Raymond Huang

Structural genomics: a pipeline for providing structures for the biologist.

Progress in understanding the organization and sequences of genes in model organisms and humans is rapidly accelerating. Although genome sequences from prokaryotes have been available for some time, only recently have the genome sequences of several eukaryotic organisms been reported, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and humans (Green 2001). A logical continuation of this line of scientific inquiry is to understand the structure and function of all genes in simple and complex organisms, including the pathways leading to the organization and biochemical function of macromolecular assemblies, organelles, cells, organs, and whole life forms. Such investigations have been variously called integrative or systems biology and -omics or high-throughput biology (Ideker et al. 2001, Greenbaum et al. 2001, Vidal 2001). These studies have blossomed because of advances in technologies that allow highly parallel examination of multiple genes and gene products as well as a vision of biology that is not purely reductionist. Although a unified understanding of biological organisms is still far in the future, new high-throughput biological approaches are having a drastic impact on the scientific mainstream. One offshoot of the high-throughput approach, which directly leverages the accumulating gene sequence information, involves mining the sequence data to detect important evolutionary relationships, to identify the basic set of genes necessary for independent life, and to reveal important metabolic processes in humans and clinically relevant pathogens. Programs such as MAGPIE (www.genomes.rockefeller.edu/magpie/magpie.html) compare organisms at a whole genome level (Gaasterland and Sensen 1996; Gaasterland and Ragan 1998) and ask what functions are conferred by the new genes that have evolved in higher organisms (Gaasterland and Oprea 2001). Concurrent with computational annotations of gene structure and function, thousands of full-length ORFs from yeast and higher eukaryotes have become available because of advances in cloning and other molecular biology techniques (Walhout et al. 2000a). Structural biologists have embraced high-throughput biology by developing and implementing technologies that will enable the structures of hundreds of protein domains to be solved in a relatively short time. Although thousands of structures are deposited annually in the Protein Data Bank (PDB), most are identical or very similar in sequence to a structure previously existing in the data bank, representing structures of mutants or different ligand bound states (Brenner et al. 1997). Providing structural information for a broader range of sequences requires a focused effort on determining structure for sequences that are divergent from those already in the database. Although structure does not always elucidate function, in many instances (including the structures of two proteins reported here) the atomic structure readily provides insight into the function of a protein whose function was previously unknown. Typically, such functional annotations are based on homologies that are not recognizable at the sequence level but that are clearly revealed on inspection of the protein fold, identification of a conserved constellation of side-chain functionalities, or by the observation of cofactors associated with function (Burley et al. 1999; Shi et al. 2001; Bonanno et al. 2002).

Center for Synchrotron Biosciences' U2B beamline: an international resource for biological infrared spectroscopy

A synchrotron infrared (IR) beamline, U2B, dedicated to the biomedical and biological sciences has been constructed and is in operation at the National Synchrotron Light Source (NSLS) of Brookhaven National Laboratory. The facility is operated by the Center for Synchrotron Biosciences of the Albert Einstein College of Medicine in cooperation with the NSLS. Owing to the broadband nature of the synchrotron beam with brightness 1000 times that of conventional sources, Fourier transform IR spectroscopy experiments are feasible on diffraction-limited sample areas at high signal-to-noise ratios and with relatively short data-acquisition times. A number of synchrotron IR microscopy experiments that have been performed in the mid-IR spectral range (500-5000 cm(-1)) are summarized, including time-resolved protein-folding studies in the microsecond time regime, IR imaging of neurons, bone and other biological tissues, as well as imaging of samples of interest in the chemical and environmental sciences. Owing to the high flux output of this beamline in the far-IR region (50-500 cm(-1)), investigations of hydrogen bonding and dynamic molecular motions of biomolecules have been carried out from 10 to 300 K using a custom-made cryostat and an evacuated box. This facility is intended as an international resource for biological IR spectroscopy fully available to outside users based on competitive proposal.

IN SITU CHEMISTRY OF OSTEOPOROSIS REVEALED BY SYNCHROTRON INFRARED MICROSPECTROSCOPY

Reduced bone density is a well-known feature of osteoporosis, yet little is known about the changes in the chemical composition of bone or the impact of such chemical changes on fracture risks. Using ovariectomized cynomolgus monkeys (Macaca fascicularis) as a model for the menopausal onset of osteoporosis, we examined the microscopic chemical changes of bone measured by synchrotron infrared microspectroscopy as a function of time after ovariectomy. The results demonstrate that cortical bone formed 1 or 2 years after ovariectomy, as identified by fluorochrome labeling, has a higher phosphate content (PO4(3-)/matrix ratio), a lower carbonate content (CO3(2-)/matrix ratio), and more mature collagen cross-links (nonreducible cross-link/reducible cross-link ratio) than that formed in sham-operated controls. Trabecular bone after ovariectomy shows no changes in phosphate content, a lower carbonate content, and immature collagen cross-linking. Treatment with a bone turnover suppressor, (nandrolone decanoate) reverses most of the ovariectomy-induced chemical changes in the cortical bone to the levels of the ovary-intact controls, but has little effect on the trabecular bone. These results demonstrate that bone newly synthesized after ovariectomy is chemically different from healthy bone within specific bone regions, which may contribute to reduced bone quality in osteoporosis.

Pitfalls in the Neuroimaging of Glioblastoma in the Era of Antiangiogenic and Immuno/Targeted Therapy – Detecting Illusive Disease, Defining Response

Glioblastoma, the most common malignant primary brain tumor in adults is a devastating diagnosis with an average survival of 14–16 months using the current standard of care treatment. The determination of treatment response and clinical decision making is based on the accuracy of radiographic assessment. Notwithstanding, challenges exist in the neuroimaging evaluation of patients undergoing treatment for malignant glioma. Differentiating treatment response from tumor progression is problematic and currently combines long-term follow-up using standard magnetic resonance imaging (MRI), with clinical status and corticosteroid-dependency assessments. In the clinical trial setting, treatment with gene therapy, vaccines, immunotherapy, and targeted biologicals similarly produces MRI changes mimicking disease progression. A neuroimaging method to clearly distinguish between pseudoprogression and tumor progression has unfortunately not been found to date. With the incorporation of antiangiogenic therapies, a further pitfall in imaging interpretation is pseudoresponse. The Macdonald criteria that correlate tumor burden with contrast-enhanced imaging proved insufficient and misleading in the context of rapid blood–brain barrier normalization following antiangiogenic treatment that is not accompanied by expected survival benefit. Even improved criteria, such as the RANO criteria, which incorporate non-enhancing disease, clinical status, and need for corticosteroid use, fall short of definitively distinguishing tumor progression, pseudoresponse, and pseudoprogression. This review focuses on advanced imaging techniques including perfusion MRI, diffusion MRI, MR spectroscopy, and new positron emission tomography imaging tracers. The relevant image analysis algorithms and interpretation methods of these promising techniques are discussed in the context of determining response and progression during treatment of glioblastoma both in the standard of care and in clinical trial context.

Characterization of bone mineral composition in the proximal tibia of cynomolgus monkeys: effect of ovariectomy and nandrolone decanoate treatment.

Life postmenopausal women, ovariectomized cynomolgus monkeys (Macaca fascicularis) experience accelerated loss of bone mass. Treatment of ovariectomized monkeys with nandrolone decanoate results in an increase in bone mass to levels comparable to those of intact animals. The changes in bone composition that occur with these treatments, however, are less well characterized. In the present study, we used synchrotron Fourier-transform infrared microspectroscopy (FT-IRM) and curve-fitting methods to monitor specific changes at cortical, subchondral, and trabecular bone regions in the proximal tibia. Four groups were studied: (1) sham-operated (sham); (2) ovariectomized and treated with placebo for 2 years (ovx); (3) ovx + nandrolone decanoate for 2 years (NAN); and (4) ovx + nandrolone decanoate beginning 1 year after ovx (dNAN). The results demonstrate that ovariectomy and nandrolone treatment did not affect the degree of mineralization as defined by the phosphate/protein ratio, but acid phosphate content (HPO(4)(2-)) in cortical and subchondral bone was increased by ovariectomy, suggesting this bone to be less mature due to increased remodeling that occurs after ovariectomy. In the subchondral and cortical bone regions, ovariectomized monkeys showed a lower total carbonate content (CO(3)(2-)/matrix ratio) than sham controls, specifically due to the decrease in labile carbonate content. In the trabecular region, no change of carbonate content was observed. Treatment with nandrolone decanoate was found to restore the loss in carbonate, where the resulting mineral had a larger quantity of type B carbonate. Finally, we correlated carbonate content with dual-energy X-ray absorptiometry measurements, and found a positive correlation between bone mineral density and type A carbonate in bone, which is stoichiometrically related to the amount of calcium in bone. Therefore, the results presented herein identify significant differences in bone chemistry after ovariectomy and nandrolone treatment, which may help explain previous findings that, although nandrolone decanoate treatment increased bone mass, it could not reverse the decrease in bone strength due to ovariectomy.

Multimodal MRI features predict isocitrate dehydrogenase genotype in high-grade gliomas

Background. High-grade gliomas with mutations in the isocitrate dehydrogenase (IDH) gene family confer longer overall survival relative to their IDH-wild-type counterparts. Accurate determination of the IDH genotype preoperatively may have both prognostic and diagnostic value. The current study used a machine-learning algorithm to generate a model predictive of IDH genotype in high-grade gliomas based on clinical variables and multimodal features extracted from conventional MRI. Methods. Preoperative MRIs were obtained for 120 patients with primary grades III (n = 35) and IV (n = 85) glioma in this retrospective study. IDH genotype was confirmed for grade III (32/35, 91%) and IV (22/85, 26%) tumors by immunohistochemistry, spectrometry-based mutation genotyping (OncoMap), or multiplex exome sequencing (OncoPanel). IDH1 and IDH2 mutations were mutually exclusive, and all mutated tumors were collapsed into one IDH-mutated cohort. Cases were randomly assigned to either the training (n = 90) or validation cohort (n = 30). A total of 2970 imaging features were extracted from pre- and postcontrast T1-weighted, T2-weighted, and apparent diffusion coefficient map. Using a random forest algorithm, nonredundant features were integrated with clinical data to generate a model predictive of IDH genotype. Results. Our model achieved accuracies of 86% (area under the curve [AUC] = 0.8830) in the training cohort and 89% (AUC = 0.9231) in the validation cohort. Features with the highest predictive value included patient age as well as parametric intensity, texture, and shape features. Conclusion. Using a machine-learning algorithm, we achieved accurate prediction of IDH genotype in high-grade gliomas with preoperative clinical and MRI features.

The Impact of T2/FLAIR Evaluation per RANO Criteria on Response Assessment of Recurrent Glioblastoma Patients Treated with Bevacizumab

Purpose: The RANO criteria have not been assessed using outcome data from prospective trials. We examined the radiologic data of patients with recurrent glioblastoma from the randomized phase II trial (AVF3708g) to determine the effect of including T2/FLAIR evaluation as per RANO criteria on measurements of objective response rates (ORRs) and progression-free survival (PFS) compared with assessment based on contrast enhancement (Macdonald criteria). Experimental Design: The ORRs and median PFS were determined using the RANO criteria and compared with those obtained using the Macdonald criteria. Landmark analyses were performed at 2, 4, and 6 months, and Cox proportional hazard models were used to determine the associations between OR and progression with subsequent survival. Results: The ORRs were 0.331 [95% confidence interval (CI), 0.260–0.409] and 0.393 (95% CI, 0.317–0.472) by RANO and Macdonald criteria, respectively (P < 0.0001). The median PFS was 4.6 months (95% CI, 4.1–5.5) using RANO criteria, compared with 6.4 months (95% CI, 5.5–7.1) as determined by Macdonald criteria (P = 0.01). At 2-, 4-, and 6-month landmarks, both OR status and PFS determined by either RANO or Macdonald criteria were predictive of overall survival [OS; hazard ratios for 4-month landmark (OR HR = 1.93, P = 0.0012; PFS HR, 4.23, P < 0.0001)]. Conclusions: The inclusion of T2/FLAIR assessment resulted in statistically significant differences in median PFS and ORRs compared with assessment of solely enhancing tumor (Macdonald criteria), although OR and PFS determined by both RANO and Macdonald criteria correlated with OS. Clin Cancer Res; 22(3); 575–81. ©2015 AACR.

Recurrent glioblastoma: Volumetric assessment and stratification of patient survival with early posttreatment magnetic resonance imaging in patients treated with bevacizumab

Despite a high radiographic response rate in patients with recurrent glioblastoma following bevacizumab therapy, survival benefit has been relatively modest. We assess whether tumor volume measurements based on baseline and early posttreatment MRI can stratify patients in terms of progression‐free survival (PFS) and overall survival (OS).

Semi-Automatic Segmentation Software for Quantitative Clinical Brain Glioblastoma Evaluation

RATIONALE AND OBJECTIVES Quantitative measurement provides essential information about disease progression and treatment response in patients with glioblastoma multiforme (GBM). The goal of this article is to present and validate a software pipeline for semi-automatic GBM segmentation, called AFINITI (Assisted Follow-up in NeuroImaging of Therapeutic Intervention), using clinical data from GBM patients. MATERIALS AND METHODS Our software adopts the current state-of-the-art tumor segmentation algorithms and combines them into one clinically usable pipeline. Both the advantages of the traditional voxel-based and the deformable shape-based segmentation are embedded into the software pipeline. The former provides an automatic tumor segmentation scheme based on T1- and T2-weighted magnetic resonance (MR) brain data, and the latter refines the segmentation results with minimal manual input. RESULTS Twenty-six clinical MR brain images of GBM patients were processed and compared with manual results. The results can be visualized using the embedded graphic user interface. CONCLUSION Validation results using clinical GBM data showed high correlation between the AFINITI results and manual annotation. Compared to the voxel-wise segmentation, AFINITI yielded more accurate results in segmenting the enhanced GBM from multimodality MR imaging data. The proposed pipeline could be used as additional information to interpret MR brain images in neuroradiology.

Retrospective study of carmustine or lomustine with bevacizumab in recurrent glioblastoma patients who have failed prior bevacizumab

BACKGROUND Currently, there are no known effective treatments for recurrent glioblastoma once patients have progressed on a bevacizumab-containing regimen. We examined the efficacy of adding nitrosoureas to bevacizumab in patients who progressed while on an initial bevacizumab-containing regimen. METHODS In this retrospective study, we identified adult patients with histologically confirmed glioblastoma (WHO grade IV) who were treated with lomustine or carmustine in combination with bevacizumab as a second or third regimen after failing an alternative initial bevacizumab-containing regimen. Response rate (RR), 6-month progression free survival (PFS6), and progression-free survival (PFS) were assessed for each treatment. RESULTS Forty-two patients were identified (28 males) with a median age of 49 years (range, 24-78 y). Of 42 patients, 28 received lomustine (n = 22) or carmustine (n = 6) with bevacizumab as their second bevacizumab-containing regimen, and 14 received lomustine (n = 11) or carmustine (n = 3) as their third bevacizumab-containing regimen. While the median PFS for the initial bevacizumab-containing regimen was 16.3 weeks, the median PFS for the nitrosourea-containing bevacizumab regimen was 6.3 weeks. Patients had an RR of 44% and a PFS6 rate of 26% during the initial bevacizumab regimen and an RR of 0% and a PFS6 rate of 3% during the nitrosourea-containing bevacizumab regimen. There was increased grade 3-4 toxicity (45% vs 19%, P = .010) during the nitrosourea-containing bevacizumab regimen relative to the initial bevacizumab regimen. Median overall survival was 18.7 weeks from initiation of the nitrosourea-containing bevacizumab regimen. CONCLUSION The addition of lomustine or carmustine to bevacizumab after a patient has already progressed on a bevacizumab-containing regimen does not appear to provide benefit for most patients and is associated with additional toxicity with the doses used in this cohort.