Automated tumour annotation and analysis for molecular pathology using TissueMark (R)

Objective: Molecular pathology relies on identifying anomalies using PCR or analysis of DNA/RNA. This is important in solid tumours where molecular stratification of patients define targeted treatment. These molecular biomarkers rely on examination of tumour, annotation for possible macro dissection/tumour cell enrichment and the estimation of % tumour. Manually marking up tumour is error prone. Method: We have developed a method for automated tumour mark-up and % cell calculations using image analysis called TissueMark® based on texture analysis for lung, colorectal and breast (cases=245, 100, 100 respectively). Pathologists marked slides for tumour and reviewed the automated analysis. A subset of slides was manually counted for tumour cells to provide a benchmark for automated image analysis. Results: There was a strong concordance between pathological and automated mark-up (100 % acceptance rate for macro-dissection). We also showed a strong concordance between manually/automatic drawn boundaries (median exclusion/inclusion error of 91.70 %/89 %). EGFR mutation analysis was precisely the same for manual and automated annotation-based macrodissection. The annotation accuracy rates in breast and colorectal cancer were 83 and 80 % respectively. Finally, region-based estimations of tumour percentage using image analysis showed significant correlation with actual cell counts. Conclusion: Image analysis can be used for macro-dissection to (i) annotate tissue for tumour and (ii) estimate the % tumour cells and represents an approach to standardising/improving molecular diagnostics.

Transcriptome analysis of a parasitic clade V nematode:comparative analysis of potential molecular anthelmintic targets in Cylicostephanus goldi

Clade V nematodes comprise several parasitic species that include the cyathostomins, primary helminth pathogens of horses. Next generation transcriptome datasets are available for eight parasitic clade V nematodes, although no equine parasites are included in this group. Here, we report next generation transcriptome sequencing analysis for the common cyathostomin species, Cylicostephanus goldi. A cDNA library was generated from RNA extracted from 17 C. goldi male and female adult parasites. Following sequencing using a 454 GS FLX pyrosequencer, a total of 475,215 sequencing reads were generated, which were assembled into 26,910 contigs. Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, 27% of the transcriptome was annotated. Further in-depth analysis was carried out by comparing the C. goldi dataset with the next generation transcriptomes and genomes of other clade V nematodes, with the Oesophagostomum dentatum transcriptome and the Haemonchus contortus genome showing the highest levels of sequence identity with the cyathostomin dataset (45%). The C. goldi transcriptome was mined for genes associated with anthelmintic mode of action and/or resistance. Sequences encoding proteins previously associated with the three major anthelmintic classes used in horses were identified, with the exception of the P-glycoprotein group. Targeted resequencing of the glutamate gated chloride channel α4 subunit (glc-3), one of the primary targets of the macrocyclic lactone anthelmintics, was performed for several cyathostomin species. We believe this study reports the first transcriptome dataset for an equine helminth parasite, providing the opportunity for in-depth analysis of these important parasites at the molecular level. Sequences encoding enzymes involved in key processes and genes associated with levamisole/pyrantel and macrocyclic lactone resistance, in particular the glutamate gated chloride channels, were identified. This novel data will inform cyathostomin biology and anthelmintic resistance studies in future.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma:analysis of a clinic-based population

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry

Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.

Meta-analysis of prostate cancer gene expression data identifies a novel discriminatory signature enriched for glycosylating enzymes

BACKGROUND: Tumorigenesis is characterised by changes in transcriptional control. Extensive transcript expression data have been acquired over the last decade and used to classify prostate cancers. Prostate cancer is, however, a heterogeneous multifocal cancer and this poses challenges in identifying robust transcript biomarkers.

METHODS: In this study, we have undertaken a meta-analysis of publicly available transcriptomic data spanning datasets and technologies from the last decade and encompassing laser capture microdissected and macrodissected sample sets.

RESULTS: We identified a 33 gene signature that can discriminate between benign tissue controls and localised prostate cancers irrespective of detection platform or dissection status. These genes were significantly overexpressed in localised prostate cancer versus benign tissue in at least three datasets within the Oncomine Compendium of Expression Array Data. In addition, they were also overexpressed in a recent exon-array dataset as well a prostate cancer RNA-seq dataset generated as part of the The Cancer Genomics Atlas (TCGA) initiative. Biologically, glycosylation was the single enriched process associated with this 33 gene signature, encompassing four glycosylating enzymes. We went on to evaluate the performance of this signature against three individual markers of prostate cancer, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) expression, prostate specific antigen (PSA) expression and androgen receptor (AR) expression in an additional independent dataset. Our signature had greater discriminatory power than these markers both for localised cancer and metastatic disease relative to benign tissue, or in the case of metastasis, also localised prostate cancer.

CONCLUSION: In conclusion, robust transcript biomarkers are present within datasets assembled over many years and cohorts and our study provides both examples and a strategy for refining and comparing datasets to obtain additional markers as more data are generated.

Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility

Small bowel accounts for only 0.5% of cancer cases in the US but incidence rates have been rising at 2.4% per year over the past decade. One-third of these are adenocarcinomas but little is known about their molecular pathology and no molecular markers are available for clinical use. Using a retrospective 28 patient matched normal-tumor cohort, next-generation sequencing, gene expression arrays and CpG methylation arrays were used for molecular profiling. Next-generation sequencing identified novel mutations in IDH1, CDH1, KIT, FGFR2, FLT3, NPM1, PTEN, MET, AKT1, RET, NOTCH1 and ERBB4. Array data revealed 17% of CpGs and 5% of RNA transcripts assayed to be differentially methylated and expressed respectively (p < 0.01). Merging gene expression and DNA methylation data revealed CHN2 as consistently hypermethylated and downregulated in this disease (Spearman -0.71, p < 0.001). Mutations in TP53 which were found in more than half of the cohort (15/28) and Kazald1 hypomethylation were both were indicative of poor survival (p = 0.03, HR = 3.2 and p = 0.01, HR = 4.9 respectively). By integrating high-throughput mutational, gene expression and DNA methylation data, this study reveals for the first time the distinct molecular profile of small bowel adenocarcinoma and highlights potential clinically exploitable markers.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009-2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

samExploreR: exploring reproducibility and robustness of RNA-seq results based on SAM files

MOTIVATION: Data from RNA-seq experiments provide us with many new possibilities to gain insights into biological and disease mechanisms of cellular functioning. However, the reproducibility and robustness of RNA-seq data analysis results is often unclear. This is in part attributed to the two counter acting goals of (a) a cost efficient and (b) an optimal experimental design leading to a compromise, e.g., in the sequencing depth of experiments.

RESULTS: We introduce an R package called samExploreR that allows the subsampling (m out of n bootstraping) of short-reads based on SAM files facilitating the investigation of sequencing depth related questions for the experimental design. Overall, this provides a systematic way for exploring the reproducibility and robustness of general RNA-seq studies. We exemplify the usage of samExploreR by studying the influence of the sequencing depth and the annotation on the identification of differentially expressed genes.

AVAILABILITY: Availability: samExploreR is available as an R package from Bioconductor (after acceptance of the paper, download link: http://www.bio-complexity.com/samExploreR_1.0.0.tar.gz).