Mutational Profiling Can Establish Clonal or Independent Origin in Synchronous Bilateral Breast and Other Tumors

Background

Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal) pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC) mutations/ # total HC mutations).

Methods

Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas.

Results

We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48) and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value <0.01), across renal cell, testicular, and colorectal cancer. The CLS test correctly classified all validation samples but one, which it appears may have been incorrectly classified in the published data. As proof-of-concept we then applied the CLS test to two new cases of invasive synchronous bilateral breast cancer at our institution, each with one hormone receptor positive (ER+/PR+/HER2-) lobular and one triple negative ductal carcinoma. High confidence mutations were identified by exome sequencing and results were validated using deep targeted sequencing. The first tumor pair had CLS of 81% (p-value < 10–15), supporting clonality. In the second pair, no common mutations of 184 variants were validated (p-value >0.99), supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair.

Conclusion

We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.     

Adaptive genetic variation mediates bottom-up and top-down control in an aquatic ecosystem

Research in eco-evolutionary dynamics and community genetics has demonstrated that variation within a species can have strong impacts on associated communities and ecosystem processes. Yet, these studies have centred around individual focal species and at single trophic levels, ignoring the role of phenotypic variation in multiple taxa within an ecosystem. Given the ubiquitous nature of local adaptation, and thus intraspecific variation, we sought to understand how combinations of intraspecific variation in multiple species within an ecosystem impacts its ecology. Using two species that co-occur and demonstrate adaptation to their natal environments, black cottonwood (Populus trichocarpa) and three-spined stickleback (Gasterosteus aculeatus), we investigated the effects of intraspecific phenotypic variation on both top-down and bottom-up forces using a large-scale aquatic mesocosm experiment. Black cottonwood genotypes exhibit genetic variation in their productivity and consequently their leaf litter subsidies to the aquatic system, which mediates the strength of top-down effects from stickleback on prey abundances. Abundances of four common invertebrate prey species and available phosphorous, the most critically limiting nutrient in freshwater systems, are dictated by the interaction between genetic variation in cottonwood productivity and stickleback morphology. These interactive effects fit with ecological theory on the relationship between productivity and top-down control and are comparable in strength to the effects of predator addition. Our results illustrate that intraspecific variation, which can evolve rapidly, is an under-appreciated driver of community structure and ecosystem function, demonstrating that a multi-trophic perspective is essential to understanding the role of evolution in structuring ecological patterns.     

Use of an in silico approach to define the gene structure of eukaryotic adenylyl cyclases.

The limited information available regarding the gene structure of adenylyl cyclases (AC), which catalyze the synthesis of cAMP, suggests a complex arrangement with many exons and large introns such that molecular techniques to define these gene structures are time- and labor-intensive. We report here the use of a computer-based approach involving the assembly of fragmented sequence data generated by the Human Genome Project and nucleic acid analysis software to decipher the gene structure of human and murine AC 6 and other human AC isoforms (ACs 3, 7, and 8). The results, which document 21 exons in human and murine AC 6, human AC 3, 18 exons in AC 8, and 24 exons in AC 7, show substantial conservation of exon organization in the AC family and in particular regions of the AC protein. Application of such in silico methods should prove useful to characterize genes for other ACs and protein families and data provided here should facilitate studies of polymorphisms in AC genes.     

Understanding the recognition of protein structural classes by amino acid composition

Knowledge of amino acid composition, alone, is verified here to be sufficient for recognizing the structural class, α, β, α+β, or α/β of a given protein with an accuracy of 81%. This is supported by results from exhaustive enumerations of all conformations for all sequences of simple, compact lattice models consisting of two types (hydrophobic and polar) of residues. Different compositions exhibit strong affinities for certain folds. Within the limits of validity of the lattice models, two factors appear to determine the choice of particular folds: 1) the coordination numbers of individual sites and 2) the size and geometry of non-bonded clusters. These two properties, collectively termed the distribution of non-bonded contacts, are quantitatively assessed by an eigenvalue analysis of the so-called Kirchhoff or adjacency matrices obtained by considering the non-bonded interactions on a lattice. The analysis permits the identification of conformations that possess the same distribution of non-bonded contacts. Furthermore, some distributions of non-bonded contacts are favored entropically, due to their high degeneracies. Thus, a competition between enthalpic and entropic effects is effective in determining the choice of a distribution for a given composition. Based on these findings, an analysis of non-bonded contacts in protein structures was made. The analysis shows that proteins belonging to the four distinct folding classes exhibit significant differences in their distributions of non-bonded contacts, which more directly explains the success in predicting structural class from amino acid composition. Proteins 29:172–185, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Goverment work and, as such, is in the public domain in the United States of America.

     

Pyrimidine-5-carbonitrile based potential anticancer agents as apoptosis inducers through PI3K/AKT axis inhibition in leukaemia K562

A novel series of 4-(4-Methoxyphenyl)-2-(methylthio)pyrimidine-5-carbonitrile was developed linked to an aromatic moiety via N-containing bridge and then evaluated for their cytotoxic activity against MCF-7 and K562 cell lines. Seven compounds exhibited the highest activity against both cell lines where compounds 4d and 7f were the most active against K562 cell line. Exploring their molecular mechanisms by enzyme inhibition assay on PI3Kδ/γ and AKT-1 showed that compound 7f was promising more than 4d with IC₅₀ = 6.99 ± 0.36, 4.01 ± 0.55, and 3.36 ± 0.17 uM, respectively. Also, flowcytometric analysis revealed that 7f caused cell cycle arrest at S-phase followed by caspase 3 dependent apoptosis induction. Mechanistically, compound 7f proved to modulate the expression of PI3K, p-PI3K, AKT, p-AKT, Cyclin D1, and NFΚβ. Furthermore, in-vivo toxicity study indicated good safety profile for 7f. These findings suggest that the trimethoxy derivative 7f has strong potential as a multi-acting inhibitor on PI3K/AKT axis targeting breast cancer and leukaemia.     

Characteristics of ammonium absorption by excised root and leaf tissues of maize

Absorption of ammonium from solutions of ammonium chloride by maize ( Zea mays L. cv. GS-2) tissue was studied. In contrast to an initial rapid phase of absorption in root tissue, a one hour lag period was recorded in leaf tissue. The maximum rate of uptake was observed at 5–10 m M NH₄Cl in both tissues. Roots had a K_m value of 1.0 m M and V_max of 24.3 μmol ammonium (g fresh weight)⁻¹ h⁻¹, whereas the leaf tissue had a higher K_m (4.1 m M ) and a lower V_max (8.7 μmol). There was a concentration dependent increase in ethanol soluble and insoluble fractions of organic nitrogen during ammonium supply. The optimum pH for ammonium absorption for both tissues was 7.4. The optimal concentration of CaCl₂ for ammonium absorption was 5 m M whereas that of KCl was only 1 m M . In both tissues, the absorption was inhibited substantially by DCMU, DNP, cycloheximide, lincomycin, sodium tungstate, sodium arsenate and to some extent also by the anions nitrate and sulfate. It is suggested that a carrier is involved in an active uptake of ammonium in the leaf tissues.     

Studies on two Serologically Distinct Raspberry Ringspot Virus Strains from Artichoke

Two raspberry ringspot virus variants, RRV-T and RRV-G, found in artichoke of Turkish and Greek origin, were compared biologically (indexing), biochemically and serologically to two strains of the same virus, RRV-S and RRV-E originating from Scotland and England, respectively. Molecular weight values of protein and nucleic acid of RRV-T and RRV-G were in good agreement with those already known for RRV. RRV-T and RRV-G appeared serologically very similar to each other (serological differentiation index = 1) and well distinguishable from RRV-S and RRV-E (serological differentiation indices varying between 3 and 6). Phaseolus vulgaris cv. La Victoire, Ocimum basilicum cv. Foglia di Lattuga, Cucumis sativus cv. Delicatezza, and Cucurbita pepo cv. Zucchetta striata d'ltalia seemed herbaceous hosts useful for differentiating each strain from the others on symptomatological basis.     

Characterization of a Tymovirus Isolated from Anagyris foetida as a Strain of Scrophularia Mottle Virus

A virus isolated from Anagyris foetida and infecting species in the Leguminosae and Solanaceae had typical properties of a tymovirus. It sedimented as two components (49 S and 103 S) and induced the formation of double membrane-bounded invaginations in the chloroplasts of infected cells. Large numbers of ‘empty shells’ were found in the nuclei. The coat protein had a molecular weight of c. 20.000 and the RNA consisted of c. 5500 nucleotides. Crystallization of the virus in laminal crystals could be achieved by precipitation with 10 % polyethylenglycol 6000 and 1 % NaCl. Serologically, the virus was closely related to Scrophularia mottle, Ononis yellow mosaic and Plantago mottle viruses. The four viruses which all infect leguminous hosts are separated by serological differentiation indices which are mostly between 1 and 3. It is therefore suggested that they all should be considered as strains of the same virus which for reasons of priority should have the name Scrophularia mottle virus (ScrMV). The proposed Anagyris strain clearly differs from the Scrophularia mottle, Ononis yellow mosaic and Plantago mottle strains of ScrMV in host range, symptomatology, electrophoretic mobility serological properties and some cytopathogenic effects. It is not clear why the Anagyris strain infects A. foetida systemically in nature, but only locally under greenhouse conditions.     

Floristic characterisation of alkali soils in northwestern India

Summary Floristic composition studied in a 40 hectares representative site of alkali soils in the Indo-gangetic plains of Northwestern India revealed a remarkably restricted spectrum of natural vegetation. Soil analysis of specific niches, occupied by aggregations of different species, was employed to identify alkali halophytes. Response functions of eight prominent species to increasing levels of soil alkalinity, studied in pot culture, showed that most of them were adapted to alkali soils. Based on the computed importance value indices and observed biomass production maxima, it has been inferred thatSporobolus marginatus, Sporobulus coromandelianus, Diplachne fusca andChloris barbata, qualify as plant indicators of high-alkali soil conditions.     

Further Studies on a Tomato Black Ring Virus Isolate from Artichoke

An isolate of tomato black ring virus from artichoke (TBRV-A) was compared biologically, physico-chemically and serologically with three strains of the virus, i.e. TBRV-potato bouquet (TBRV-BU), TBRV-beet ringspot (TBRV-W), and TBRV-celery yellow vein (TBRV-Ce). Cytopathic effects of TBRV-A infection in C. quinoa and its relationships with two strains of artichoke Italian latent virus (AILV-S and AILV-G) were also investigated. Physical properties in vitro, sedimentation coefficients and molecular weight of protein subunits and nucleic acid species of TBRV-A were very similar to those known for TBRV. In serological tests, TBRV-A appeared more closely related to TBRV-W (SDI = 1) than to TBRV-Ce and TBRV-BU (SDI = 2–3). Finally, TBRV-A was very, distantly related to AILV-S and AILV-G (SDI = 11–12).