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Endoluminal surgery for the treatment of colorectal neoplasia is typically carried out using electrocautery tools which imply limited precision and the risk of harm through collateral thermal damage to the adjacent healthy tissue. As a potential alternative, we present the successful colonic epithelial laser ablation by means of picosecond laser pulses. Laser ablation studies performed in ex-vivo colon tissue result in cavities with comparable thickness to early stage colorectal cancers. The corresponding histology sections exhibit only minimal collateral damage to the surrounding tissue and the depth of the ablation can be controlled precisely by means of the pulse energy. High-speed imaging has been used for the first time to visualize picosecond laser ablation of cancerous tissue in a clinically relevant model. This information was correlated with histopathology and optical surface profilometry revealing the dynamic nature of the laser tissue interaction and the need for temporal or spatial separation of pulses for optimum efficacy with regards to tissue removal. Overall, the application of picosecond laser pulses to ablate endoluminal bowel lesions demonstrates significantly improved precision and reduced thermal damage to the adjacent tissue in comparison to conventional procedures and hence will enable more precise surgical treatment of cancers.

203 Background: AREG and EREG are ligands of EGFR. AI assisted IHC evaluation of tumor AREG/EREG expression predicted benefit from anti-EGFR therapy in a retrospective analysis of the PICCOLO trial of second-line irinotecan ± panitumumab. Here, we sought to validate those findings through an analysis of patients who received anti-EGFR therapy during routine care for mCRC. Methods: Patients (pts) with available archival FFPE tumor tissue who received panitumumab or cetuximab ± chemotherapy at any time for treatment of mCRC at 8 UK cancer centers were eligible. Central RAS testing by next generation sequencing (NGS) was performed for pts where extended RAS testing had not been previously undertaken. RAS-mutant (mut) and RAS-unknown pts were excluded from the primary analysis population. AREG and EREG positive tumor cells were identified by IHC, performed locally at 6 of 8 sites. Pathologists annotated tumor areas on digital images of glass slides. AI algorithms calculated the percentage of tumor cells staining positive for AREG and EREG within tumor areas. More than 20% AREG and/or EREG tumor cell positivity was regarded as high biomarker expression, the optimal cut-point identified in PICCOLO. Study endpoints were progression-free survival (PFS), overall survival (OS), and locally reported response rate (RR) and disease control rate (DCR). Results: Of 541 pts recruited, 494 (91.3%) had adequate archival tissue for analysis. Central RAS testing was successfully performed in 255 of 393 (64.9%) pts without existing extended RAS results, leading to 45 exclusions, leaving 449 pts in the primary analysis population. 26 (5.8%) pts were BRAF-mut. 110 (24.5%) received concomitant FOLFOX and 304 (67.7%) FOLFIRI. After adjustment for additional prognostic factors, high AREG/EREG expression (n = 360; 80.2%) was associated with significantly prolonged PFS (HR 0.73; 95% CI, 0.56-0.95; p = 0.02), OS (HR 0.66 [0.50-0.86]; p = 0.002), and DCR (OR 1.92 [1.05-3.54]; p = 0.04), but not RR (unadjusted OR 1.39 [0.83-2.33]; p = 0.21). Median PFS in the high vs low biomarker groups was 8.5 vs 4.4 months; median OS 16.4 vs 8.9 months. The significant difference in OS (high vs low) was maintained in the subgroup with right-sided primary tumor location (n = 107; 23.8%) (HR 0.56 [0.37-0.86]; p = 0.007). Conclusions: High tumor AREG/EREG expression was associated with significantly prolonged PFS, OS and DCR among a cohort of pts treated with anti-EGFR therapy during routine care of mCRC. The prognostic effect observed validates the predictive effect of AREG/EREG seen in the PICCOLO trial, where AREG/EREG had no prognostic effect in patients receiving chemotherapy alone. A prospective biomarker-led trial is in set-up to support the use of AREG/EREG IHC in clinical practice.

204 Background: High CD3+ (all) and CD8+ (cytotoxic) T cell densities in the core (CT) and invasive margin (IM) of primary CRCs have been shown to be associated with superior prognosis at all stages of disease. Their predictive effect on benefit from adjuvant chemotherapy in early stage CRC has not been tested. Methods: FFPE samples from participants (pts) in the QUASAR trial (adjuvant fluorouracil/folinic acid vs observation in stage 2/3 CRC) were analysed for CD3 and CD8 immunohistochemistry (IHC). Pathologists annotated the core and peritumor areas on digital slide images. Artificial intelligence (AI) algorithms delineated the CT and IM, and calculated the densities (cells/mm2) of each marker in each region (CD3-CT, CD3-IM, CD8-CT, CD8-IM). Pts were randomly partitioned into test and validation sets (1:1). In the test set, each measure’s prognostic effect on recurrence-free interval (RFI) (primary endpoint), colorectal cancer mortality (CCM) and overall survival (OS) in each trial arm was assessed. Maximum likelihoods methods were used to develop optimal cut-points. Analyses were repeated in the validation set. Analysis of 425 pts in each set would give > 95% power (α = 0.05, 2-sided) to detect a twofold difference in recurrence risk. In predictive analyses, 2-year recurrence rate was the primary outcome; biomarker-treatment interactions were assessed. Results: Tumor tissue from 868 pts (797 [92%] stage 2; 531 [61%] colon) was analysed, with evaluable results for CD3-CT in 851 (98.0%), CD3-IM in 833 (96.0%), CD8-CT in 849 (97.0%) and CD8-IM in 820 (94.5%) pts. In the test set, optimal cut-points of 318, 798, 81 and 186 cells/mm2 were defined for CD3-CT, CD3-IM, CD8-CT and CD8-IM respectively. The recurrence rate in the high-risk group was twice that in the low-risk group for all measures (CD3-CT: rate ratio [RR] 2.00, [95%CI 1.33-2.94], p = 0.0008; CD3-IM: 2.38, [1.59-3.57], p < 0.00001; CD8-CT: 2.17, [1.59-3.57], p = 0.0001; CD8-IM: 2.13 [1.43-3.23], p = 0.0001), which was closely replicated in the validation set (CD3-CT: RR 1.96, [1.30-2.94], p = 0.002; CD3-IM: 1.79, [1.18-2.70], p = 0.005; CD8-CT: 1.72, [1.18-2.56], p = 0.005; CD8-IM: 1.72 [1.15-2.56], p = 0.008). In multivariate analyses, prognostic effects were similar in colon and rectal cancers, and in stage 2 and 3 disease. CD3/8 counts were not predictive of benefit from adjuvant chemotherapy, with similar efficacy in the high and low risk groups. Conclusions: AI-assisted CD3 and CD8 counts were strongly associated with tumor recurrence rates. With no biomarker-treatment interactions, proportional reductions in recurrence with chemotherapy were similar in high and low-risk disease. Hence, numbers of high-risk patients needed to treat to prevent one recurrence were about half the number for low-risk patients.

Background Occupational and environmental airborne asbestos concentrations are too low and variable for lifetime exposures to be estimated reliably, and building workers and occupants may suffer higher exposure when asbestos in older buildings is disturbed or removed. Mesothelioma risks from current asbestos exposures are therefore not known. Methods We interviewed and measured asbestos levels in lung samples from 257 patients treated for pneumothorax and 262 with resected lung cancer, recruited in England and Wales. Average lung burdens in British birth cohorts from 1940 to 1992 were estimated for asbestos-exposed workers and the general population. Results Regression analysis of British mesothelioma death rates and average lung burdens in birth cohorts born before 1965 suggests a lifetime mesothelioma risk of approximately 0.01% per fibre/mg of amphiboles in the lung. In those born since 1965, the average lung burden is ∼1 fibre/mg among those with no occupational exposure. Conclusions The average lifetime mesothelioma risk caused by recent environmental asbestos exposure in Britain will be about 1 in 10 000. The risk is an order of magnitude higher in a subgroup of exposed workers and probably in occupants in the most contaminated buildings. Further data are needed to discover whether asbestos still present in buildings, particularly schools, is a persistent or decreasing hazard to workers who disturb it and to the general population, and whether environmental exposure occurs predominantly in childhood or after beginning work. Similar studies are needed in other countries to estimate continuing environmental and occupational mesothelioma hazards worldwide, including the contribution from chrysotile.

Colorectal cancer is the fourth-most prominent cause of cancer related fatalities across the globe. Conventional electrocautery techniques used in the resection of colon tissue cause a relatively high degree of collateral damage to the healthy tissues bordering the target sites. Ultrafast infrared lasers offer significantly improved localisation in the ablation of such biological tissues arising from a plasma-mediated ablation mechanism. This improved localisation is two-fold, with lateral confinement and precise depth control being advantageous in minimising thermal necrosis and avoiding bowel perforation respectively. Various laser scanning strategies and optical elements have been investigated, with the intent to exploit the inherent advantages offered from applying photonics to these procedures. Evaluation of the corresponding ablation characteristics was carried out using three-dimensional optical profilometry and histological analysis. If adopted in operating theatres, surgeons could benefit from more control when carrying out resection of neoplasia in the mucosal or submucosal layers of colon tissue, compared to previous electrocautery methods.

111 Background: High tumor mRNA levels of the EGFR ligands, AREG and EREG are associated with anti-EGFR agent response in patients (pts) with RAS-wt mCRC, regardless of tumor location. However, ligand RNA assays have not been adopted into routine clinical practice due to issues with analytical precision and practicality. Here we test whether AREG and EREG expression assessed by IHC can predict benefit from Pan. Methods: A retrospective biomarker study within the PICCOLO trial (NCT00389870; irinotecan [Ir] ± Pan in fluoropyrimidine-resistant RAS-wt mCRC). AREG and EREG positive tumor cells were assessed by IHC in all RAS-wt patients with available tumor tissue. Pathologists annotated tumor areas on digital images of glass slides. Artificial intelligence (AI) algorithms calculated the percentage of tumor cells staining positive for AREG and EREG within the tumor areas. More than 50% AREG and/or EREG tumor cell positivity was regarded as high ligand expression. The primary endpoint was progression-free survival (PFS) and secondary endpoints were RECIST response rate (RR) and overall survival (OS). Results: 274 RAS-wt pts had available tumor tissue. High ligand expression (n = 132) was associated with significant PFS benefit from IrPan compared with Ir (8.0 vs 3.2 months; HR 0.54 [0.37-0.79]; p = 0.001); whereas low ligand expression (n = 142) was not (3.4 vs 4.4 months; HR 1.05 [95% CI, 0.74-1.49]; p = 0.78). The ligand-treatment interaction was significant (p = 0.02) and independent of BRAF-mutation status and primary tumor location. Likewise RR was significantly improved in pts with high ligand expression (IrPan vs Ir: 48% vs 6%; risk ratio, 7.8 [2.90-20.69]; p < 0.0001) but not those with low ligand expression (IrPan vs Ir: 25% vs 14%; risk ratio, 1.8 [95% CI, 0.89-3.65]; p = 0.10) (interaction p = 0.01). Lesser effect was seen on OS. Conclusions: IHC assessment of AREG and EREG identified pts who did or did not benefit from Pan, as has been previously demonstrated through mRNA quantification. IHC represents a more practicable technique as it can be provided at the point of care and is associated with shorter turn-around times. AREG and EREG IHC may be of use in routine practice to identify patients who would benefit from anti-EGFR therapy and those for whom alternative treatment strategies should be explored.

Local resection of early stage tumors in the large bowel via colonoscopy has been a widely accepted surgical modality for colon neoplasm treatment. The conventional electrocautery techniques used for the resection of neoplasia in the mucosal or submucosal layer of colon tissue has been shown to create obvious thermal necrosis to adjacent healthy tissues and lacks accuracy in resection. Ultrafast picosecond (ps) laser ablation using a wavelength of 1030 or 515 nm is a promising surgical tool to overcome the limitations seen with conventional surgical techniques. The purpose of this initial study is to analyze the depth of ablation or the extent of coagulation deployed by the laser as a function of pulse energy and fluence in an ex-vivo porcine model. Precise control of the depth of tissue removal is of paramount importance for bowel surgery where bowel perforation can lead to morbidity or mortality. Thus we investigate the regimes that are optimal for tissue resection and coagulation through plasma mediated ablation of healthy colon tissue. The ablated tissue samples were analyzed by standard histologic methods and a three dimensional optical profilometer technique. We demonstrate that ultrafast laser resection of colonic tissue can minimize the region of collateral thermal damage (

Foveal hypoplasia and optic nerve misrouting are developmental defects of the visual pathway and only co-occur in connection with albinism; to date, they have only been associated with defects in the melanin-biosynthesis pathway. Here, we report that these defects can occur independently of albinism in people with recessive mutations in the putative glutamine transporter gene SLC38A8. Nine different mutations were identified in seven Asian and European families. Using morpholino-mediated ablation of Slc38a8 in medaka fish, we confirmed that pigmentation is unaffected by loss of SLC38A8. Furthermore, by undertaking an association study with SNPs at the SLC38A8 locus, we showed that common variants within this gene modestly affect foveal thickness in the general population. This study reveals a melanin-independent component underpinning the development of the visual pathway that requires a functional role for SLC38A8.

Three-dimensional (3D) reconstruction and examination of tissue at microscopic resolution have significant potential to enhance the study of both normal and disease processes, particularly those involving structural changes or those in which the spatial relationship of disease features is important. Although other methods exist for studying tissue in 3D, using conventional histopathological features has significant advantages because it allows for conventional histopathological staining and interpretation techniques. Until now, its use has not been routine in research because of the technical difficulty in constructing 3D tissue models. We describe a novel system for 3D histological reconstruction, integrating whole-slide imaging (virtual slides), image serving, registration, and visualization into one user-friendly package. It produces high-resolution 3D reconstructions with minimal user interaction and can be used in a histopathological laboratory without input from computing specialists. It uses a novel method for slice-to-slice image registration using automatic registration algorithms custom designed for both virtual slides and histopathological images. This system has been applied to >300 separate 3D volumes from eight different tissue types, using a total of 5500 virtual slides comprising 1.45 TB of primary image data. Qualitative and quantitative metrics for the accuracy of 3D reconstruction are provided, with measured registration accuracy approaching 120 μm for a 1-cm piece of tissue. Both 3D tissue volumes and generated 3D models are presented for four demonstrator cases.

Infantile myopathies with diaphragmatic paralysis are genetically heterogeneous, and clinical symptoms do not assist in differentiating between them. We used phased haplotype analysis with subsequent targeted exome sequencing to identify MEGF10 mutations in a previously unidentified type of infantile myopathy with diaphragmatic weakness, areflexia, respiratory distress and dysphagia. MEGF10 is highly expressed in activated satellite cells and regulates their proliferation as well as their differentiation and fusion into multinucleated myofibers, which are greatly reduced in muscle from individuals with early onset myopathy, areflexia, respiratory distress and dysphagia.

Anterior segment dysgenesis describes a group of heterogeneous developmental disorders that affect the anterior chamber of the eye and are associated with an increased risk of glaucoma. Here, we report homozygous mutations in peroxidasin (PXDN) in two consanguineous Pakistani families with congenital cataract-microcornea with mild to moderate corneal opacity and in a consanguineous Cambodian family with developmental glaucoma and severe corneal opacification. These results highlight the diverse ocular phenotypes caused by PXDN mutations, which are likely due to differences in genetic background and environmental factors. Peroxidasin is an extracellular matrix-associated protein with peroxidase catalytic activity, and we confirmed localization of the protein to the cornea and lens epithelial layers. Our findings imply that peroxidasin is essential for normal development of the anterior chamber of the eye, where it may have a structural role in supporting cornea and lens architecture as well as an enzymatic role as an antioxidant enzyme in protecting the lens, trabecular meshwork, and cornea against oxidative damage.

Tamoxifen (TAM) remains the adjuvant therapy of choice for pre-menopausal women with ERα-positive breast cancer. Resistance and recurrence remain, however, a major challenge with many women relapsing and subsequently dying. The insulin-like growth factor (IGF) axis is involved in breast cancer pathogenesis and progression to endocrine resistant disease, but there is very little data on the expression and potential role of IGF-binding proteins (IGFBP) during acquisition of the resistant phenotype. The aim of this study was to determine the expression and functional role of IGFBP-2 and -5 in the development of TAM resistance (TamR) in vitro and to test retrospectively whether they were predictive of resistance in a tissue microarray of 77 women with primary breast cancers who relapsed on/after endocrine therapy and 193 who did not with long term follow up. Reciprocal expression of IGFBP-2 and IGFBP-5 was observed at both mRNA and protein level in TamR cells. IGFBP-2 expression was increased by 10-fold while IGFBP-5 was decreased by 100-fold, compared to TAM-sensitive control cells. shRNA-mediated silencing of IGFBP-2 in TamR cells restored TAM sensitivity suggesting a causal role for this gene in TamR. While silencing of IGFBP-5 in control cells had no effect on TAM sensitivity, it significantly increased the migratory capacity of these cells. Quantitative image analysis of immunohistochemical data failed, however, to demonstrate an effect of IGFBP2 expression in endocrine-relapsed patients. Likewise, IGFBP-2 and IGFBP-5 expression failed to show any significant associations with survival either in patients relapsing or those not relapsing on/after endocrine therapy. By contrast, in silico mining of a separate published dataset showed that in patients who received endocrine treatment, loss of expression of IGBP-5 was significantly associated with worse survival. Overall these data suggest that co-ordinated and reciprocal alteration in IGFBP-2 and -5 expression may play a role in the acquisition of endocrine resistance.

Objective To investigate qualitative and quantitative differences in the structure of the posterior segment of the eye in 1-day post-hatch and 12-month-old retinal dysplasia and degeneration (rdd) and wild-type chickens. Animal studied Retinal dysplasia and degeneration and wild-type chickens. Procedure Using a commercially available spectral domain optical coherence tomography (OCT) system, 15° horizontal line scans were performed in both eyes of 24 live birds. Qualitative differences in retinal lamination and choroidal structure were investigated, and retinal and choroidal thickness were measured. Results Progressive retinal thinning with loss of outer retinal lamination and changes in the appearance of the choroid were seen in the rdd birds. Mean total retinal thickness was 202 μm (SD 7.8) and 251 μm (SD 8.8) in the rdd and wild-type chicks and 154 μm (SD 18) and 280 μm (SD 10.8) in the adult birds. Much of the difference was the result of loss of outer retinal lamination and thickness in the rdd birds. Mean choroidal thickness was 76 μm (SD 19.6) and 112 μm (SD 36.9) in the rdd and wild-type chicks and 85 μm (SD 23.7) and 228 μm (SD 44.1) in the rdd and wild-type adult birds, respectively. Conclusions Differences in retinal and choroidal structure and thickness between rdd and wild-type birds were evident on spectral domain OCT imaging at 1-day post-hatch and more marked at 1 year. Spectral domain OCT may provide a reliable end point for therapeutic intervention in this animal model of inherited retinal degeneration.

614 Background: Trial data, while valuable, does not reflect the importance of a biomarker or treatment in the general population as trials generally exclude patients (pts) due to age or comorbidities. We used merged large population datasets to validate a linked anonymized stage II CRC pt cohort as representative of the population to identify the prognostic and predictive value of deficient mismatch repair (MMR) and a new biomarker Ga-interacting vesicle-associated protein (GIV). Methods: English national data on stage II CRC pts surgically treated from 2001-2015, n = 92,147, were analyzed for survival and clinicopathological parameters. Anonymized data were then linked to pathology and a subset of 405 unselected pts surgically treated from 1990-2003 at the Leeds Teaching Hospitals NHS Trust. These were investigated for 5 antibodies. 4 identified deficient MMR status and one was a new marker; GIV. Results: Population data vs the cohort of 405 pts showed a median age of 73 vs 74 yrs, M:F 55%:45% vs. 53%:47%. These are older than seen in most clinical trials and more reflective of the general stage II CRC population. The median survivals of 108 vs 92 months are as expected based on relative time periods covered. 374 patients yielded valid MMR status on immunohistochemistry (92%); 17% were dMMR and 83% were proficient. dMMR vs pMMR pts did not differ in age (median age 75 vs 74.5) or distribution of T3 (69% vs 66%), but were more likely to be female (71% vs 42%), sited in the right colon (76% vs 30%) and poorly differentiated (42% vs 8%). 11% of dMMR and 8% of pMMR received adjuvant chemotherapy in this cohort. GIV scoring status was also evaluated (405): 30% (121) were negative, 65% (264) positive and 5% (20) were not scored. Survival and risk scores by MMR and GIV status will be presented. Conclusions: Population based data has been created to validate the representativeness of a stage II biomarker cohort. This approach using large, population based data-sets provides opportunities to understand the generalizability of biomarker cohorts. The creation and expansion of such cohorts will more effectively validate existing and new biomarkers and treatments in real-world populations.

Purpose. To identify the defective gene in the sex-linked, recessively inherited retinal dysplasia and degeneration (rdd) chicken and to search for the human equivalent disease. Methods. Microsatellites from chicken chromosome Z were genotyped in 77 progeny of a carrier male (rdd/+) and an affected female (rdd/W), and candidate genes were sequenced. Retinal cross-sections from rdd and wild-type birds were analyzed by immunohistology. The human orthologous gene was screened in a panel of archival DNAs from 276 patients with retinitis pigmentosa (RP) or Leber congenital amaurosis (LCA) using melting curve analysis and DNA sequencing. Results. The rdd locus was refined to an approximately 3-Mb region on chromosome Z. Sequence analysis identified a C→T change in the mpdz gene that created a premature stop codon (c.1372C→T, p.R458X), which segregated with the disease phenotype. As expected, the full-length mpdz protein was absent in rdd retinas, but in wild-type birds, it localized to the retinal outer limiting membrane, where it may have a role in the interactions between photoreceptors and Muller glia cells. The screen to identify the human equivalent disease found 10 heterozygous variants in the orthologous gene in patients with RP (three missense and two null alleles) and LCA (four missense and one null allele). Conclusions. These findings reveal that MPDZ is essential for normal development of the retina and may have a role in maintaining photoreceptor integrity. The identification of human mutations suggests that MPDZ plays a role in human retinal disease, but the precise nature of this role remains to be determined.

Primary congenital glaucoma (PCG) is an autosomal-recessive condition characterized by high intraocular pressure (IOP), usually within the first year of life, which potentially could lead to optic nerve damage, globe enlargement, and permanent loss of vision. To date, PCG has been linked to three loci: 2p21 (GLC3A), for which the responsible gene is CYP1B1, and 1p36 (GLC3B) and 14q24 (GLC3C), for which the genes remain to be identified. Here we report that null mutations in LTBP2 cause PCG in four consanguineous families from Pakistan and in patients of Gypsy ethnicity. LTBP2 maps to chromosome 14q24.3 but is around 1.3 Mb proximal to the documented GLC3C locus. Therefore, it remains to be determined whether LTBP2 is the GLC3C gene or whether a second adjacent gene is also implicated in PCG. LTBP2 is the largest member of the latent transforming growth factor (TGF)-beta binding protein family, which are extracellular matrix proteins with multidomain structure. It has homology to fibrillins and may have roles in cell adhesion and as a structural component of microfibrils. We confirmed localization of LTBP2 in the anterior segment of the eye, at the ciliary body, and particularly the ciliary process. These findings reveal that LTBP2 is essential for normal development of the anterior chamber of the eye, where it may have a structural role in maintaining ciliary muscle tone.