Tissue microarray study for classification of breast tumors

Clinical and pathological heterogeneity of breast cancer hinders selection of appropriate treatment for individual cases. Molecular profiling at gene or protein levels may elucidate the biological variance of tumors and provide a new classification system that correlates better with biological, clinical and prognostic parameters. We studied the immunohistochemical profile of a panel of seven important biomarkers using tumor tissue arrays. The tumor samples were then classified with a monothetic (binary variables) clustering algorithm. Two distinct groups of tumors are characterized by the estrogen receptor (ER) status and tumor grade (p = 0.0026). Four biomarkers, c-erbB2, Cox-2, p53 and VEGF, were significantly overexpressed in tumors with the ER-negative (ER-) phenotype. Eight subsets of tumors were further identified according to the expression status of VEGF, c-erbB2 and p53. The malignant potential of the ER-/VEGF+ subgroup was associated with the strong correlations of Cox-2 and c-erb132 with VEGF. Our results indicate that this molecular classification system, based on the statistical analysis of immunohistochemical profiling, is a useful approach for tumor grouping. Some of these subgroups have a relative genetic homogeneity that may allow further study of specific genetically-controlled metabolic pathways. This approach may hold great promise in rationalizing the application of different therapeutic strategies for different subgroups of breast tumors. (C) 2003 Elsevier Inc. All rights reserved.

Applied aspects of Rhodococcus genetics

Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.

Exploring statistical and population aspects of network complexity

The characterization and the definition of the complexity of objects is an important but very difficult problem that attracted much interest in many different fields. In this paper we introduce a new measure, called network diversity score (NDS), which allows us to quantify structural properties of networks. We demonstrate numerically that our diversity score is capable of distinguishing ordered, random and complex networks from each other and, hence, allowing us to categorize networks with respect to their structural complexity. We study 16 additional network complexity measures and find that none of these measures has similar good categorization capabilities. In contrast to many other measures suggested so far aiming for a characterization of the structural complexity of networks, our score is different for a variety of reasons. First, our score is multiplicatively composed of four individual scores, each assessing different structural properties of a network. That means our composite score reflects the structural diversity of a network. Second, our score is defined for a population of networks instead of individual networks. We will show that this removes an unwanted ambiguity, inherently present in measures that are based on single networks. In order to apply our measure practically, we provide a statistical estimator for the diversity score, which is based on a finite number of samples.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective FFA2 agonists that interact with the orthosteric binding site. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.

Classification of fundus autofluorescence patterns in early age-related macular disease

PURPOSE. To describe and classify patterns of abnormal fundus autofluorescence (FAF) in eyes with early nonexudative age-related macular disease (AMD). METHODS. FAF images were recorded in eyes with early AMD by confocal scanning laser ophthalmoscopy (cSLO) with excitation at 488 nm (argon or OPSL laser) and emission above 500 or 521 nm (barrier filter). A standardized protocol for image acquisition and generation of mean images after automated alignment was applied, and routine fundus photographs were obtained. FAF images were classified by two independent observers. The ? statistic was applied to assess intra- and interobserver variability. RESULTS. Alterations in FAF were classified into eight phenotypic patterns including normal, minimal change, focal increased, patchy, linear, lacelike, reticular, and speckled. Areas with abnormal increased or decreased FAF signals may or may not have corresponded to funduscopically visible alterations. For intraobserver variability, ? of observer I was 0.80 (95% confidence interval [CI]0.71-0.89) and of observer II, 0.74. (95% CI, 0.64-0.84). For interobserver variability, ? was 0.77 (95% CI, 0.67-0.87). CONCLUSIONS. Various phenotypic patterns of abnormal FAF can be identified with cSLO imaging. Distinct patterns may reflect heterogeneity at a cellular and molecular level in contrast to a nonspecific aging process. The results indicate that the classification system yields a relatively high degree of intra- and interobserver agreement. It may be applicable for determination of novel prognostic determinants in longitudinal natural history studies, for identification of genetic risk factors, and for monitoring of future therapeutic interventions to slow the progression of early AMD. Copyright © Association for Research in Vision and Ophthalmology.

Molecular Phylogenies Support Homoplasy of Multiple Morphological Characters Used in the Taxonomy of Heteroscleromorpha (Porifera: Demospongiae)

Sponge classification has long been based mainly on morphocladistic analyses but is now being greatly challenged by more than 12 years of accumulated analyses of molecular data analyses. The current study used phylogenetic hypotheses based on sequence data from 18S rRNA, 28S rRNA, and the CO1 barcoding fragment, combined with morphology to justify the resurrection of the order Axinellida Lévi, 1953. Axinellida occupies a key position in different morphologically derived topologies. The abandonment of Axinellida and the establishment of Halichondrida Vosmaer, 1887 sensu lato to contain Halichondriidae Gray, 1867, Axinellidae Carter, 1875, Bubaridae Topsent, 1894, Heteroxyidae Dendy, 1905, and a new family Dictyonellidae van Soest et al., 1990 was based on the conclusion that an axially condensed skeleton evolved independently in separate lineages in preference to the less parsimonious assumption that asters (star-shaped spicules), acanthostyles (club-shaped spicules with spines), and sigmata (C-shaped spicules) each evolved more than once. Our new molecular trees are congruent and contrast with the earlier, morphologically based, trees. The results show that axially condensed skeletons, asters, acanthostyles, and sigmata are all homoplasious characters. The unrecognized homoplasious nature of these characters explains much of the incongruence between molecular-based and morphology-based phylogenies. We use the molecular trees presented here as a basis for re-interpreting the morphological characters within Heteroscleromorpha. The implications for the classification of Heteroscleromorpha are discussed and a new order Biemnida ord. nov. is erected.

RAS testing of colorectal carcinoma—a guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Group

Analysis of colorectal carcinoma (CRC) tissue for KRAS codon 12 or 13 mutations to guide use of anti-epidermal growth factor receptor (EGFR) therapy is now considered mandatory in the UK. The scope of this practice has been recently extended because of data indicating that NRAS mutations and additional KRAS mutations also predict for poor response to anti-EGFR therapy. The following document provides guidance on RAS (i.e., KRAS and NRAS) testing of CRC tissue in the setting of personalised medicine within the UK and particularly within the NHS. This guidance covers issues related to case selection, preanalytical aspects, analysis and interpretation of such RAS testing.

Abstract 1905: Defining a therapeutic classification of breast cancer by actionable targets

Breast cancer remains a frequent cause of female cancer death despite the great strides in elucidation of biological subtypes and their reported clinical and prognostic significance. We have defined a general cohort of breast cancers in terms of putative actionable targets, involving growth and proliferative factors, the cell cycle, and apoptotic pathways, both as single biomarkers across a general cohort and within intrinsic molecular subtypes.

We identified 293 patients treated with adjuvant chemotherapy. Additional hormonal therapy and trastuzumab was administered depending on hormonal and HER2 status respectively. We performed immunohistochemistry for ER, PR, HER2, MM1, CK5/6, p53, TOP2A, EGFR, IGF1R, PTEN, p-mTOR and e-cadherin. The cohort was classified into luminal (62%) and non-luminal (38%) tumors as well as luminal A (27%), luminal B HER2 negative (22%) and positive (12%), HER2 enriched (14%) and triple negative (25%). Patients with luminal tumors and co-overexpression of TOP2A or IGF1R loss displayed worse overall survival (p=0.0251 and p=0.0008 respectively). Non-luminal tumors had much greater heterogeneous expression profiles with no individual markers of prognostic significance. Non-luminal tumors were characterised by EGFR and TOP2A overexpression, IGF1R, PTEN and p-mTOR negativity and extreme p53 expression.

Our results indicate that only a minority of intrinsic subtype tumors purely express single novel actionable targets. This lack of pure biomarker expression is particular prevalent in the triple negative subgroup and may allude to the mechanism of targeted therapy inaction and myriad disappointing trial results. Utilising a combinatorial biomarker approach may enhance studies of targeted therapies providing additional information during design and patient selection while also helping decipher negative trial results.

Molecular Medicine and Molecular Pathology for Hematologists:II. Lets go Techno; Principles of the Molecular Technologies and their Applications in Haematology

Molecular techniques have a key role to play in laboratory and clinical haematology. Restriction enzymes allow nucleic acids to be reduced in size for subsequent analysis. In addition they allow selection of specific DNA or RNA sequences for cloning into bacterial plasmids. These plasmids are naturally occuring DNA molecules which reside in bacterial cells. They can be manipulated to act as vehicles or carriers for biologically and medically important genes, allowing the production of large amounts of cloned material for research purposes or to aid in the production of medically important recombinant molecules such as insulin. As acquired or inherited genetic changes are implicated in a wide range of haematological diseases, it is necessary to have highly specific and sensitive assays to detect these mutations. Most of these techniques rely on nucleic acid hybridisation, benefitting from the ability of DNA or RNA to bind tighly to complimentary bases in the nucleic acid structure. Production of artificial DNA molecules called probes permits nucleic acid hybridiation assays to be performed, using the techniques of southern blotting or dot blot analysis. In addition the base composition of any gene or region of DNA can be determined using DNA sequencing technology. The advent of the polymerase chain reaction (PCR) has revolutionised all aspects of medicine, but has particular relevance in haematology where easy access to biopsy material provides a wealth of material for analysis. PCR permits quick and reliable manipulation of sample material and its ability to be automated makes it an ideal tool for use in the haematology laboratory.

Challenging the cancer molecular stratification dogma: Intratumoral heterogeneity undermines consensus molecular subtypes and potential diagnostic value in colorectal cancer

Purpose:
A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer (CRC) with potential diagnostic utility, culminating in publication of a CRC Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal-derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled.

Experimental Design:
We performed multi-region tissue RNA extraction/transcriptomic analysis using Colorectal Specific Arrays on invasive front, central tumor and lymph node regions selected from tissue samples from 25 CRC patients.

Results:
We identified a consensus 30 gene list which represents the intratumoral heterogeneity within a cohort of primary CRC tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential (HR=2.914 (CI 0.9286-9.162) in stage II/III CRC patients, but in addition we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread Epithelial Mesenchymal Transition (EMT). Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analysed.

Conclusions:
Gene expression profiles derived from the non-malignant stromal region can influence assignment of CRC transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision-making in CRC.

The Clinical Impact and Molecular Biology of del(17p) in Multiple Myeloma Treated with Conventional or Thalidomide-Based Therapy

Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide-based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P <0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group. (C) 2011 Wiley-Liss, Inc.