was implanted in the sulcus. Postoperative examination included best corrected visual acuity (BCVA), anterior segment biomicroscopy, IOL centralization and position, intraocular pressure (IOP), and fundus biomicroscopy. The follow-up was 6 months. The most common postoperative complications were transient corneal edema in 12 eyes and increased IOP in 11 eyes. Slight asymptomatic decentralization from the center of the pupil (1.0 to 2.0 mm) and pseudophacodonesis were observed in 5 eyes each. Friction of the IOL with the iris occurred in 1 eye. Clinically significant cystoid macular edema occurred in 3 eyes. The final BCVA was between 20/15 and 20/25 in 18 patients, 20/30 in 2, between 20/40 and 20/50 in 4, and between 20/60 and 20/200 in 4. AcrySof IOL implantation in the sulcus during phacoemulsification complicated by posterior capsule rupture preserved the advantages of small-incision surgery. The postoperative behavior and centralization of the IOLs resembled those of poly(methyl methacrylate) lenses.

VEGF-Trap: a VEGF blocker with potent antitumor effects.

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.