A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment.

BACKGROUND: In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available 'Immunoscore®' exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. METHODS: A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. RESULTS: Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I-II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. CONCLUSION: A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.

Prevalence of PD-L1 expression is associated with EMAST, density of peritumoral T-cells and recurrence-free survival in operable non-metastatic colorectal cancer.

INTRODUCTION: Microsatellite instability (MSI) predict response to anti-PD1 immunotherapy in colorectal cancer (CRC). CRCs with MSI have higher infiltration of immune cells related to a better survival. Elevated Microsatellite Alterations at Tetranucleotides (EMAST) is a form of MSI but its association with PD-L1 expression and immune-cell infiltration is not known. METHODS: A consecutive, observational cohort of patients undergoing surgery for CRC. EMAST and clinicopathological characteristics were investigated against PD-L1, as well as CD3 and CD8 expression in the invasive margin or tumour centre (Immunoscore). Difference in survival between groups was assessed by log rank test. RESULTS: A total of 149 stage I-III CRCs patients, with a median follow up of 60.1 months. Patients with PD-L1+ tumours (7%) were older (median 79 vs 71 years, p = 0.045) and had EMAST+ cancers (OR 10.7, 95% CI 2.2-51.4, p = 0.001). Recurrence-free survival was longer in cancers with PD-L1+ immune cells (HR 0.35, 95% CI 0.16-0.76, p = 0.008, independent of EMAST) and high Immunoscore (HR 0.10, 95% CI 0.01-0.72, p = 0.022). Patients expressing PD-L1 in immune cells had longer disease-specific survival (HR 0.28, 95% CI 0.10-0.77, p = 0.014). CONCLUSIONS: Higher Immunoscore (CD3/CD8 cells) and expression of tumour PD-L1 is found in CRCs with EMAST. Lymphocytic infiltrate and peritumoral PD-L1 expression have prognostic value in CRC.

Elevated Microsatellite Alterations at Selected Tetranucleotides (EMAST) in Colorectal Cancer is Associated with an Elderly, Frail Phenotype and Improved Recurrence-Free Survival.

BACKGROUND: Elevated microsatellite alterations at selected tetranucleotides (EMAST) is a poorly investigated form of microsatellite instability (MSI) in colorectal cancer (CRC). OBJECTIVE: The aim of this study was to investigate the clinicopathological features of EMAST in CRC and its relation to outcome. METHODS: A population-based, consecutive cohort of surgically treated stage I-III CRC patients investigated for high-frequency MSI (MSI-H) and EMAST. Clinicopathological differences were reported as odds ratios (OR) and survival was presented as hazard ratios (HR) with 95% confidence intervals (CIs). RESULTS: Of 161 patients included, 25% were aged > 79 years. There was a large overlap in the prevalence of EMAST (31.7%) and MSI-H (27.3%) [82.4% of EMAST were also MSI-H]. EMAST had the highest prevalence in the proximal colon (OR 15.9, 95% CI 5.6-45.1; p < 0.001) and in women (OR 4.1, 95% CI 1.9-8.6; p < 0.001), and were poorly differentiated (OR 5.0, 95% CI 2.3-10.7; p < 0.001). Compared with EMAST-negative patients, EMAST-positive patients were older (median age 77 vs. 69 years; p < 0.001), leaner (median weight 67.5 vs. 77 kg; p = 0.001), had significantly higher rates of hypoalbuminemia (24% vs. 6%; OR 2.3, 95% CI 1.5-3.6; p = 0.002) and anemia (45% vs. 20%; OR 3.3, 95% CI 1.6-6.8; p = 0.001), and had elevated preoperative C-reactive protein (CRP) levels (51% vs. 34%; OR 1.9, 95% CI 1.0-3.9; p = 0.046). Improved recurrence-free survival was found in both MSI-H and EMAST subtypes. In multivariable analysis, node status (pN +), together with elevated CRP and MSI-positive, were the strongest prognostic factors for recurrence-free survival. CONCLUSIONS: EMAST in CRC is associated with an older, leaner, and frailer phenotype with a lower risk of recurrence. The relevance of, and putative mechanisms to, EMAST warrants further investigation.

The prognostic yield of biomarkers harvested in chemotherapy-naive stage II colon cancer: can we separate the wheat from the chaff?

The TNM-system fails to accurately predict disease recurrence in a considerate number of patients. While node-negative (stage II) colon cancer is considered to have an overall good prognosis, the 5-year cancer-specific survival is reported at 81-83% in patients who did not have adjuvant chemotherapy. Thus, reliance on node-status alone has lead to under-treatment in a subgroup of stage II patients with an unfavorable prognosis. The search for new and better prognosticators in stage II colon cancer has suggested several proposed biomarkers of better prognostication and prediction. However, few such biomarkers have reached widespread clinical utility. For the clinician swimming in the sea of emerging biomarkers, it may be hard to recognize the true floating aid from the surrounding debris in the search for more precise decision-making. Proposed markers include microsatellite instability (MSI), KRAS mutations and BRAF mutations, but a number of gene panels and consensus molecular subtypes are proposed for clinical prediction and prognostication as well. While several studies suggest such biomarkers or panels to have a prognostic role in subgroups of patients, a number of studies are reported in heterogeneous groups with in part discordant findings, which again distorts the predictive and prognostic ability of each marker. Lack of homogeneous cohorts, underpowered studies in strict subgroups and challenges in analytical and clinical validity may hamper the progress towards widespread clinical utility. The harvest of prognostic biomarkers in colon cancer has yielded a huge number of candidates for which it is now time to separate the wheat from the chaff.

Assessment of clinically related outcomes and biomarker analysis for translational integration in colorectal cancer (ACROBATICC): study protocol for a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastasis.

BACKGROUND: More accurate predictive and prognostic biomarkers for patients with colorectal cancer (CRC) primaries or colorectal liver metastasis (CLM) are needed. Outside clinical trials, the translational integration of emerging pathways and novel techniques should facilitate exploration of biomarkers for improved staging and prognosis. METHODS: An observational study exploring predictive and prognostic biomarkers in a population-based, consecutive cohort of surgically treated colorectal cancers and resected colorectal liver metastases. Long-term outcomes will be cancer-specific survival, recurrence-free survival and overall survival at 5 years from diagnosis. Beyond routine clinicopathological and anthropometric characteristics and laboratory and biochemistry results, the project allows for additional blood samples and fresh-frozen tumour and normal tissue for investigation of circulating tumour cells (CTCs) and novel biomarkers (e.g. immune cells, microRNAs etc.). Tumour specimens will be investigated by immunohistochemistry in full slides. Extracted DNA/RNA will be analysed for genomic markers using specific PCR techniques and next-generation sequencing (NGS) panels. Flow cytometry will be used to characterise biomarkers in blood. Collaboration is open and welcomed, with particular interest in mutual opportunities for validation studies. STATUS AND PERSPECTIVES: The project is ongoing and recruiting at an expected rate of 120-150 patients per year, since January 2013. A project on circulating tumour cells (CTCs) has commenced, with analysis being prepared. Investigating molecular classes beyond the TNM staging is under way, including characteristics of microsatellite instability (MSI) and elevated microsatellite alterations in selected tetranucleotides (EMAST). Hot spot panels for known mutations in CRC are being investigated using NGS. Immune-cell characteristics are being performed by IHC and flow cytometry in tumour and peripheral blood samples. The project has ethical approval (REK Helse Vest, #2012/742), is financially supported with a Ph.D.-Grant (EMAST project; Folke Hermansen Cancer Fund) and a CTC-project (Norwegian Research Council; O. Nordgård). The ACROBATICC clinical and molecular biobank repository will serve as a long-term source for novel exploratory analysis and invite collaborators for mutual validation of promising biomarker results. The project aims to generate results that can help better discern prognostic groups in stage II/III cancers; explore prognostic and predictive biomarkers, and help detail the biology of colorectal liver metastasis for better patient selection and tailored treatment. The project is registered at http://www.ClinicalTrials.gov NCT01762813.

Validity and reliability of an alternative method for measuring power output during six-second all-out cycling.

In a laboratory setting where both a mechanically-braked cycling ergometer and a motion analysis (MA) system are available, flywheel angular displacement can be estimated by using MA. The purpose of this investigation was to assess the validity and reliability of a MA method for measuring maximal power output (Pmax) in comparison with a force transducer (FT) method. Eight males and eight females undertook three identical sessions, separated by 4 to 6 days; the first being a familiarization session. Individuals performed three 6-second sprints against 50% of the maximal resistance to complete two pedal revolutions with a 3-minute rest between trials. Power was determined independently using both MA and FT analyses. Validity: MA recorded significantly higher Pmax than FT (P < .05). Bland-Altman plots showed that there was a systematic bias in the difference between the measures of the two systems. This difference increased as power increased. Repeatability: Intraclass correlation coefficients were on average 0.90 ± 0.05 in males and 0.85 ± 0.08 in females. Measuring Pmax by MA, therefore, is as appropriate for use in exercise physiology research as Pmax measured by FT, provided that a bias between these measurements methods is allowed for.

Retrospective analysis on the outcomes of contact lens-associated keratitis in a tertiary centre: an evidence-based management protocol to optimise resource allocation.

BACKGROUND/AIMS: Contact lens-associated keratitis (CLAK) is a common sight-threatening complication of contact lens use. Current management protocols in the UK are based on historical practice and necessitate a review for every patient within 48 hours regardless of severity, increasing the treatment burden on a resource-limited healthcare service. Our study aims to identify the different risk factors associated with CLAK, categorise CLAK using a novel grading system and recommend modifications to current management protocols based on the outcomes in the individual subgroups. METHODS: The retrospective cohort study identified 161 eyes from 153 patients with CLAK from the electronic patient records of a tertiary eye centre between 1 July 2021 and 28 February 2022. Patients were categorised based on epithelial defect size (grade 1: <1.0 mm, grade 2: 1.0-2.0 mm, grade 3: >2.0 mm) and their risk factors, clinical features, treatments and outcomes were analysed. RESULTS: The most significant risk factors for CLAK include extended-wear contact lens, poor hygiene and prolonged duration of wear. Grades 1 and 2 CLAKs have excellent outcomes following an empirical treatment regime with topical moxifloxacin with 96% discharged within 48 hours and 94.1% discharged in 2 weeks, respectively. Grade 3 CLAKs require prolonged average duration of treatment. CONCLUSION: We recommend typical grade 1 and 2 CLAKs can be discharged with empirical fluoroquinolone treatment. Grade 3 and all CLAKs with atypical features require monitoring for resolution, further diagnostics or treatment. We provide an evidence-based approach to reduce unnecessary patient visits and optimise resource allocation in an urban setting.

Backbone 1H, 13C and 15N resonance assignment of the ubiquitin specific protease 7 catalytic domain (residues 208-554) in complex with a small molecule ligand.

The backbone 1H, 13C and 15N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1H-15N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2H, 13C, 15N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography.

Do blood growth factors offer additional benefit in refractory lateral epicondylitis? A prospective, randomized pilot trial of dry needling as a stand-alone procedure versus dry needling and autologous conditioned plasma.

OBJECTIVE: To evaluate whether autologous conditioned plasma offers any therapeutic advantage over ultrasound-guided dry needling as a stand-alone procedure in the treatment of refractory lateral epicondylitis. MATERIALS AND METHODS: Prospective, randomized pilot study of 28 patients (11 men, 17 women, mean age, 49.1  years) with refractory lateral epicondylitis (mean symptom duration, 19.1 months) who underwent either dry needling (n = 13) or dry needling combined with autologous conditioned plasma (ACP) injection (n = 15). Each patient received two separate injections (0 weeks and 1 month) and analysis of visual analogue pain scores (VAS) and Nirschl scores were performed pre-procedure, at 2 months and final evaluation at 6 months. Successful treatment was defined as more than a 25 % reduction in pain scores without re-intervention. Data was analyzed using the Mann-Whitney test and local research ethics committee approval was obtained. RESULTS: At 2 months, the mean VAS improvement was 0.85 (12.3 %) in the dry needling group compared to 2.19 (27.1 %) in the ACP group (p = 0.76) and there was a 5.83-point and 20.3-point Nirschl score improvement respectively (p = 0.72). At the final follow-up of 6 months, the mean VAS improvement was 2.37 (34 %) in the dry needling group compared to 3.92 (48.5 %) in the ACP group (p = 0.74) and there was a 22.5-point and 40-point Nirschl score improvement, respectively (p = 0.82). CONCLUSIONS: There is a trend to greater clinical improvement in the short term for patients treated with additional ACP, however no significant difference between the two treatment groups was demonstrated at each follow-up interval. A larger, multicenter, randomized controlled trial is required to corroborate the results of this pilot study.

Cost and clinical effectiveness of MRI in occult scaphoid fractures: a randomised controlled trial.

BACKGROUND: Clinical and radiographic diagnoses of scaphoid fractures are often challenging at the time of injury. Patients are therefore usually reassessed which has cost implications. Various investigations exist but MRI has been suggested as effective in diagnosing these injuries early. AIM: To determine whether early MRI in suspected occult scaphoid fractures is more clinically and cost effective than conventional management with immobilisation and reassessment. METHODS: All patients presenting to the Emergency Department at a district general hospital with a suspected occult scaphoid fracture were randomised into two groups, MRI (early scan of the wrist, discharged if no injury) and control (reassessment in clinic). RESULTS: 84 patients were randomised into MRI (45) and control (39) groups. There were no baseline differences apart from greater dominant hand injuries in the MRI group (62% (26) vs 36% (14), p=0.02). There were three (6.7%) scaphoid fractures in the MRI group and four (10.3%) in the control group (p=0.7). More fractures (15.6% (7) vs 5.1% (2), p=0.9) and other injuries were detected in the MRI group who had fewer mean clinic appointments (1.1 ± 0.5 vs 2.3 ± 0.8, p=0.001) and radiographs (1.2 ± 0.8 vs 1.7 ± 1.1, p=0.03). Mean management costs were £504.13 (MRI) and £532.87 (control) (p=0.9). The MRI group had better pain and satisfaction scores (not significant) with comparable time off work and sporting activities. CONCLUSION: Early MRI in occult scaphoid fractures is marginally cost saving compared with conventional management and may reduce potentially large societal costs of unnecessary immobilisation. It enables early detection and appropriate treatment of scaphoid and other injuries.

Dynamic ultrasound of the subacromial-subdeltoid bursa in patients with shoulder impingement: a comparison with normal volunteers.

OBJECTIVE: The purpose of the study was to determine if the thickness of the subacromial-subdeltoid (SASD) bursa during dynamic ultrasound and on static views differs between patients with shoulder impingement syndrome and healthy volunteers. MATERIALS AND METHODS: Twenty-two patients with a clinical diagnosis of shoulder impingement syndrome and 23 healthy volunteers were recruited. A subset of patients showing an immediate symptom response following intrabursal injection was identified as "injection responders". Ultrasound of the shoulder was performed on all participants using three standard static views and two dynamic views (before and after arm abduction). The thickness of both the intrabursal fluid and the superficial peribursal fat was measured on all views. The bursal thickness measurements in the two groups were compared using a t test for significance. RESULTS: The mean increase in SASD bursal fluid thickness following arm abduction was not statistically different among all patients (0.39 ± 0.41 mm) and controls (0.35 ± 0.32 mm), p = 0.72. The same was true comparing injection responders (0.46 ± 0.49 mm) with controls, p = 0.41. On static views, greater bursal fluid thickness was found in patients (1.01 ± 0.48 mm) compared with controls (0.67 ± 0.32 mm) when using the short axis view of the supraspinatus, p = 0.006. No statistically significant difference was found between injection responders and controls when measuring peribursal fat thickness on any view. CONCLUSIONS: Gathering of the SASD bursa demonstrated during dynamic ultrasound does not necessarily indicate painful impingement of the bursa as it is found to a similar degree in patients with a clinical diagnosis of impingement and healthy volunteers.

Early magnetic resonance imaging in acute knee injury: a cost analysis.

PURPOSE: Acute knee injury is common, and MRI is often only used when non-operative management fails because of limited availability. We investigated whether early MRI in acute knee injury is more clinically and cost-effective compared to conventional physiotherapy and reassessment. METHODS: All patients with acute indirect soft tissue knee injury referred to fracture clinic were approached. Recruited patients were randomised to either the MRI group: early MRI within 2 weeks or the control group: conventional management with physiotherapy. Patients were assessed in clinic initially, at 2 weeks and 3 months post-injury. Management costs were calculated for all patients until surgical treatment or discharge. RESULTS: Forty-six patients were recruited: 23 in the MRI and 23 in the control group. Male sex and mean age were similar in the two groups. The total management cost of the MRI group was £16,127 and control group was £16,170, with a similar mean cost per patient (NS). The MRI group had less mean physiotherapy (2.5 ± 1.9 vs. 5.1 ± 3.5, p < 0.01) and outpatient appointments (NS). Median time to surgery and time off work was less in the MRI group (NS). The MRI group had less pain (p < 0.05), less activity limitation (p = 0.04) and better satisfaction (p = 0.04). CONCLUSIONS: Early MRI in acute knee injury facilitates faster diagnosis and management of internal derangement at a cost comparable to conventional treatment. Moreover, patients had significantly less time off work with improved pain, activity limitation and satisfaction scores. LEVEL OF EVIDENCE: II.

The Co-Creation of a Patient Information Leaflet for Patients With the Rare Eye Infection Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a rare but severe eye disease. A research engagement event, "The Cornea Day," in London, UK in 2013, identified the lack of credible information about AK and a need for practical day to day management strategies. Experiences of 15 AK patients attending The Cornea Day were distilled into a survey that was administered to a wider group of 76 patients, carers, researchers, and clinicians. A Patient Information Leaflet was cocreated and then represented to additional patients for final modification. The AK Patient Leaflet (revised 2019) is available in several languages and used globally.

Evaluation and management of a spontaneous corneal rupture secondary to pellucid marginal degeneration, using swept-source anterior segment optical coherence tomography.

We describe a case of bilateral spontaneous corneal perforation secondary to pellucid marginal degeneration and present the associated swept-source anterior segment optical coherence tomography (SS-ASOCT) findings and management principles used. A 47-year-old woman presented with ocular pain, redness, foreign body sensation and clear discharge in the right eye in 2017 and with very similar symptoms in 2019 in the left eye. Clinically she had a corneal perforation at the inferior cornea with associated loss of anterior chamber volume. Corneal topography demonstrated peripheral thinning and steepening in the contralateral eye. ASOCT images revealed full-thickness perforation, iridocorneal touch and iris stranding. The patient was managed with a combination of contact bandaging and corneal gluing. SS-ASOCT is a useful adjunctive tool in the clinical assessment and evaluation of spontaneous corneal perforation. Alongside the clinical evaluation, it can be used to monitor the clinical response.

The Impact of COVID-19 on Acute and Elective Corneal Surgery at Moorfields Eye Hospital London.

PURPOSE: Moorfields Eye Hospital sits as a major tertiary centre for ophthalmic care in the United Kingdom and became a central hub to provide safe and effective ophthalmic care across London and surrounding regions during the COVID-19 pandemic. We explore the impact on both the acute and elective corneal services during the first wave of this pandemic. METHODS: A retrospective review of the proportion of corneal transplants and anterior segment trauma repairs was performed during the period of March 23rd to July 1st 2020 compared with an identical period in 2019. Data were acquired from our in-house electronic patient records. RESULTS: A 92% reduction in corneal elective work was observed during the lockdown period compared with an identical period in 2019, with only 10 elective cases in total being performed. In addition, 91 corneal cross-linking and 76 therapeutic lasers were cancelled. There were 15 cases of primary repair for anterior segment trauma compared with 6 cases pre-COVID-19. A similar scenario occurs with removal of foreign body (4 cases during COVID-19 period versus no cases during pre-COVID-19 era) and with traumatic lens aspirations (6 cases during COVID-19 compared with 2 pre-COVID-19). Interestingly, a statistical difference (p=0.03) was found in the time interval from presentation of symptoms to emergency corneal surgery. During the COVID-19 period, a delay of 1.5 days ± 2.29 (range 0-10 days) occurred compared with 0.8 days ± 1.54 (range 0-6 days) pre-COVID-19. CONCLUSION: Stringent risk stratification reduced elective corneal surgery capacity during the lockdown thereby preserving social distancing requirements. However, an apparent increase in emergency corneal surgery seen is likely attributed to centralisation of ophthalmic services during the pandemic crisis, alongside increased domestic injuries. Despite the challenges posed, successful delivery of corneal surgery occurred whilst helping to identify lessons in preparations for future pandemics and current inefficiencies in healthcare delivery.

Phenotypic plasticity underlies local invasion and distant metastasis in colon cancer.

Phenotypic plasticity represents the most relevant hallmark of the carcinoma cell as it bestows it with the capacity of transiently altering its morphological and functional features while en route to the metastatic site. However, the study of phenotypic plasticity is hindered by the rarity of these events within primary lesions and by the lack of experimental models. Here, we identified a subpopulation of phenotypic plastic colon cancer cells: EpCAMlo cells are motile, invasive, chemo-resistant, and highly metastatic. EpCAMlo bulk and single-cell RNAseq analysis indicated (1) enhanced Wnt/ß-catenin signaling, (2) a broad spectrum of degrees of epithelial to mesenchymal transition (EMT) activation including hybrid E/M states (partial EMT) with highly plastic features, and (3) high correlation with the CMS4 subtype, accounting for colon cancer cases with poor prognosis and a pronounced stromal component. Of note, a signature of genes specifically expressed in EpCAMlo cancer cells is highly predictive of overall survival in tumors other than CMS4, thus highlighting the relevance of quasi-mesenchymal tumor cells across the spectrum of colon cancers. Enhanced Wnt and the downstream EMT activation represent key events in eliciting phenotypic plasticity along the invasive front of primary colon carcinomas. Distinct sets of epithelial and mesenchymal genes define transcriptional trajectories through which state transitions arise. pEMT cells, often earmarked by the extracellular matrix glycoprotein SPARC together with nuclear ZEB1 and ß-catenin along the invasive front of primary colon carcinomas, are predicted to represent the origin of these (de)differentiation routes through biologically distinct cellular states and to underlie the phenotypic plasticity of colon cancer cells.

Patients' experiences of unilateral spatial neglect between stroke onset and discharge from inpatient rehabilitation: a thematic analysis of qualitative interviews.

Purpose: The purpose of this qualitative study was to explore how stroke patients with unilateral spatial neglect experience; (a) performance in activities of daily living; (b) alterations in bodily perceptions; and (c) personal hopes and expectations, looking at the period between stroke onset and discharge from inpatient rehabilitation.Materials and methods: We conducted individual semi-structured interviews with 7 (5 men, 2 women, mean age 69 years) consecutively sampled participants. All interviews were transcribed verbatim and analyzed using thematic analysis.Findings: Participants' experiences were captured in three over-arching themes: "unawareness of neglect," "emergent awareness for neglect-related difficulties," and "comparing the new life with the old one." Findings showed that participants progressed from initial unawareness to emergent awareness for their neglect-related difficulties over the course of rehabilitation. Comparing their current life situation with the one before their stroke triggered feelings of uncertainty and regret, with associated decreased pleasure in meaningful activities.Conclusions: This study informs health professionals regarding personal experiences of orientation in and reorganization of life of stroke patients with unilateral spatial neglect. The findings highlight that being aware of neglect-related deficits is a prerequisite for using coping strategies and incorporating them in daily life. Possible therapeutic strategies that fit the current stage of recovery and level of awareness are discussed.IMPLICATIONS FOR REHABILITATIONSymptoms of unilateral spatial neglect and anosognosia are most common after right hemispheric stroke, both being predictors of poorer functional outcome during rehabilitation.Patients' regaining some awareness of their impairments is a prerequisite for successful treatment and for engagement in neglect-specific interventions.Health care providers should carefully examine the "state of awareness" of the patient and adapt their therapeutic approach accordingly.This check should frequently be repeated over the course of rehabilitation, as awareness changes at different paces for each patient.

Elevated Microsatellite Alterations at Selected Tetranucleotides (EMAST) Is Not Attributed to MSH3 Loss in Stage I-III Colon cancer: An Automated, Digitalized Assessment by Immunohistochemistry of Whole Slides and Hot Spots.

INTRODUCTION: EMAST is a poorly understood form of microsatellite instability (MSI) in colorectal cancer (CRC) for which loss of MSH3 has been proposed as the underlying mechanism, based on experimental studies. We aimed to evaluate whether MSH3 loss is associated with EMAST in CRC. METHODS: A consecutive cohort of patients with stage I-III CRC. Digital image analysis using heatmap-derived hot spots investigated MSH3 expression by immunohistochemistry. Fragment analysis of multiplex PCR was used to assess MSI and EMAST, and results cross-examined with MSH3 protein expression. RESULTS: Of 152 patients, EMAST was found in 50 (33%) and exclusively in the colon. Most EMAST-positive cancers had instability at all 5 markers, and EMAST overlapped with MSI-H in 42/50 cases (84%). The most frequently altered tetranucleotide markers were D8S321 (38.2% of tumors) and D20S82 (34.4%). Subjective evaluation of MSH3 expression by IHC in tumor found ≤10% negative tumor cells in all samples, most being ≤5% negative. Digital analysis improved the detection but showed a similar spread of MSH3 loss (range 0.1-15.7%, mean 2.2%). Hotspot MSH3 negativity ranged between 0.1 to 95.0%, (mean 8.6%) with significant correlation with the whole slide analysis (Spearman's rho=0.677 P<.001). Loss of MSH3 expression did not correlate with EMAST. CONCLUSIONS: In a well-defined cohort of patients with CRC, loss of MSH3 was not associated with EMAST. Further investigation into the mechanisms leading to EMAST in CRC is needed.