Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes

Background

The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results

We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions

Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.     

Women Commencing Anastrozole,Letrozole or Tamoxifen for Early Breast Cancer: The Impact of Comorbidity and Demographics on Initial Choice

Background

Australian clinical guidelines recommend endocrine therapy for all women with hormone-dependent early breast cancer. Guidelines specify tamoxifen as first-line therapy for pre-menopausal women, and tamoxifen or an aromatase inhibitor (AI) for post-menopausal women depending on the risk of recurrence based on tumour characteristics including size. Therapies have different side effect profiles; therefore comorbidity may also influence choice. We examined comorbidity, and the clinical and demographic characteristics of women commencing different therapies.

Patients and Methods

We identified the first dispensing of tamoxifen, anastrozole or letrozole for women diagnosed with invasive breast cancer in the 45 and Up Study from 2004–2009 (N = 1266). Unit-level pharmacy and medical service claims, hospital, Cancer Registry, and self-reported data were linked to determine menopause status at diagnosis, tumour size, age, comorbidities, and change in subsidy restrictions. Chi-square tests and generalised regression models were used to compare the characteristics of women commencing different therapies.

Results

Most pre-menopausal women commenced therapy with tamoxifen (91%). Anastrozole was the predominant therapy for post-menopausal women (57%), followed by tamoxifen (28%). Women with osteoporosis were less likely to commence anastrozole compared with tamoxifen (anastrozole RR = 0.7, 95% CI = 0.5–0.9). Women with arthritis were 1.6-times more likely to commence letrozole than anastrozole (95% CI = 1.1–2.1). Tamoxifen was more often initiated in women with tumours >1 cm, who were also ≥75 years. Subsidy restriction changes were associated with substantial increases in the proportion of women commencing AIs (anastrozole RR = 4.3, letrozole RR = 8.3).

Conclusions

The findings indicate interplay of comorbidity and therapy choice for women with invasive breast cancer. Most post-menopausal women commenced therapy with anastrozole; however, letrozole and tamoxifen were more often initiated for women with comorbid arthritis and osteoporosis, respectively. Tamoxifen was also more common for women with tumours >1 cm and aged ≥75 years. Subsidy restrictions appear to have strongly influenced therapy choice.     

Under what conditions do climate‐driven sex ratios enhance versus diminish population persistence?

For many species of reptile, crucial demographic parameters such as embryonic survival and individual sex (male or female) depend on ambient temperature during incubation. While much has been made of the role of climate on offspring sex ratios in species with temperature‐dependent sex determination (TSD), the impact of variable sex ratio on populations is likely to depend on how limiting male numbers are to female fecundity in female‐biased populations, and whether a climatic effect on embryonic survival overwhelms or interacts with sex ratio. To examine the sensitivity of populations to these interacting factors, we developed a generalized model to explore the effects of embryonic survival, hatchling sex ratio, and the interaction between these, on population size and persistence while varying the levels of male limitation. Populations with TSD reached a greater maximum number of females compared to populations with GSD, although this was often associated with a narrower range of persistence. When survival depended on temperature, TSD populations persisted over a greater range of temperatures than GSD populations. This benefit of TSD was greatly reduced by even modest male limitation, indicating very strong importance of this largely unmeasured biologic factor. Finally, when males were not limiting, a steep relationship between sex ratio and temperature favoured population persistence across a wider range of climates compared to the shallower relationships. The opposite was true when males were limiting – shallow relationships between sex ratio and temperature allowed greater persistence. The results highlight that, if we are to predict the response of populations with TSD to climate change, it is imperative to 1) accurately quantify the extent to which male abundance limits female fecundity, and 2) measure how sex ratios and peak survival coincide over climate.     

Evaluation of microcapsule trunk injections for the control of apple scab and powdery mildew

A 3‐year field trial was conducted using established apple cv. Crown Gold and English oak (Quercus robur L.) to assess the efficacy of eight fungicides applied via microcapsule trunk injection against the foliar pathogens apple scab (Venturia inaequalis) and powdery mildew (Phyllactinia sp). In both apple cv. Crown Gold and English oak, the fungicide myclobutanil was not taken up when microcapsules were inserted into the tree vascular system at the root flare. Disease severity in injected trees, excluding myclobutanil, was lower over the following two growing seasons compared to water‐injected controls indicating seven of the eight fungicides used in this study provided a significant degree of protection against scab and powdery mildew infection. A difference in the magnitude of pathogen control achieved was recorded between fungicides. Of the fungicides tested, penconazole, pyrifenox and carbendazim significantly reduced disease severity and significantly increased leaf chlorophyll (Fv/Fm) and SPAD values as a measure of tree vitality and chlorophyll content, respectively, in both apple cv. Crown Gold and English oak over two growing seasons after microcapsule injection. Based on the results of this investigation, it is suggested that these three fungicides be used in preference to thiabendazole, fosetyl‐aluminium, triadimefon and propiconazole for the control of apple scab and powdery mildew where outbreaks of these foliar pathogens are problematic.     

Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone

Xenopus oocytes have been shown to faithfully translate, process, and secrete a number of secretory proteins after the injection of heterologous mRNAs. The oocyte has the capacity to perform a variety of posttranslational protein modifications but has been reported to be incapable of carrying out certain two-step cleavages which proceed via propeptide intermediates. We examined the ability of the oocyte to process preproPTH after the injection of parathyroid mRNA. Microinjected oocytes secreted material which could be detected in a sensitive cytochemical bioassay for PTH. This activity paralleled that of the PTH standard in the assay and was entirely eliminated by a competitive inhibitor of PTH binding, by preincubation with an anti-PTH antiserum, and by coinjecting oocytes with an oligonucleotide mixture complementary to PTH sequences. Immunoprecipitable proPTH and PTH were present in oocyte homogenates, but oocyte-conditioned medium contained only mature PTH(1-84). We conclude that the Xenopus oocyte is capable of accurately processing preproPTH to the mature secretory form of the peptide.     

The Bxs6 locus of BXSB mice is sufficient for high-level expression of gp70 and the production of gp70 immune complexes

High levels of the retroviral envelope protein gp70 and gp70 immune complexes have been linked to a single locus on chromosome 13 (Bxs6) in the BXSB model, to which linkage of nephritis was also seen. Congenic lines containing the BXSB Bxs6 interval on a non-autoimmune C57BL/10 background were bred in the presence or absence of the BXSB Y chromosome autoimmune accelerator gene (Yaa), which accelerates disease in male mice. In these mice, we have shown that Bxs6 is sufficient to cause high-level expression of gp70 and the production of gp70 autoantibodies, independently of Yaa, with gp70 immune complex levels enhanced by Yaa. In the presence of Yaa, Bxs6 also causes mild nephritis, and interestingly the sporadic production of high levels of anti-DNA Abs in some mice. Fine mapping using rare recombinant mice suggested that Bxs6 lies between 59.7 and 74.8 megabases (Mb), although the interval of 0.6 Mb between 73.6 and 78.6 Mb on chromosome 13 cannot be excluded in this study.     

Unexpected molecular and morphological diversity of hemichordate larvae from the Neotropics

The diversity of tropical marine invertebrates is poorly documented, especially those groups for which collecting adults is difficult. We collected the planktonic tornaria larvae of hemichordates (acorn worms) to assess their hidden diversity in the Neotropics. Larvae were retrieved in plankton tows from waters of the Pacific and Caribbean coasts of Panama, followed by DNA barcoding of mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal DNA to estimate their diversity in the region. With moderate sampling efforts, we discovered six operational taxonomic units (OTUs) in the Bay of Panama on the Pacific coast, in contrast to the single species previously recorded for the entire Tropical Eastern Pacific. We found eight OTUs in Bocas del Toro province on the Caribbean coast, compared to seven species documented from adults in the entire Caribbean. All OTUs differed from each other and from named acorn worm sequences in GenBank by >10% pairwise distance in COI and >2% in 16S. Two of our OTUs matched 16S hemichordate sequences in GenBank: one was an unidentified or unnamed Balanoglossus from the Caribbean of Panama, and the other was an unidentified ptychoderid larva from the Bahamas. The species accumulation curves suggest that nearly all the species have been collected and only one more species might still remain undetected in the Pacific. In contrast, the Caribbean species accumulation curve suggests that further sampling could yield more than 10 additional OTUs. Tornaria from the 14 OTUs exhibited typical planktotrophic morphologies, and, in some cases, may be distinguished by differences in pigmentation and by the number of telotrochal ciliary bands, but in general, few diagnostic differences were detected.     

How many came home? Evaluating ex situ conservation of green turtles in the Cayman Islands

Ex situ management is an important conservation tool that allows the preservation of biological diversity outside natural habitats while supporting survival in the wild. Captive breeding followed by re‐introduction is a possible approach for endangered species conservation and preservation of genetic variability. The Cayman Turtle Centre Ltd was established in 1968 to market green turtle (Chelonia mydas) meat and other products and replenish wild populations, thought to be locally extirpated, through captive breeding. We evaluated the effects of this re‐introduction programmme using molecular markers (13 microsatellites, 800‐bp D‐loop and simple tandem repeat mitochondrial DNA sequences) from captive breeders (N = 257) and wild nesting females (N = 57) (sampling period: 2013–2015). We divided the captive breeders into three groups: founders (from the original stock), and then two subdivisions of F₁ individuals corresponding to two different management strategies, cohort 1995 (“C1995”) and multicohort F₁ (“MCF1”). Loss of genetic variability and increased relatedness was observed in the captive stock over time. We found no significant differences in diversity among captive and wild groups, and similar or higher levels of haplotype variability when compared to other natural populations. Using parentage and sibship assignment, we determined that 90% of the wild individuals were related to the captive stock. Our results suggest a strong impact of the re‐introduction programmme on the present recovery of the wild green turtle population nesting in the Cayman Islands. Moreover, genetic relatedness analyses of captive populations are necessary to improve future management actions to maintain genetic diversity in the long term and avoid inbreeding depression.