HER2-HER3 heterodimer quantification by FRET-FILM and patient subclass analysis of the COIN colorectal trial

BACKGROUND: The phase 3 MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. METHODS: HER2-HER3 dimerization was quantified by 'FLIM Histology' in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy +/-cetuximab. Bayesian latent class analysis (LCA) and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation and cetuximab on progression-free survival (PFS) and overall survival (OS). All statistical tests were two-sided. RESULTS: LCA on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS: 1624 days [95%CI=1466-1816] vs 461 [95%CI=431-504]): Class 1 (15.6%) showed a benefit from cetuximab in OS (HR = 0.43 [95%CI=0.25-0.76]; p = 0.004). Class 2 showed an association of increased HER2-HER3 with better OS (HR = 0.64 [95%CI=0.44-0.94]; p = 0.02). A class prediction signature was formed and tested on an independent validation cohort (N = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (N = 1,630) based on 10 baseline clinicopathological and genetic covariates. CONCLUSIONS: Our work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment

Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated Viruses

We report the initial characterization of adeno-associated virus type 5 (AAV5) RNAs generated following viral infection and the construction of a replicating infectious clone of AAV5. While the basic transcription profile of AAV5 was similar to that of AAV2, there were also significant differences. Mapping of the AAV5 transcripts demonstrated an efficient transcription initiation site within the AAV5 inverted terminal repeat (ITR), and mapping of the AAV5 intron revealed that it is considerably smaller than that of AAV2. Furthermore, in contrast to the case for AAV2, neither the Rep protein nor additional adenovirus gene products were required to achieve efficient promoter activity and pre-mRNA splicing following transfection of an AAV5 rep/cap plasmid clone lacking the ITRs into 293 cells. Perhaps most surprisingly, RNAs generated from both the AAV5 P7 and P19 promoters were efficiently polyadenylated at a site lying within the intronic region in the center of the genome. Because P7- and P19-generated transcripts are polyadenylated at this site and not spliced, Rep78 and Rep52 were the only Rep proteins detected during AAV5 infection

Neural precursors isolated from the developing cat brain show retinal integration following transplantation to the retina of the dystrophic cat

The cat has served as an important nonrodent research model for neurophysiology and retinal degenerative disease processes, yet very little is known about feline neural precursor cells. To culture these cells and evaluate marker expression, brains were dissected from 45-day-old fetuses, enzymatically dissociated, and grown in the presence of EGF, bFGF and PDGF. Expanded cells widely expressed nestin, Sox2, Ki-67, fusin (CXCR4) and vimentin, while subpopulations expressed A2B5, GFAP, or beta-III tubulin. Precursors prelabeled with BrdU and/or transduced with a recombinant lentivirus that expresses GFP were transplanted subretinally in five dystrophic Abyssinian cats. Two to 4 weeks following surgery, histology showed survival of grafted cells in three of the animals. Labeled cells were found in the neuroretina and RPE layer, as well as in the vitreous and the vicinity of Bruch's membrane. There was no evidence of an immunologic response in any of the eyes. Neural precursor cells can therefore be cultured from the developing cat brain and survive as allografts for up to 4 weeks without immune suppression. The feasibility of deriving and transplanting feline neural precursor cells, combined with the availability of the dystrophic Abyssinian cat, provide a new feline model system for the study of retinal repair

Imaging tumour heterogeneity of the consequences of a PKCα-substrate interaction in breast cancer patients

Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein–protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC–ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically <10 nm range to address the oncological challenge of tumour heterogeneity, is discussed