The Escherichia coli protein DbpA is a member of the ‘DEAD box’ family of putative RNA‐dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp‐Glu‐Ala‐Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in‐frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA‐dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA.

The RNA helicases p68 and p72 are highly related members of the DEAD box family of proteins, sharing 90% identity across the conserved core, and have been shown to be involved in both transcription and mRNA processing. We previously showed that these proteins co-localise in the nucleus of interphase cells. In this study we show that p68 and p72 can interact with each other and self-associate in the yeast two-hybrid system. Co-immunoprecipitation experiments confirmed that p68 and p72 can interact in the cell and indicated that these proteins preferentially exist as hetero-dimers. In addition, we show that p68 can interact with NFAR-2, a protein that is also thought to function in mRNA processing. Moreover, gel filtration analysis suggests that p68 and p72 can exist in a variety of complexes in the cell (ranging from approximately 150 to approximately 400 kDa in size), with a subset of p68 molecules being in very large complexes (>2 MDa). The potential to exist in different complexes that may contain p68 and/or p72, together with a range of other factors, would provide the potential for these proteins to interact with different RNA substrates and would be consistent with recent reports implying a wide range of functions for p68/p72.

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BACKGROUND: Interim changes in breast tumour volume at magnetic resonance imaging (MRI) can predict ultimate response to neoadjuvant chemotherapy (NAC), but there is little data on the best measure of volumetric change. PURPOSE: To assess whether changes in measurements of semi-automated enhancing tumour volume (ETV) or fully automated functional tumour volume (FTV) between baseline and interim contrast-enhanced MRI are equivalent in predicting ultimate pathological response to neoadjuvant chemotherapy (NAC) for primary breast cancer, assessed using the residual cancer burden (RCB) score. MATERIALS & METHODS: 78 patients undergoing treatment with NAC for primary breast cancer underwent contrast-enhanced MRI on a 1.5T or 3.0T MRI scanner using a dedicated bilateral breast coil before and after two or three cycles of NAC. Image analysis was performed using either semi-automated, user-defined thresholding (ITK-Snap; ETV) or fully-automated (Siemens SyngoVia BreVis; FTV) approaches. For ETV, the two-minute post-contrast subtracted volumes were analysed, with enhancing pixels thresholded to define tumour volume. FTV was measured using a manufacturer default setting of 50% enhancement threshold, relative to pre-contrast signal intensity, to define tumour volume. ETV intra-observer reproducibility was assessed by repeat analysis one month after initial analysis and a second observer also repeated the measure. Coefficient of reproducibility (CoR) and intraclass correlation coefficents (ICC) were calculated for intra- and inter-observer repeatability. ETV and FTV percentage reduction between baseline and interim examinations was compared with final pathological response, as assessed using the residual cancer burden (RCB) score on resected cancer specimens. Correlation of the two volumetric measures was performed using a Pearson Intra-class Correlation Coefficient (ICC) and pair-wise comparisons of ETV and FTV changes between RCB groups carried out using a Mann-Whitney U test. All statistical assessment was performed using SPSS, v21, with p<0.05 considered significant. RESULTS: There was significant correlation between ETV and FTV (ICC= 0.744, p<0.05). Intra and inter observer reproducibility for ETV was excellent, with ICC 0.940 and 0.861 respectively and corresponding CoRs of 11.6% and 14.8%. Average percentage reductions in ETV for each pathological response category were: pCR 96.4% (n=12), RCB-I 66.6% (n=10), RCB-II 62.9% (n=39) and RCB-III 27.3% (n=17). Corresponding values for FTV were 88.8%, 70.6%, 54.6% and 20.8%. Significant differences in percentage ETV changes were found for pCR vs. RCB-I (p<0.008), II (p<0.001) & III (p<0.001) and RCB-II vs. RCB-III (p<0.001). For FTV, significant differences were measured only for pCR vs. RCB-II & III (p<0.001). CONCLUSION: changes in the semi-automated ETV measuement between baseline and interim MRI may provide more useful predictive information on final pathological response to NAC than FTV, as the changes are better able to discriminate between pCR and minimal residual disease (RCB-I). The ability to confidently predict pCR versus all other residual disease categories could facilitate planning of enhanced approaches to surgical management. Citation Format: Thompson AM, Vinnicombe SJ, Waugh SA, Purdie CA, Evans AJ, Brunton T, Fuller-Pace FV. Which measure of the interim changes in breast tumoral volume at breast MRI in response to neoadjuvant chemotherapy best predicts final pathological response? [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD3-04.

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