AIMS:To analyse microRNA (miR)-21 distribution and expression at the cellular level in non-small cell lung cancer (NSCLC). MiR-21 is an oncogenic microRNA overexpressed in NSCLC. In previous studies, overexpression of miR-21 was evaluated from the tumour bulk by quantitative reverse transcription PCR with results expressed on average across the entire cell population.METHODS:We used in situ hybridisation and immunohistochemistry to assess the correlation between miR-21 levels and the expression of markers that may be possible targets (epidermal growth factor reaction) or may be involved in its upregulation (phosphatase and tensin homolog (PTEN), p53). The Pearson's χ2 tests was used to assess correlation with clinicopathological data and with miR-21 expression both in tumour and tumour stroma.RESULTS:Cytoplasmic staining and expression of Mir-21 were detected in the tumours and in associated stromal cells. Expression was highest in the stroma immediately surrounding the tumour cells and decreased as the distance from the tumour increased. No expression of miR-21 was found in normal lung parenchyma and a significant association was found between tumour localised miR-21 and PTEN.CONCLUSIONS:Presence of miR-21 in both cell tumour and stromal compartments of NSCLC and the relationship with PTEN confirms miR-21 as a microenvironment signalling molecule, possibly inducing epithelial mesenchymal transition and invasion by targeting PTEN in the stromal compartment possibly through exosomal transport. In situ immunohistochemical studies such as ours may help shed light on the complex interactions between miRNAs and its role in NSCLC biology.

Analyzing immunoglobulin gene sequences, especially in the high throughput sequencing age, raises many challenges. The diverse repertoire and somatic hypermutation they undergo differ the immunoglobulin sequences from other genes, making the commonly used tools unfit for their analysis. Unlike the other genes, immunoglobulins lack a reference gene, raising problems in cleaning the high throughput sequences and defining the germline sequence that will be used as such. Clustering into clones and repertoire representation are nonexistent issues when dealing with uniform genes, but are key analyzes while dealing with immunoglobulin's. This chapter is a thorough review of the informatics tools that were designed to address the unique nature of the immunoglobulins.