Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.

Mediterranean populations of the lesser white-toothed shrew (Crocidura suaveolens group): an unexpected puzzle of Pleistocene survivors and prehistoric introductions.

An earlier study revealed the strong phylogeographical structure of the lesser white-toothed shrew (Crocidura suaveolens group) within the northern Palaearctic. Here, we aim to reconstruct the colonization history of Mediterranean islands and to clarify the biogeography and phylogeographical relationships of the poorly documented Middle East region with the northern Palaearctic. We performed analyses on 998-bp-long haplotypes of the mitochondrial cytochrome b gene of 143 samples collected around the Mediterranean basin, including islands and the Middle East. The analyses suggest that the Cypriot shrew belongs to the rare group of relict insular Pleistocene mammal taxa that have survived to the present day. In contrast, the Cretan, Corsican and Menorcan populations were independently introduced from the Middle East during the Holocene. The phylogeographical structure of this temperate Palaearctic species within the Middle East appears to be complex and rich in diversity, probably reflecting fragmentation of the area by numerous mountain chains. Four deeply divergent clades of the C. suaveolens group occur in the area, meaning that a hypothetical contact zone remains to be located in central western Iran.

The HER2 amplicon in breast cancer: Topoisomerase IIA and beyond

HER2 gene amplification is observed in about 15% of breast cancers. The subgroup of HER2-positive breast cancers appears to be heterogeneous and presents complex patterns of gene amplification at the locus on chromosome 17q12-21. The molecular variations within the chromosome 17q amplicon and their clinical implications remain largely unknown. Besides the well-known TOP2A gene encoding Topoisomerase IIA, other genes might also be amplified and could play functional roles in breast cancer development and progression. This review will focus on the current knowledge concerning the HER2 amplicon heterogeneity, its clinical and biological impact and the pitfalls associated with the evaluation of gene amplifications at this locus, with particular attention to TOP2A and the link between TOP2A and anthracycline benefit. In addition it will discuss the clinical and biological implications of the amplification of ten other genes at this locus (MED1, STARD3, GRB7, THRA, RARA, IGFPB4, CCR7, KRT20, KRT19 and GAST) in breast cancer.

Combined clonotyping and gene signatures of individual tumor-reactive CD8 T-cells define their functional relevance following peptide vaccination in melanoma patients

Novel cancer vaccines are capableto efficiently induce and boost humantumor antigen specific T-cells. However,the properties of these CD8T-cells are only partially characterized.For in depth investigation ofT-cells following Melan-A/MART-1peptide vaccination in melanoma patients,we conducted a detailed prospectivestudy at the single cell level.We first sorted individual human naiveand effector CD8 T-cells from peripheralblood by flow cytometry, andtested a modified RT-PCR protocolincluding a global amplification ofexpressed mRNAs to obtain sufficientcDNAfromsingle cells.We successfullydetected the expression ofseveral specific genes of interest evendown to 106-fold dilution (equivalentto 10-5 cell). We then analyzed tumor-specific effector memory (EM)CD8T-cell subpopulations ex vivo, assingle cells from vaccinated melanomapatients. To elucidate the hallmarksof effective immunity the genesignatures were defined by a panel ofgenes related to effector functions(e.g. IFN-, granzyme B, perforin),and individual clonotypes were identifiedaccording to the expression ofdistinct T-cell receptors (TCR). Usingthis novel single cell analysis approach,we observed that T-cell differentiationis clonotype dependent,with a progressive restriction in TCRBV clonotype diversity from EMCD28pos to EMCD28neg subsets. However,the effector function gene imprintingis clonotype-independent,but dependent on differentiation,since it correlates with the subset oforigin (EMCD28pos or EMCD28neg). We also conducted a detailedcomparative analysis after vaccinationwith natural vs. analog Melan-Apeptide. We found that the peptideused for vaccination determines thefunctional outcome of individualT-cell clonotypes, with native peptideinducing more potent effector functions.Yet, selective clonotypic expansionwith differentiation was preservedregardless of the peptide usedfor vaccination. In summary, the exvivo single cell RT-PCR approach ishighly sensitive and efficient, andrepresents a reliable and powerfultool to refine our current view of molecularprocesses taking place duringT-cell differentiation.