Development and Validation of a Gene Signature Classifier for Consensus Molecular Subtyping of Colorectal Carcinoma in a CLIA-Certified Setting.

PURPOSE: Consensus molecular subtyping (CMS) of colorectal cancer has potential to reshape the colorectal cancer landscape. We developed and validated an assay that is applicable on formalin-fixed, paraffin-embedded (FFPE) samples of colorectal cancer and implemented the assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. EXPERIMENTAL DESIGN: We performed an in silico experiment to build an optimal CMS classifier using a training set of 1,329 samples from 12 studies and validation set of 1,329 samples from 14 studies. We constructed an assay on the basis of NanoString CodeSets for the top 472 genes, and performed analyses on paired flash-frozen (FF)/FFPE samples from 175 colorectal cancers to adapt the classifier to FFPE samples using a subset of genes found to be concordant between FF and FFPE, tested the classifier's reproducibility and repeatability, and validated in a CLIA-certified laboratory. We assessed prognostic significance of CMS in 345 patients pooled across three clinical trials. RESULTS: The best classifier was weighted support vector machine with high accuracy across platforms and gene lists (>0.95), and the 472-gene model outperforming existing classifiers. We constructed subsets of 99 and 200 genes with high FF/FFPE concordance, and adapted FFPE-based classifier that had strong classification accuracy (>80%) relative to "gold standard" CMS. The classifier was reproducible to sample type and RNA quality, and demonstrated poor prognosis for CMS1-3 and good prognosis for CMS2 in metastatic colorectal cancer (P < 0.001). CONCLUSIONS: We developed and validated a colorectal cancer CMS assay that is ready for use in clinical trials, to assess prognosis in standard-of-care settings and explore as predictor of therapy response.

Diffusion characteristics of human annulus fibrosus-a study documenting the dependence of annulus fibrosus on end plate for diffusion.

BACKGROUND: Intervertebral disc being avascular depends on nutrition from either the end plate or the annulus fibrosus (AF). The role of the end plate on disc diffusion had been extensively studied. However, diffusion of human AF remains poorly understood because of the lack of reliable techniques to study AF in vivo and non-invasively. The present study for the first time evaluates the 24-hour diffusion characteristics of AF in radial, axial, and circumferential directions. PURPOSE: The study aimed to document the 24-hour diffusion characteristics of human AF. STUDY DESIGN: This is an in vivo human serial post-contrast magnetic resonance image study. METHODS: Twenty-five discs from five healthy volunteers (age <20 years) were studied. Diffusion over 24 hours following intravenous gadodiamide injection (0.3 mmol/kg) was studied at 10 minutes, and at 2, 4, 6, 12, and 24 hours. Axial images of the cranial, middle, and caudal zones of the discs were obtained. The vertebral body and end plate signal intensities were measured in sagittal sections. Thirty-nine regions of interest (24 in AF, 15 in nucleus pulposus) in each disc were analyzed. The peak enhancement percentage (EPmax) and the time to attain EPmax (Tmax) were calculated. Radial (outer vs. inner AF), axial (cranial vs. caudal vs. middle zone), and circumferential diffusions were analyzed. (The study received research grant from AOSpine India for US$6,000). RESULTS: Annulus fibrosus showed a biphasic pattern of diffusion with a characteristic "double peak." Early peak was seen at 10 minutes (coinciding with Tmax of the vertebral body) and delayed peak was seen at 6 hours (coinciding with Tmax of the nucleus pulposus), and characteristically noted after Tmax of the end plate (2 hours). The inner AF showed significant regional differences both at the early and delayed peaks, but the outer AF had no regional differences in the early peak. In axial direction, both outer and inner AF showed maximum enhancement percentage in the middle zone, followed by the caudal zone and least in the cranial zone. CONCLUSIONS: Annulus fibrosus characteristically showed a "double-peak" pattern of diffusion. Both the peaks had different characteristics, confirming two different sources of nutrition. The initial peak was contributed by periannular vascularity and the delayed one via the end plate from the vertebral body. The fact that even AF depends on the end plate for nutrition helps us better understand the complex nutritional pathways of intervertebral discs.

Optical analysis of RE(3+) (RE = Pr(3) (+) , Er(3) (+) and Nd(3) (+) ):cadmium lead boro tellurite glasses.

This article reports on the optical characterization of Pr(3) (+) -, Er(3) (+) - and Nd(3) (+) -doped cadmium lead boro tellurite (CLBT) glasses prepared using the melt quenching method. The visible-near infrared (Vis-NIR) absorption spectra of these glasses were analyzed systematically. On measuring the NIR emission spectra of Er(3) (+) :CLBT glasses, a broad emission band centered at 1536 nm ((4) I13 /2  â†’ (4) I15 /2 ) was observed, as were three NIR emission bands at 900 nm ((4) F3 /2  â†’ (4) I9 /2 ), 1069 nm ((4) F3 /2  â†’ (4) I11 /2 ) and 1338 nm ((4) F3 /2  â†’ (4) I13 /2 ) from Nd(3) (+) :CLBT glasses and an NIR emission band at 1334 nm ((1) G4  â†’ (3) H5 ) from Pr(3) (+) :CLBT glasses at an excitation wavelength (λex ) of 514.5 nm (Ar(+) laser). Copyright © 2016 John Wiley & Sons, Ltd.

Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-ß was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

Increasing the specimen adequacy of transpedicular vertebral body biopsies. Role of intraoperative scrape cytology.

BACKGROUND CONTEXT: Obtaining adequate representative material has been a problem in transpedicular vertebral body biopsies resulting in inconclusive diagnosis and delayed management. By rapidly confirming the adequacy of the material intraoperatively, the rate of negative biopsies can be minimized. Present study evaluates the role of intraoperative cytology in increasing the diagnostic yield of vertebral biopsies. PURPOSE: To evaluate the role of intraoperative cytological confirmation in increasing the specimen adequacy of vertebral biopsies. STUDY DESIGN: A prospective cytological and histopathologic study. PATIENT SAMPLE: Thirty-nine patients undergoing transpedicular biopsy were included. OUTCOME MEASURES: Adequacy and accuracy along with sensitivity and specificity of transpedicular biopsies with and without adopting scrape cytological screening were analyzed. METHODS: After obtaining transpedicular specimens, they were initially screened by intraoperative cytology before submitting for histopathologic examination. Cytological smears were prepared by rapid hematoxylin and eosin technique. Additional specimens were obtained if cytology showed inadequate or inconclusive cells. RESULTS: Of 39 patients who underwent transpedicular biopsy, the intraoperative cytology confirmed 32 (82.1%) specimens as adequate, 5 (12.8%) inadequate, and 2 (5.1%) as inconclusive. Adequacy of biopsy specimens with intraoperative cytology was found to have a sensitivity of 96.9%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 87.5%. If the intraoperative cytology was not adopted, six of 39 (15.4%) patients would have required repeat biopsy. Of the 47 specimens from 39 lesions, 46 intraoperative cytological diagnoses correlated well with the histopathology, with an accuracy of 95.7%. The average time taken to report the adequacy was 8.9 ± 1.7 minutes. CONCLUSIONS: Onsite intraoperative cytology is a rapid and an inexpensive technique to obtain accurate and adequate vertebral body tissue specimen with 100% diagnostic yield. The technique can be adopted easily into day-to-day practice and requires only few glass slides and regular reagents.

Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics.

Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.

Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring.

Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, a next-generation sequencing platform to screen mutational hotspots in 54 cancer-related genes including genes relevant in acute myeloid leukemia (NRAS, KRAS, FLT3, NPM1, DNMT3A, IDH1/2, JAK2, KIT and EZH2). We sequenced 63 samples from patients with acute myeloid leukemia/myelodysplastic syndrome using MiSeq and compared the results with those obtained using another next-generation sequencing platform, Ion-Torrent Personal Genome Machine and other conventional testing platforms. MiSeq detected a total of 100 single nucleotide variants and 23 NPM1 insertions that were confirmed by Ion Torrent or conventional platforms, indicating complete concordance. FLT3-internal tandem duplications (n=10) were not detected; however, re-analysis of the MiSeq output by Pindel, an indel detection algorithm, did detect them. Dilution studies of cancer cell-line DNA showed that the quantitative accuracy of mutation detection was up to an allelic frequency of 1.5% with a high level of inter- and intra-run assay reproducibility, suggesting potential utility for monitoring response to therapy, clonal heterogeneity and evolution. Examples demonstrating the advantages of MiSeq over conventional platforms for disease monitoring are provided. Easy work-flow, high throughput multiplexing capability, 4-day turnaround time and simultaneous assessment of routinely tested and emerging markers make MiSeq highly applicable for clinical molecular testing in acute myeloid leukemia.

Clinical validation of a next-generation sequencing screen for mutational hotspots in 46 cancer-related genes.

Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments-certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.

Predictors of primary imatinib resistance in chronic myelogenous leukemia are distinct from those in secondary imatinib resistance.

PURPOSE: A subset of patients with chronic myelogenous leukemia (CML) do not respond to the tyrosine kinase inhibitor (TKI) imatinib mesylate. Such primary imatinib resistance is distinguished from secondary resistance which reemerges after attainment of cytogenetic remission. PATIENTS AND METHODS: We studied gene expression patterns in total WBCs using a panel of 21 genes previously implicated in TKI handling, resistance, or progression comparing patients who had newly diagnosed TKI-naive CML that had optimal (n = 41), or suboptimal (n = 7) responses to imatinib, or primary resistance (n = 20). Expression patterns were compared to those in secondary TKI-resistant chronic phase CML without ABL1 kinase domain mutations (n = 29), and to lymphoid (n = 15) or myeloid blast phase disease (n = 12). RESULTS: Fifteen genes in the panel distinguished blast phase from chronic phase disease, and 12 genes distinguished newly diagnosed CML from TKI-resistant CML without ABL1 kinase domain mutations, but only a single gene, prostaglandin-endoperoxide synthase 1/cyclooxgenase 1 (PTGS1/COX1; P = .005), differentiated imatinib-responsive from primary imatinib-resistant CML. The association of primary imatinib resistance with higher transcript levels of the drug metabolism gene PTGS1 was confirmed in a separate data set of 68 newly diagnosed, imatinib-treated CML (P = .008). In contrast, up to 11 different genes were identified in a multivariate model that optimally discriminated secondary imatinib resistance lacking ABL1 kinase domain mutation from imatinib-responsive cases, likely related to the more complex pathogenesis of secondary resistance. CONCLUSION: Gene expression profiling of CML at diagnosis for PTGS1 may be useful in predicting imatinib response and in selecting alternate therapy.

Vrksayurveda of parasara--an ancient treatise on plant science.

Vrksayurveda of Parasara is a great contribution to the Botany in ancient India. N.N. Sircar and Roma sarkar edited this text with English translation. Notes with comparative references of modern botany were added. This book can be placed in all probability in between 1st century B.C to 4th century A.D by its linguistic style. Many scientific branches of Botany including origin of life, ecology, distribution of forests, morphology, classification, nomenclature, histology and physiology were dealt in this ancient work. Though it is presumed that this book was written by Parasara to teach Botany to preparatory to Ayurveda studies to ancient Ayurveda students, it is true to the Ayurveda personals and other disciplines related to Botany of present day as well. Aim of this article is to attract the attention of all scholars who are related to Ayurveda and Botany and to feel the depth of the knowledge of ancient Indian botany.

A review of the acute pain service in Hospital Kuala Lumpur.

This study is a review of the Acute Pain Service in Hospital Kuala Lumpur for the years 1998 to 2001. 5042 records from post-operative patients were analysed. The majority of patients (81.8%) had satisfactory pain control. Eighty-two percent of patients experienced only mild pain at rest on the first post-operative day. The highest pain score occurred on the first day in 68.3% of patients. Nausea or vomiting occurred in 23.2% of the patients. Eight patients had respiratory depression. The low pain scores recorded by most patients and the low incidence of side effects reflect the efficiency of the service provided.