Simulating a base population in honey bee for molecular genetic studies

<p>Abstract</p> <p>Background</p> <p>Over the past years, reports have indicated that honey bee populations are declining and that infestation by an ecto-parasitic mite (<it>Varroa destructor</it>) is one of the main causes. Selective breeding of resistant bees can help to prevent losses due to the parasite, but it requires that a robust breeding program and genetic evaluation are implemented. Genomic selection has emerged as an important tool in animal breeding programs and simulation studies have shown that it yields more accurate breeding value estimates, higher genetic gain and low rates of inbreeding. Since genomic selection relies on marker data, simulations conducted on a genomic dataset are a pre-requisite before selection can be implemented. Although genomic datasets have been simulated in other species undergoing genetic evaluation, simulation of a genomic dataset specific to the honey bee is required since this species has a distinct genetic and reproductive biology. Our software program was aimed at constructing a base population by simulating a random mating honey bee population. A forward-time population simulation approach was applied since it allows modeling of genetic characteristics and reproductive behavior specific to the honey bee.</p> <p>Results</p> <p>Our software program yielded a genomic dataset for a base population in linkage disequilibrium. In addition, information was obtained on (1) the position of markers on each chromosome, (2) allele frequency, (3) χ² statistics for Hardy-Weinberg equilibrium, (4) a sorted list of markers with a minor allele frequency less than or equal to the input value, (5) average r² values of linkage disequilibrium between all simulated marker loci pair for all generations and (6) average r² value of linkage disequilibrium in the last generation for selected markers with the highest minor allele frequency.</p> <p>Conclusion</p> <p>We developed a software program that takes into account the genetic and reproductive biology specific to the honey bee and that can be used to constitute a genomic dataset compatible with the simulation studies necessary to optimize breeding programs. The source code together with an instruction file is freely accessible at <url>http://msproteomics.org/Research/Misc/honeybeepopulationsimulator.html</url></p

Heterogeneity of Estrogen Receptor Expression in Circulating Tumor Cells from Metastatic Breast Cancer Patients

<div><p>Background</p><p>Endocrine treatment is the most preferable systemic treatment in metastatic breast cancer patients that have had an estrogen receptor (ER) positive primary tumor or metastatic lesions, however, approximately 20% of these patients do not benefit from the therapy and demonstrate further metastatic progress. One reason for failure of endocrine therapy might be the heterogeneity of ER expression in tumor cells spreading from the primary tumor to distant sites which is reflected in detectable circulating tumor cells (CTCs).</p><p>Methods</p><p>A sensitive and specific staining protocol for ER, keratin 8/18/19, CD45 was established. Peripheral blood from 35 metastatic breast cancer patients with ER-positive primary tumors was tested for the presence of CTCs. Keratin 8/18/19 and DAPI positive but CD45 negative cells were classified as CTCs and evaluated for ER staining. Subsequently, eight individual CTCs from four index patients (2 CTCs per patient) were isolated and underwent whole genome amplification and <i>ESR1</i> gene mutation analysis.</p><p>Results</p><p>CTCs were detected in blood of 16 from 35 analyzed patients (46%), with a median of 3 CTCs/7.5 ml. In total, ER-negative CTCs were detected in 11/16 (69%) of the CTC positive cases, including blood samples with only ER-negative CTCs (19%) and samples with both ER-positive and ER-negative CTCs (50%). No correlation was found between the intensity and/or percentage of ER staining in the primary tumor with the number and ER status of CTCs of the same patient. <i>ESR1</i> gene mutations were not found.</p><p>Conclusion</p><p>CTCs frequently lack ER expression in metastatic breast cancer patients with ER-positive primary tumors and show a considerable intra-patient heterogeneity, which may reflect a mechanism to escape endocrine therapy. Provided single cell analysis did not support a role of <i>ESR1</i> mutations in this process.</p></div

The established triple staining protocol for detection and characterization of ER expression on CTC.

<p>ER – estrogen receptor; CTC – circulating tumor cell; NBT/BCIP – nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate; PBS – phosphate buffered saline; TBS – tris buffered saline.</p

Occurrence of ER-positive and ER-negative CTCs in the peripheral blood of patients with breast carcinomas classified as ER-positive.

<p>Circulating tumor cells disseminating from an ER-positive breast tumor can be ER-positive or ER-negative. ER-positive CTCs can have normal functional ER machinery and be sensitive to endocrine therapy (cell A) or have dysfunctional ER machinery and therefore be resistant to endocrine therapy (cell B). ER-negative CTCs might disseminate from ER-negative subclones in tumors classified as ER-positive (diagnostic cut-off value: 1% of ER-stained tumor cells) (cell C) or disseminate from ER-positive subclones that lost ER expression during the metastatic cascade or as a result of systemic therapy (cell D).</p

CTC status in respect to clinical status of the patients.

<p>CTC – circulating tumor cell.</p

Kaplan–Meier estimate of survival function.

<p>Kaplan–Meier estimate of survival function of metastatic breast cancer patients separated on CTC-positive (red line) and CTC-negative (blue line) groups. The survival period in month of the corresponding patient. Censored patients are indicated by vertical bars (|). Statistical significance determined by log-rank test. Shorter survival correlates with presence of CTCs in blood (<i>P:</i> 0.0332, HR: 7.38, (CI = 0.84-64.09)).</p

Overview of studies on ER status of CTC in metastatic breast cancer patients.

<p>CTC – circulating tumor cell; ER – estrogen receptor; IF – immunofluorescence; RT-PCR – real-time PCR.</p>*<p>RT-PCR approach does not allow to assess intrapatient heterogeneity of ER-status of CTCs.</p

Number of detected CTCs and corresponding ER status.

*<p>ER positive group includes CTCs with weak, moderate, and strong uniform ER staining. For more detailed information see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075038#pone.0075038.s001" target="_blank">Table S1</a>.</p><p>ER – estrogen receptor; CTC – circulating tumor cell.</p