DNA copy number profiling reveals extensive genomic loss in hereditary BRCA1 and BRCA2 ovarian carcinomas.

BACKGROUND: Few studies have attempted to characterise genomic changes occurring in hereditary epithelial ovarian carcinomas (EOCs) and inconsistent results have been obtained. Given the relevance of DNA copy number alterations in ovarian oncogenesis and growing clinical implications of the BRCA-gene status, we aimed to characterise the genomic profiles of hereditary and sporadic ovarian tumours. METHODS: High-resolution array Comparative Genomic Hybridisation profiling of 53 familial (21 BRCA1, 6 BRCA2 and 26 non-BRCA1/2) and 15 sporadic tumours in combination with supervised and unsupervised analysis was used to define common and/or specific copy number features. RESULTS: Unsupervised hierarchical clustering did not stratify tumours according to their familial or sporadic condition or to their BRCA1/2 mutation status. Common recurrent changes, spanning genes potentially fundamental for ovarian carcinogenesis, regardless of BRCA mutations, and several candidate subtype-specific events were defined. Despite similarities, greater contribution of losses was revealed to be a hallmark of BRCA1 and BRCA2 tumours. CONCLUSION: Somatic alterations occurring in the development of familial EOCs do not differ substantially from the ones occurring in sporadic carcinomas. However, some specific features like extensive genomic loss observed in BRCA1/2 tumours may be of clinical relevance helping to identify BRCA-related patients likely to respond to PARP inhibitors.

Expression signature in peripheral blood cells for molecular diagnosis of head and neck squamous cell carcinoma.

Patients with head and neck squamous cell carcinoma (HNSCC) have a poor prognosis due to the development of locoregional recurrences, distant metastases, and second primary tumors. There is an urgent need for biomarkers that enable detection and monitoring of the disease to provide adequate therapeutic strategies. In this study, we have investigated markers in peripheral blood cells (PBC) of 28 HNSCC patients who underwent surgery by means of expression profiling. Our hypothesis is that nucleated blood cells circulate continuously, also pass the tumor, and change their expression profile in response to tumor cell factors. For comparison, we enrolled a control group of 11 patients who underwent surgery in the head and neck region for non-HNSCC reasons. A set of 2949 genes was found to be statistically different between the groups (P < 0.05, false discovery rate-corrected) and the most prominently different pathways were EIF2, EIF4, and mTOR signaling. These preliminary results are promising and warrant further studies on the definitive role of PBC gene expression as a biomarker for HNSCC detection and monitoring.

Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene.

We show here that quantitative measurement of DNA copy number across amplified regions using array comparative genomic hybridization (CGH) may facilitate oncogene identification by providing precise information on the locations of both amplicon boundaries and amplification maxima. Using this analytical capability, we resolved two regions of amplification within an approximately 2-Mb region of recurrent aberration at 20q13.2 in breast cancer. The putative oncogene ZNF217 (ref. 5) mapped to one peak, and CYP24 (encoding vitamin D 24 hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other.

Assembly of microarrays for genome-wide measurement of DNA copy number.

We have assembled arrays of approximately 2,400 BAC clones for measurement of DNA copy number across the human genome. The arrays provide precise measurement (s.d. of log2 ratios=0.05-0.10) in cell lines and clinical material, so that we can reliably detect and quantify high-level amplifications and single-copy alterations in diploid, polyploid and heterogeneous backgrounds.

Small bowel adenocarcinoma copy number profiles are more closely related to colorectal than to gastric cancers.

BACKGROUND: Small bowel adenocarcinoma (SBA) is a rare cancer and consequently, the options for clinical trials are limited. As they are treated according to either a colorectal or a gastric cancer regimen and the molecular biology of a tumor is a pivotal determinant for therapy response, chromosomal copy number aberrations were compared with the colorectal and gastric adenocarcinomas. MATERIALS AND METHODS: A total of 85 microsatellite stable (MSS) adenocarcinomas from the stomach, colorectum and small bowel were selected from existing array comparative genomic hybridization (aCGH) datasets. We compared the aCGH profiles of the three tumor sites by supervised analysis and hierarchical clustering. RESULTS: Hierarchical clustering revealed substantial overlap of 27 SBA copy number profiles with matched colorectal adenocarcinomas but less overlap with profiles of gastric adenocarcinomas. DNA copy number aberrations located at chromosomes 1p36.3-p34.3, 4p15.3-q35.2, 9p24.3-p11.1, 13q13.2-q31.3 and 17p13.3-p13.2 were the strongest features discriminating SBAs and colorectal adenocarcinomas from gastric adenocarcinomas. CONCLUSIONS: We show that MSS SBAs are more similar to colorectal than to gastric cancer, based on the 27 genome-wide DNA copy number profiles that are currently available. These molecular similarities provide added support for treatment of MSS small bowel cancers according to colorectal cancer regimens.

High lymph node yield is related to microsatellite instability in colon cancer.

BACKGROUND: Lymph node (LN) yield in colon cancer resection specimens is an important indicator of treatment quality and has especially in early-stage patients therapeutic implications. However, underlying disease mechanisms, such as microsatellite instability (MSI), may also influence LN yield, as MSI tumors are known to exhibit more prominent lymphocytic antitumor reactions. The aim of the present study was to investigate the association of LN yield, MSI status, and recurrence rate in colon cancer. METHODS: Clinicopathological data and tumor samples were collected from 332 stage II and III colon cancer patients. DNA was isolated and PCR-based MSI analysis performed. LN yield was defined as "high" when 10 or more LNs were retrieved and "low" in case of fewer than 10 LNs. RESULTS: Tumors with high LN yield were significantly associated with the MSI phenotype (high LN yield: 26.3% MSI tumors vs low LN yield: 15.1% MSI tumors; P=.01), mainly in stage III disease. Stage II patients with high LN yield had a lower recurrence rate compared with those with low LN yield. Patients with MSI tumors tended to develop fewer recurrences compared with those with MSS tumors, mainly in stage II disease. CONCLUSIONS: In the present study, high LN yield was associated with MSI tumors, mainly in stage III patients. Besides adequate surgery and pathology, high LN yield is possibly a feature caused by biologic behavior of MSI tumors.

Integrative analysis of array-comparative genomic hybridisation and matched gene expression profiling data reveals novel genes with prognostic significance in oesophageal adenocarcinoma.

BACKGROUND AND AIMS: The incidence of oesophageal adenocarcinoma (OAC) has been increasing rapidly with a dismal survival rate of less than 20%. Understanding the genomic aberrations and biology of this cancer may enhance disease interventions. This study aimed to use genome-wide genomic and expression data to enhance the understanding of OAC pathogenesis and identify groups with differential outcomes. METHODS: Array-comparative genomic hybridisation (aCGH) analysis was carried out on 56 fresh frozen OAC resection samples with long-term clinical follow-up data. Samples with aberrations were further analysed with whole-genome single-nucleotide polymorphism arrays to confirm aCGH findings. Matched gene expression microarray data were used to identify genes with high copy number-expression correlations. Nested-multiplex PCR on DNA from microdissected specimens and fluorescence in situ hybridisation assays were used for target validation. Immunohistochemistry on the same cohort and independent samples (n=371) was used for subsequent validation. Kaplan-Meier survival analyses were performed based on aCGH data after unsupervised K-means clustering (K=5, 50 iterations) and immunohistochemistry data. RESULTS: aCGH identified 17 common regions (>5% samples) of gains and 11 common regions of losses, including novel regions in OAC (loci 11p13 and 21q21.2). Integration of aCGH data with matched gene expression microarray data highlighted genes with high copy number-expression correlations: two deletions (p16/CDKN2A, MBNL1) and four gains (EGFR, WT1, NEIL2, MTMR9). Immunohistochemistry demonstrated protein over-expression of targets with gains: EGFR (10%), WT1 (20%), NEIL2 (14%) and MTMR9 (25%). These targets individually (p<0.060) and in combination had prognostic significance (p=0.008). On the genomic level, K-means clustering identified a cluster (32% of cohort) with differential log(2) ratios of 16 CGH probes (p<4×10(-7)) and a worse prognosis (median survival=1.37 years; p=0.015). CONCLUSIONS: Integration of aCGH and gene expression data identified copy number aberrations and novel genes with prognostic potential in OAC.

Gene expression microarray analysis of heat stress in the soil invertebrate Folsomia candida.

Sudden temperature changes in soil can induce stress in soil-dwelling invertebrates. Hyperthermic conditions have an impact on gene expression as one of the first steps. We use a transcriptomics approach using microarrays to identify expression changes in response to heat in the springtail Folsomia candida. An elevation of temperature (Delta 10 degrees C) altered the expression of 142 genes (116 up-, 26 down-regulated). Many up-regulated genes encoded heat shock proteins, enzymes involved in ATP synthesis, oxidative stress responsive enzymes and anion-transporting ATPases. Down-regulated were glycoside hydrolases, involved in catalysis of disaccharides. The small number of altered transcripts suggest a mild response to heat in this soil invertebrate, but further research is needed to confirm this. This study presents candidate genes for future functional studies concerning thermal stress in soil-dwelling invertebrates.

Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression.

OBJECTIVE: This study aimed to identify the oncogenes at 20q involved in colorectal adenoma to carcinoma progression by measuring the effect of 20q gain on mRNA expression of genes in this amplicon. METHODS: Segmentation of DNA copy number changes on 20q was performed by array CGH (comparative genomic hybridisation) in 34 non-progressed colorectal adenomas, 41 progressed adenomas (ie, adenomas that present a focus of cancer) and 33 adenocarcinomas. Moreover, a robust analysis of altered expression of genes in these segments was performed by microarray analysis in 37 adenomas and 31 adenocarcinomas. Protein expression was evaluated by immunohistochemistry on tissue microarrays. RESULTS: The genes C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5, mapping at 20q, were significantly overexpressed in carcinomas compared with adenomas as a consequence of copy number gain of 20q. CONCLUSION: This approach revealed C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5 genes to be important in chromosomal instability-related adenoma to carcinoma progression. These genes therefore may serve as highly specific biomarkers for colorectal cancer with potential clinical applications.

DNA copy number profiles of primary tumors as predictors of response to chemotherapy in advanced colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is biologically a heterogeneous disease, which may affect response to drug therapy. We investigated the correlation of genome-wide DNA copy number profiles of primary tumors with response to systemic chemotherapy in advanced CRC. PATIENTS AND METHODS: DNA was isolated from formaldehyde-fixed paraffin-embedded primary tumors of 32 patients with advanced CRC, which were selected based on either a good response (n = 16) or a poor response (n = 16) to first-line combination therapy with capecitabine and irinotecan. High-resolution DNA copy number profiles were obtained by means of 30 K oligonucleotide-based array comparative genomic hybridization (aCGH). RESULTS: Unsupervised hierarchical cluster analysis of the aCGH data revealed two clusters of 19 and 13 tumors, respectively, and cluster membership showed a significant correlation with response status (P < 0.03). The nonresponders had fewer chromosomal alterations compared with the responders, in particular less losses were found (P < 0.03). Most prominent differences between the two groups were losses of regions 18p11.32-q11.2 (P < 0.02) and 18q12.1-q23 (P < 0.03), which were more frequently observed in responders. CONCLUSIONS: Differences in DNA copy number profiles of primary CRCs are associated with response to systemic combination chemotherapy with capecitabine and irinotecan. Responders overall had more chromosomal alterations, especially loss of chromosome 18.

Sugar sweet springtails: on the transcriptional response of Folsomia candida (Collembola) to desiccation stress.

Several species of Collembola survive stressful desiccating conditions by absorbing water vapour from the environment. To obtain insight into the transcriptomic responses underlying this 'water vapour absorption' mechanism we subjected Folsomia candida (Collembola) to transcriptome profiling. We show that ecologically relevant desiccation stress leads to strong time-dependent transcriptomic changes. Exposure of F. candida to 98.2% relative humidity over an interval of 174 h resulted in a high number of gene transcripts being differentially expressed (up to 41%; P-value < 0.05). Additional Gene Ontology analyses suggest that carbohydrate transport, sugar catabolism and cuticle maintenance are biological processes involved in combating desiccation. However, many additional pathways seem to be affected; additional experiments are needed to elucidate which responses are primarily linked to desiccation resistance.

Cross-platform array comparative genomic hybridization meta-analysis separates hematopoietic and mesenchymal from epithelial tumors.

A series of studies have been published that evaluate the chromosomal copy number changes of different tumor classes using array comparative genomic hybridization (array CGH); however, the chromosomal aberrations that distinguish the different tumor classes have not been fully characterized. Therefore, we performed a meta-analysis of different array CGH data sets in an attempt to classify samples tested across different platforms. As opposed to RNA expression, a common reference is used in dual channel CGH arrays: normal human DNA, theoretically facilitating cross-platform analysis. To this aim, cell line and primary cancer data sets from three different dual channel array CGH platforms obtained by four different institutes were integrated. The cell line data were used to develop preprocessing methods, which performed noise reduction and transformed samples into a common format. The transformed array CGH profiles allowed perfect clustering by cell line, but importantly not by platform or institute. The same preprocessing procedures used for the cell line data were applied to data from 373 primary tumors profiled by array CGH, including controls. Results indicated that there is no apparent feature related to the institute or platform and that array CGH allows for unambiguous cross-platform meta-analysis. Major clusters with common tissue origin were identified. Interestingly, tumors of hematopoietic and mesenchymal origins cluster separately from tumors of epithelial origin. Therefore, it can be concluded that chromosomal aberrations of tumors from hematopoietic and mesenchymal origin versus tumors of epithelial origin are distinct, and these differences can be picked up by meta-analysis of array CGH data. This suggests the possibility of prospectively using combined analysis of diverse copy number data sets for cancer subtype classification.

Genome-wide DNA copy number alterations in head and neck squamous cell carcinomas with or without oncogene-expressing human papillomavirus.

Oncogene-expressing human papillomavirus type 16 (HPV16) is found in a subset of head and neck squamous cell carcinomas (HNSCC). HPV16 drives carcinogenesis by inactivating p53 and pRb with the viral oncoproteins E6 and E7, paralleled by a low level of mutations in TP53 and allelic loss at 3p, 9p, and 17p, genetic changes frequently found in HNSCCs of nonviral etiology. We hypothesize that two pathways to HNSCC exist: one determined by HPV16 and the other by environmental carcinogens. To define the critical genetic events in these two pathways, we now present a detailed genome analysis of HNSCC with and without HPV16 involvement by employing high-resolution microarray comparative genomic hybridization. Four regions showed alterations in HPV-negative tumors that were absent in HPV-positive tumors: losses at 3p11.2-26.3, 5q11.2-35.2, and 9p21.1-24, and gains/amplifications at 11q12.1-13.4. Also, HPV16-negative tumors demonstrated loss at 18q12.1-23, in contrast to gain in HPV16-positive tumors. Seven regions were altered at high frequency (>33%) in both groups: gains at 3q22.2-qter, 5p15.2-pter, 8p11.2-qter, 9q22-34.1, and 20p-20q, and losses at 11q14.1-qter and 13q11-33. These data show that HNSCC arising by environmental carcinogens are characterized by genetic alterations that differ from those observed in HPV16-induced HNSCC, and most likely occur early in carcinogenesis. A number of genetic changes are shared in both tumor groups and can be considered crucial in the later stages of HNSCC progression.

Expression profiling and prediction of distant metastases in head and neck squamous cell carcinoma.

BACKGROUND: For breast and prostate cancer, a gene expression signature of the tumour is associated with the development of distant metastases. Regarding head and neck squamous cell carcinoma (HNSCC), the only known risk factor is the presence of > or =3 tumour-positive lymph nodes. AIM: To evaluate whether a HNSCC gene expression signature can discriminate between the patients with and without distant metastases. METHODS: Patients with HNSCC with and without distant metastases had >3 tumour-positive lymph nodes, and did not differ with respect to other risk factors. Statistical analysis was carried out using Student's t test, as well as statistical analysis of microarrays (SAM), to assess the false discovery rate for each gene. These analyses were supplemented with a newly developed method that computed deviations from gaussian-order statistics (DEGOS). To validate the platform, normal mucosa of the head and neck was included as control. RESULTS: 2963 genes were differently expressed between HNSCC and normal mucosa (t test; p<0.01). More rigorous statistical analysis with SAM confirmed the differential expression of most genes. The comparison of genes in HNSCC with and without metastases showed 150 differently expressed genes (t test; p<0.01), none of which, however, could be confirmed using SAM or DEGOS. CONCLUSIONS: No evidence for a metastasis signature is found, and gene expression profiling of HNSCC has seemingly no value in determining the risk of developing distant metastases. The absence of such a signature can be understood when it is realised that, for HNSCC in contrast with breast cancer, the lymph nodes are a necessary in-between station for haematogenous spread.

Micro-array analysis of resistance for gemcitabine results in increased expression of ribonucleotide reductase subunits.

To study in detail the relation between gene expression and resistance against gemcitabine, a cell line was isolated from a tumor for which gemcitabine resistance was induced in vivo. Similar to the in vivo tumor, resistance in this cell line, C 26-G, was not related to deficiency of deoxycytidine kinase (dCK). Micro-array analysis showed increased expression of ribonucleotide reductase (RR) subunits M1 and M2 as confirmed by real time PCR analysis (28- and 2.7-fold, respectively). In cell culture, moderate cross-resistance (about 2-fold) was observed to 1-ss-D-arabinofuranosylcytosine (ara-C), 2-chloro-2'deoxyadenosine (CdA), LY231514 (ALIMTA), and cisplatin (CDDP), and pronounced cross-resistance (>23-fold) to 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxyguanosine (dFdG). Culture in the absence of gemcitabine reduced resistance as well as RRM1 RNA expression, demonstrating a direct relationship of RRM1 RNA expression with acquired resistance to gemcitabine.

Analysis of mRNA stabilities during pollen development and in BY2 cells

RNA stability is important in post-transcriptional gene expression. However, its role during plant male gametophyte development has not been studied in detail, probably because of technical difficulties and because earlier work suggested that, in general, mRNAs expressed during late pollen development would be stable. We determined mRNA stabilities of 12 pollen-expressed genes. We measured pollen mRNA decay during in planta development and in vitro maturation after transcriptional inhibition with ActinomycinD (ActD). Because five of these genes were also expressed in somatic cells we also measured RNA decay in BY2 cells after transcriptional inhibition with ActD. Ten of the mRNAs were highly stable during pollen development (t1/2 > 30 h), including all seven pollen-specific mRNAs. However, two mRNAs showed a logarithmic decrease as pollen development proceeded, both in planta and in vitro. In fact, for one of these two, GUT8-2b, the mRNA level decreased significantly within 3 h. Our results show that many pollen-expressed mRNAs are indeed long-lived but that at least some mRNAs undergo decay during pollen maturation. Thus, there is no overall cytoplasmic condition in pollen that stabilizes mRNAs and specific mRNA turnover mechanisms must exist. We detected reciprocal mRNA stabilities with two genes that are expressed in both BY2 cells and pollen. GUT15 mRNA, known to be unstable (t1/2 < 3 h) in BY2 cells, was highly stable in pollen. In contrast, GRP2 mRNA decayed in pollen but was highly stable in BY2 cells. Therefore, we conclude that mRNA stability can be tissue-specific.

Steroid Hormones Stimulate Germination and Tube Growth of in Vitro Matured Tobacco Pollen.

A study of the effects of different steroids on germination and tube growth of tobacco pollen (Nicotiana tabacum L. cv Petit Havana SR1) matured in vitro is presented. Application of the mammalian steroid sex hormones (testosterone, progesterone, and estradiol) resulted in a stimulation of pollen germination and tube elongation. The presence of both steroids and flavonols in the germination medium strongly enhanced the growth of tobacco male gametophytes.

DNA copy number changes at 8q11-24 in metastasized colorectal cancer.

BACKGROUND: C-Myc, a well-known oncogene located on 8q24.12-q24.23, is often amplified and over-expressed in both primary and metastasizing colorectal cancer. In addition, PRL-3 (also known as PTP4A3), a tyrosine phosphatase located on 8q24.3, is amplified in colorectal cancer metastasis. Beside PRL-3 and c-myc, other oncogenes located on the 8q23-24 region might be involved in this process. Therefore, the present study aims to correlate DNA copy number status of a series of genes at 8q23-24 in colorectal cancer at high resolution in correlation to metastatic disease. MATERIALS AND METHODS: Thirty-two cases of colorectal cancer, 10 stage B1, 10 B2 and 12 D (Astler-Coller) with their corresponding liver metastasis and one colorectal cell line (colo205, previously analyzed by array-CGH), were included in this study. A chromosome 8 specific MLPA probe mixture was used to analyze the presence of DNA copy number changes. The probe mixture contained 29 probes covering 25 genes on chromosome 8, as well as 6 control probes on other chromosomes. RESULTS AND DISCUSSION: MLPA results obtained of the colo205 colorectal cell line were comparable with previous array-CGH results, thus validating the MLPA probe mixture. Astler-Coller B1 and B2 colorectal cancers differed significantly in DNA copy number of the genes, MOS (p=0.04), MYC (p=0.007), DDEF1 (p=0.004), PTK2 (p=0.02) and PTP4A3 (p=0.04). When comparing these with Astler-Coller D primary tumors, significant differences were seen for several genes as well (MYC (p<0.000), DDEF1 (p<0.000), SLA (p<0.000), PTK2 (p<0.000), PTP4A3 (p=0.002), and RECQL4 (p=0.01)). When comparing primary Astler-Coller D tumors and their corresponding liver metastases, a similar pattern of gains and losses was observed. Most of the liver metastases showed higher DNA copy number ratios than the corresponding primary tumors, but this difference was only significant for TPD52 (p=0.02) and EIF3S6 (p=0.007). CONCLUSION: In addition to c-myc, multiple genes on chromosome 8 differed significantly between primary colorectal cancers with and without liver metastases. This observation is consistent with the concept that clinical behaviour, like risk of liver metastasis, is determined by the genomic profile that is already present in the primary tumor.

Novel variant of EATL evolving from mucosal γδ-T-cells in a patient with type I RCD.

OBJECTIVES: Enteropathy associated T-cell lymphoma (EATL) is a rare non-Hodgkin lymphoma that may complicate coeliac disease and typically occurs in patients with refractoriness to the gluten-free diet. The majority of these patients harbour a clonal expansion of intraepithelial lymphocytes (IELs) with an aberrant phenotype in the small intestine which are thus considered as the 'precursor' lymphoma cells. We describe a 51-year-old female patient with refractory coeliac disease (RCD) who developed an EATL with manifestations in the proximal small intestine and in a mesenteric lymph node that did not evolve from regular type 'aberrant' αß-T-cells but rather from a clonal expansion of γδ-T-cells. METHODS: Duodenal biopsies and lymphoma tissue from a patient with refractory coeliac disease whom developed an EATL were extensively studied by immunophenotypical, T-cell receptor immunogenetic and chromosomal analysis. RESULTS: Flow cytometric analysis of duodenal IELs revealed an unusual large clonal expansion of CD30 negative γδ-T-cells in a patient with RCD. When the patient clinically deteriorated 18 months later, a substantial part (30%) of this cell population did express CD30. In addition, identical immunogenetic aberrancies had developed in a prehepatic lymph node. CONCLUSIONS: We here report on a case of extraintestinal EATL that originated from a clonal γδ-IEL population rather than from aberrant IEL. This EATL displayed a distinctive pattern of immunophenotypical, T-cell receptor immunogenetic and chromosomal aberrancies as compared to classical EATL, defining this lymphoma as a novel variant of EATL.

Collembolan Transcriptomes Highlight Molecular Evolution of Hexapods and Provide Clues on the Adaptation to Terrestrial Life.

BACKGROUND: Collembola (springtails) represent a soil-living lineage of hexapods in between insects and crustaceans. Consequently, their genomes may hold key information on the early processes leading to evolution of Hexapoda from a crustacean ancestor. METHOD: We assembled and annotated transcriptomes of the Collembola Folsomia candida and Orchesella cincta, and performed comparative analysis with protein-coding gene sequences of three crustaceans and three insects to identify adaptive signatures associated with the evolution of hexapods within the pancrustacean clade. RESULTS: Assembly of the springtail transcriptomes resulted in 37,730 transcripts with predicted open reading frames for F. candida and 32,154 for O. cincta, of which 34.2% were functionally annotated for F. candida and 38.4% for O. cincta. Subsequently, we predicted orthologous clusters among eight species and applied the branch-site test to detect episodic positive selection in the Hexapoda and Collembola lineages. A subset of 250 genes showed significant positive selection along the Hexapoda branch and 57 in the Collembola lineage. Gene Ontology categories enriched in these genes include metabolism, stress response (i.e. DNA repair, immune response), ion transport, ATP metabolism, regulation and development-related processes (i.e. eye development, neurological development). CONCLUSIONS: We suggest that the identified gene families represent processes that have played a key role in the divergence of hexapods within the pancrustacean clade that eventually evolved into the most species-rich group of all animals, the hexapods. Furthermore, some adaptive signatures in collembolans may provide valuable clues to understand evolution of hexapods on land.