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OBJECTIVE: Rearranged ROS1, present in 1%-2% of non-small cell lung cancer (NSCLC) patients, usually young, never or light smokers, is assessed by fluorescence in situ hybridization (FISH) to determine eligibility for tyrosine kinase inhibitors (TKI). Immunohistochemistry (IHC) for the protein product of ROS1 rearrangement, a cost-effective alternative, is validated on cytology and small biopsy samples.METHODS: From 1 March to 31 December 2019, cytology cell blocks and small biopsy samples from a selected cohort of NSCLC patients were concurrently tested for ROS1 gene rearrangement by Vysis 6q22 Break Apart FISH probe and IHC using Cell Signalling D4D6 antibody. Mismatch cases were tested by an RNA fusion next generation sequencing (NGS) panel.RESULTS: In a prospective population of 95 cases, 91 were negative and two were positive by both FISH and IHC. Both dual positive cases were female never smokers and benefited from TKI treatment. Another two cases were positive by FISH but negative by IHC and repeat by NGS showed one to be negative but one failed. Turnaround time for IHC was 0 to 8 days from request to authorisation, whilst that of FISH was 9 to 42 days at a cost of £51 and £159 respectively.CONCLUSION: IHC to assess for the protein product of ROS1 gene rearrangement on cytology cell blocks and small biopsy samples in a routine setting is a promising screening method to assess eligibility for TKI treatment with positive and indeterminate cases confirmed by FISH or NGS as it has good negative predictive value, faster turnaround time and is cost effective, with proven technical and clinical validation.

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism for such protection remains elusive. One of the variants in this allele block is coding, changing cysteine to arginine at position 151 of BAG3 (rs2234962-BAG3C151R). Here, we use induced pluripotent stem cells (iPSC) to test if the BAG3C151Rvariant alters protein and cellular function in human cardiac myocytes. Quantitative protein interaction network analysis identified specific changes in BAG3C151Rprotein interaction partners in cardiomyocytes but not in iPSCs or an immortalized cell line. Knockdown of BAG3 interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151Rassociates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151Rvariant have improved response to proteotoxic stress in an allele dose-dependent manner. This study suggests that the BAG3C151Rvariant increases cardiomyocyte protection from stress by enhancing the recruitment of factors critical to the maintenance of myofibril integrity, hinting that this variant could be responsible for the cardioprotective effect of the haplotype block. By revealing specific changes in preferential binding partners of the BAG3C151Rprotein variant, we also identify potential targets for the development of novel cardioprotective therapies.

A subset of lung adenocarcinomas is driven by the EML4‐ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on‐target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4‐ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti‐apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first‐, second‐ and third‐generation ALK inhibitors., Acquired drug resistance to ALK inhibitors prevents the effective management of lung cancer. This study shows that treatment with CDK inhibitors may be tested as an alternative upon development of resistance.

The revision rate of unicompartmental knee arthroplasty (UKA) is higher than in total knee arthroplasty (TKA), and implant positioning may play a role. In combination with a pre-operative CT, robotic UKA may provide the ability to position the implants more precisely. The aim of this study was to investigate the influence of component prominence relative to the native joint surface on early outcomes and revisions. The hypothesis was that aiming for restoration of joint space to 0.5–1.5 mm will improve outcomes. Retrospective analysis of prospectively collected data of 94 patients undergoing robotic-assisted UKA (Mako, Stryker) was performed. The ‘prominence’ of the implant surface relative to the native bony surface in sagittal plane, hip–knee–ankle (HKA) correction in coronal plane was documented intraoperatively. The mean achieved gap between two components under valgus stress captured in at least 5 different flexion angles was calculated. These were then analysed for impact on early revision rate and outcomes, stratified by gender. Median HKA correction was 3.5° (range 0°–9.5°). Median femoral prominence was 1.5 mm (range − 0.6 to 4 mm) and median tibial prominence was 4.3 mm (2–7 mm). The median achieved gap was 1.0 mm (− 1.2 to 2.8 mm). There was no difference in achieved correction between men and women (p = n.s.) but men had a higher achieved combined prominence than women (p

Neisseria meningitidis inhibits the alternative pathway (AP) of complement using diverse mechanisms, including expression of capsule (select serogroups), Neisserial surface protein A (NspA), factor H (fH) binding protein (fHbp), and lipooligosaccharide (LOS) sialylation. The contribution of the latter three molecules in AP regulation in encapsulated meningococci was studied using isogenic mutants. When LOS was unsialylated, deleting NspA alone from group A strain A2594 (low fHbp/high NspA) significantly increased AP-mediated C3 deposition. C3 deposition further increased ∼2-fold in a ΔfHbpΔNspA double mutant, indicating cooperative fHbp function. LOS sialylation of A2594 ΔfHbpΔNspA decreased the rate of C3 deposition, revealing AP inhibition by LOS sialic acid. Maximal C3 deposition on group B strain H44/76 (high fHbp/low NspA) occurred when all three molecules were absent; again, LOS sialylation attenuated the AP in the absence of both fHbp and NspA. When H44/76 LOS was unsialylated, both fHbp and NspA independently inhibited the AP. LOS sialylation enhanced binding of fH C-terminal domains 18–20 to C3 fragments deposited on bacteria. Interaction of meningococci with nonhuman complement is relevant for animal models and vaccine evaluation studies that use nonhuman complement. Consistent with their human-specific fH binding, neither fHbp nor NspA regulated the rat AP. However, LOS sialylation inhibited the rat AP and, as with human serum, enhanced binding of rat fH to surface-bound C3. These data highlight the cooperative roles of meningococcal NspA and fHbp in regulating the human AP and broaden the molecular basis for LOS sialylation in AP regulation on meningococci in more than one animal species.

Purpose: Cancer progression is mediated by processes that are also important in wound repair. As a result, cancers have been conceptualized as overhealing wounds or wounds that do not heal, and gene expression signatures reflective of wound repair have shown value as predictors of breast cancer survival. Despite the widespread acknowledgment of commonalities between host responses to wounds and host responses to cancer, the gene expression responses of normal tissue adjacent to cancers have not been well characterized. Experimental Design: Using RNA extracted from histologically normal breast tissue from 107 patients, including 60 reduction mammoplasty patients and 47 cancer patients, we measured whole genome expression profiles and identified a gene expression signature that is induced in response to breast cancer. Results: This signature represents an in vivo wound response signature that is differentially expressed in the normal tissue of breast cancer patients compared with those without disease and is highly accurate (at least 92 sensitivity and 98 specificity) in distinguishing diseased and nondiseased. The in vivo wound response signature is highly prognostic of breast cancer survival, and there is a strong association between the groups identified by this signature and those identified using serum-treated fibroblasts and other microenvironment-derived or microenvironment-related signatures. Conclusions: The prevalence of the wound response signature in histologically normal tissue adjacent to breast cancer suggests that microenvironment response is an important variable in breast cancer progression and may be an important target for clinical interventions. (Clin Cancer Res 2009;15(22):70208)

B115 Parity is associated with a significant reduction in breast cancer risk. Identification of key pathways, which are altered by parity, will provide important information for the development of chemoprotective agents against breast cancer. Radiation is a potent carcinogen in the mammary gland. It appears to have mutagenic effects through its ability to induce DNA damage, as well as through its ability to induce “bystander effects”. These effects have been shown to be tumor promoting in immortalized cells and involve modulation of various growth factor pathways for stem cell maintenance. We hypothesized that we would be able to obtain important information on parity-induced changes by examining gene expression responses to radiation exposure from the two types of tissue. Methods: Women undergoing elective breast reduction surgery were asked to enroll and donate their excised tissue. Portions of tissue were cultured and then either exposed or not to 5 Gy radiation. The tissue remained in culture for six hours to allow for changes in gene expression. Tissue was fixed for immunohistochemical analysis or RNA was isolated for microarray analysis. Results: A SAM analysis indicated a loose 200 gene signature, which responded to radiation differentially. The breast tissue from nulliparous women showed the most striking changes with increased expression of 80% genes in response to radiation whereas tissue from parous women illustrated no change or decreased expression of these same genes. The other 20% of the genes were strongly down regulated in tissue from nulliparous women in response to radiation, while they were up-regulated or not changed in parous tissue. The major pathways that appeared to be inversely affected in these tissues indicate parity-induced changes in signaling to tissue homeostasis and wound repair, as well as DNA replication and repair. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B115.