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Purpose: To assess the performance of a perimetric strategy using structure-function predictions from a deep learning (DL) model., Methods: Visual field test-retest data from 146 eyes (75 patients) with glaucoma with (median [5th-95th percentile]) 10 [7, 10] tests per eye were used. Structure-function predictions were generated with a previously described DL model using cicumpapillary optical coherence tomography (OCT) scans. Structurally informed prior distributions were built grouping the observed measured sensitivities for each predicted value and recalculated for each subject with a leave-one-out approach. A zippy estimation by sequential testing (ZEST) strategy was used for the simulations (1000 per eye). Ground-truth sensitivities for each eye were the medians of the test-retest values. Two variations of ZEST were compared in terms of speed (average total number of presentations [NP] per eye) and accuracy (average mean absolute error [MAE] per eye), using either a combination of normal and abnormal thresholds (ZEST) or the calculated structural distributions (S-ZEST) as prior information. Two additional versions of these strategies employing spatial correlations were tested., Results: S-ZEST was significantly faster, with a mean average NP of 213.87 (SD = 28.18), than ZEST, with a mean average NP of 255.65 (SD = 50.27) (P < 0.001). The average MAE was smaller for S-ZEST (1.98; SD = 2.37) than ZEST (2.43; SD = 2.69) (P < 0.001). Spatial correlations further improved both strategies (P < 0.001), but the differences between ZEST and S-ZEST remained significant (P < 0.001)., Conclusions: DL structure-function predictions can significantly improve perimetric tests., Translational Relevance: DL structure-function predictions from clinically available OCT scans can improve perimetry in glaucoma patients.

Introduction: The pressure of the COVID-19 pandemic on healthcare systems led to limited roles of infectious diseases services, increased rates of irrational use of antimicrobials, and incidence of infections by multidrug-resistant microorganisms. The aim of the present study is to evaluate the incidence of antimicrobial resistance and the management of bloodstream infections before and during the COVID-19 pandemic at the University General Hospital of Alexandroupolis (Greece)., Materials and Methods: This is a retrospective study conducted from January 2018 to December 2022. Data were collected from the University Microbiology Laboratory per semester regarding the isolated strains of Gram-positive and -negative bacteria in blood cultures and respiratory samples in hospitalized patients in medical and surgical wards and in the intensive care unit (ICU). Additionally, bloodstream infections with requested infectious disease consultations were reported (n = 400), determining whether these were carried out via telephone contact or at the patient's bedside. Demographic data, comorbidities, focus of infection, antimicrobial regimen, duration of treatment, length of hospitalization, and clinical outcome were analyzed., Results: A total of 4569 strains of Gram-positive and -negative bacteria were isolated. An increasing trend was reported compared to the pre-pandemic period in the incidence of resistant Gram-negative bacteria, particularly in ICUs. Prior antimicrobial use and the rate of hospital-acquired infections were increased significantly during the pandemic. In the pre-pandemic period 2018-2019, a total of 246 infectious disease consultations were carried out, while during the period 2020-2022, the number was 154, with the percentage of telephone consultations 15% and 76%, respectively. Detection of the source of infection and timely administration of appropriate antimicrobial agents were more frequently recorded before the pandemic, and 28-day mortality was significantly reduced in cases with bedside consultations., Conclusion: The empowering of infectious disease surveillance programs and committees, rational use of antimicrobials agents, and bedside infectious disease consultations are vital in order to reduce the impact of infections caused by multidrug-resistant strains.

Purpose: To develop and validate a deep learning method of predicting visual function from spectral domain optical coherence tomography (SD-OCT)-derived retinal nerve fiber layer thickness (RNFLT) measurements and corresponding SD-OCT images., Design: Development and evaluation of diagnostic technology., Methods: Two deep learning ensemble models to predict pointwise VF sensitivity from SD-OCT images (model 1: RNFLT profile only; model 2: RNFLT profile plus SD-OCT image) and 2 reference models were developed. All models were tested in an independent test-retest data set comprising 2181 SD-OCT/VF pairs; the median of ∼10 VFs per eye was taken as the best available estimate (BAE) of the true VF. The performance of single VFs predicting the BAE VF was also evaluated. The training data set comprised 954 eyes of 220 healthy and 332 glaucomatous participants, and the test data set, 144 eyes of 72 glaucomatous participants. The main outcome measures included the pointwise prediction mean error (ME), mean absolute error (MAE), and correlation of predictions with the BAE VF sensitivity., Results: The median mean deviation was -4.17 dB (-14.22 to 0.88). Model 2 had excellent accuracy (ME 0.5 dB, SD 0.8) and overall performance (MAE 2.3 dB, SD 3.1), and significantly (paired t test) outperformed the other methods. For single VFs predicting the BAE VF, the pointwise MAE was 1.5 dB (SD 0.7). The association between SD-OCT and single VF predictions of the BAE pointwise VF sensitivities was R 2  = 0.78 and R 2  = 0.88, respectively., Conclusions: Our method outperformed standard statistical and deep learning approaches. Predictions of BAEs from OCT images approached the accuracy of single real VF estimates of the BAE., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The oxidation processes of white wines can occur during storage and commercialization due to several factors, and these can negatively affect the color, aroma, and quality of the wine. Wineries should have faster and simpler methods that provide valuable information on oxidation stability of wines and allow fast decision-making procedures, able to trigger suitable technological interventions. Using a portable prototype instrument for light irradiations at different wavelengths and times was considered and evaluated on sensorial, spectrophotometric, and colorimetric parameters of white wines. The sensorial analysis revealed that white and light blue were the most significant, after only 1 h of irradiation. The experimental results showed that hydrogen peroxide could enhance the effect of light treatment, allowing a contemporary evaluation of the oxidation stability of wine against light and chemical stresses. As expected, a good correlation (R 2 > 0.89) between optical density at 420 nm and b* parameter was highlighted. The synergic effect of light and H 2 O 2 was also studied on the hydrolyzable and condensed tannins' additions to white wine. The proposed methodology could be used to evaluate the oxidative stability of white wines, but also to evaluate the effect of some oenological adjuvants on wine stability.

Purpose: To establish whether deep learning methods are able to improve the signal-to-noise ratio of time-domain (TD) OCT images to approach that of spectral-domain (SD) OCT images., Design: Method agreement study and progression detection in a randomized, double-masked, placebo-controlled, multicenter trial for open-angle glaucoma (OAG), the United Kingdom Glaucoma Treatment Study (UKGTS)., Participants: The training and validation cohort comprised 77 stable OAG participants with TD OCT and SD OCT imaging at up to 11 visits within 3 months. The testing cohort comprised 284 newly diagnosed OAG patients with TD OCT images from a cohort of 516 recruited at 10 United Kingdom centers between 2007 and 2010., Methods: An ensemble of generative adversarial networks (GANs) was trained on TD OCT and SD OCT image pairs from the training dataset and applied to TD OCT images from the testing dataset. Time-domain OCT images were converted to synthesized SD OCT images and segmented via Bayesian fusion on the output of the GANs., Main Outcome Measures: Bland-Altman analysis assessed agreement between TD OCT and synthesized SD OCT average retinal nerve fiber layer thickness (RNFLT) measurements and the SD OCT RNFLT. Analysis of the distribution of the rates of RNFLT change in TD OCT and synthesized SD OCT in the two treatment arms of the UKGTS was compared. A Cox model for predictors of time-to-incident visual field (VF) progression was computed with the TD OCT and the synthesized SD OCT images., Results: The 95% limits of agreement were between TD OCT and SD OCT were 26.64 to -22.95; between synthesized SD OCT and SD OCT were 8.11 to -6.73; and between SD OCT and SD OCT were 4.16 to -4.04. The mean difference in the rate of RNFLT change between UKGTS treatment and placebo arms with TD OCT was 0.24 (P = 0.11) and with synthesized SD OCT was 0.43 (P = 0.0017). The hazard ratio for RNFLT slope in Cox regression modeling for time to incident VF progression was 1.09 (95% confidence interval [CI], 1.02-1.21; P = 0.035) for TD OCT and 1.24 (95% CI, 1.08-1.39; P = 0.011) for synthesized SD OCT., Conclusions: Image enhancement significantly improved the agreement of TD OCT RNFLT measurements with SD OCT RNFLT measurements. The difference, and its significance, in rates of RNFLT change in the UKGTS treatment arms was enhanced and RNFLT change became a stronger predictor of VF progression., (Copyright © 2020 American Academy of Ophthalmology. All rights reserved.)

Albeit spectral-domain OCT (SDOCT) is now in clinical use for glaucoma management, published clinical trials relied on time-domain OCT (TDOCT) which is characterized by low signal-to-noise ratio, leading to low statistical power. For this reason, such trials require large numbers of patients observed over long intervals and become more costly. We propose a probabilistic ensemble model and a cycle-consistent perceptual loss for improving the statistical power of trials utilizing TDOCT. TDOCT are converted to synthesized SDOCT and segmented via Bayesian fusion of an ensemble of GANs. The final retinal nerve fibre layer segmentation is obtained automatically on an averaged synthesized image using label fusion. We benchmark different networks using i) GAN, ii) Wasserstein GAN (WGAN) (iii) GAN + perceptual loss and iv) WGAN + perceptual loss. For training and validation, an independent dataset is used, while testing is performed on the UK Glaucoma Treatment Study (UKGTS), i.e. a TDOCT-based trial. We quantify the statistical power of the measurements obtained with our method, as compared with those derived from the original TDOCT. The results provide new insights into the UKGTS, showing a significantly better separation between treatment arms, while improving the statistical power of TDOCT on par with visual field measurements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: To develop a method for automated detection and progression analysis of chorioretinal atrophic lesions using the combined information of standard infrared (IR) and autofluorescence (AF) fundus images., Methods: Eighteen eyes (from 16 subjects) with punctate inner choroidopathy were analyzed. Macular IR and blue AF images were acquired in all eyes with a Spectralis HRA+OCT device (Heidelberg Engineering, Heidelberg, Germany). Two clinical experts manually segmented chorioretinal lesions on the AF image. AF images were aligned to the corresponding IR. Two random forest models were trained to classify pixels of lesions, one based on the AF image only, the other based on the aligned IR-AF. The models were validated using a leave-one-out cross-validation and were tested against the manual segmentation to compare their performance. A time series from one eye was identified and used to evaluate the method based on the IR-AF in a case study., Results: The method based on the AF images correctly classified 95% of the pixels (i.e., in vs. out of the lesion) with a Dice's coefficient of 0.80. The method based on the combined IR-AF correctly classified 96% of the pixels with a Dice's coefficient of 0.84., Conclusions: The automated segmentation of chorioretinal lesions using IR and AF shows closer alignment to manual segmentation than the same method based on AF only. Merging information from multimodal images improves the automatic and objective segmentation of chorioretinal lesions even when based on a small dataset., Translational Relevance: Merged information from multimodal images improves segmentation performance of chorioretinal lesions., Competing Interests: Disclosure: G. Ometto, None; G. Montesano, None; S.S. Afgeh, None; G. Lazaridis, None; X. Liu, None; P.A. Keane, None; D.P. Crabb, None; A.K. Denniston, None, (Copyright 2020 The Authors.)

Background: Cabazitaxel is a novel taxane that might be active in breast cancer resistant to first-generation taxanes., Methods: The purpose of the current multicentre phase II trial was to evaluate the activity and safety of cabazitaxel, as second-line treatment, in patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) previously treated with taxanes. The primary endpoint was objective response rate (ORR)., Results: Eighty-four patients were enrolled between October 2012 and November 2016. Taxane resistance to previous treatment was detected in 43 cases. The ORR was 22.6% in the intent-to-treat population, 23.3% in taxane-resistant and 20.5% in taxane-non-resistant cases. At a median follow-up of 39.6 months, the median progression-free survival and overall survival were 3.7 months (95% CI 2.2-4.4) and 15.2 months (95% CI 11.3-19.4), respectively. Regarding toxicity, grade 3-4 neutropenia was reported in 22.6% and febrile neutropenia in 6% of the patients, respectively. Two fatal events (one febrile neutropenia and one sepsis) were reported as being related to study treatment., Conclusions: This phase II trial suggests that cabazitaxel is active as second-line treatment in taxane-pretreated patients with HER2-negative MBC, with manageable toxicity.

Purpose: Neuromodulation, such as vagal nerve stimulation and intestinal electrical stimulation, has been introduced for the treatment of obesity and diabetes. Ideally, neuromodulation should be applied automatically after food intake. The purpose of this study was to develop a method of automatic food intake detection through dynamic analysis of heart rate variability (HRV)., Materials and Methods: Two experiments were conducted: (1) a small sample series with a standard test meal and (2) a large sample series with varying meal size. Electrocardiograms (ECGs) were collected in the fasting and postprandial states. Each ECG was processed to compute the HRV. For each HRV segment, time- and frequency-domain features were derived and used as inputs to train and test an artificial neural network (ANN). The ANN was trained and tested with different cross-validation methods., Results: The highest classification accuracy reached with leave-one-subject-out-leave-one-sample-out cross-validation was 0.93 in experiment 1 and 0.88 in experiment 2. Retraining the ANN on recordings of a subject drastically increased the achieved accuracy for that subject to values of 0.995 and 0.95 in experiments 1 and 2, respectively., Conclusions: Automatic food intake detection by ANNs, using features from the HRV, is feasible and may have a great potential for neuromodulation-based treatments of meal-related disorders.

Introduction: We sought to determine the level of activation of the critical components of the cyclin D1-mediated pathway and to evaluate their prognostic significance across the different molecular subtypes of advanced breast cancer., Patients and Methods: The study population comprised 219 female patients with advanced breast cancer who had been found to have human epidermal growth factor receptor 2 (HER2)-positive disease by local testing and were all treated with trastuzumab-based regimens. For all tumours, central testing for HER2 was performed, and cyclin D1 gene ( CCND1 ) amplification, mRNA and protein expression were assessed by FISH, quantitative real-time-PCR and immunohistochemistry, respectively. Prognostic impact on clinical endpoints was evaluated with Cox regression analyses., Results: After central testing, only 134 (61.2%) of 219 patients were confirmed to have HER2 gene amplification by FISH and/or 3+ HER2 protein expression by immunohistochemistry. After a median follow-up time of 136.0 months (95% CI 123.3 to 148.9), 105 (78.4%) HER2-positive patients and 76 (89.4%) HER2-negative patients had died, while 80% of the former and 87.1% of the latter had experienced a disease relapse. Patients with positive oestrogen receptor/progesterone receptor status presented with higher cyclin D1 mRNA expression. In the HER2-negative subgroup, patients with negative cyclin D1 protein expression were at higher risk of progression (HR= 1.66, 95%CI 1.01 to 2.72, Wald's p=0.045). Among de novo metastatic patients, the risk of progression was higher for patients with non-amplified CCND1 tumours (HR= 2.00, 95% CI 1.03 to 3.90, p=0.041)., Conclusion: Aberrant activation of the cyclin D1-mediated pathway appears to reduce the risk of progression in HER2-negative tumours, but not in HER2-positive ones., Competing Interests: Competing interests: GM: honoraria: Roche, AstraZeneca, Bristol-Myers Squibb; consulting or advisory role: MSD, Pfizer, Roche, Boehringer Ingelheim. GL: honoraria: BMS, MSD, Roche, Amgen, LEO Pharma; consultant, advisory role: Merck. CC: advisory role: Merck, Genesis Pharmaceuticals, Pfizer, Novartis, Roche, AstraZeneca, Bristol-Myers Squibb; honoraria: Roche, Bristol-Myers Squibb; travel: AZ, Sanofi. GP: advisory role: Roche; honoraria: Roche; speaker bureau: Roche; grants: Amgen. AK: consulting fees (eg, advisory boards): AstraZeneca; other, travel: Sanofi-Aventis, Astellas, Genesis, BMS, Amgen. ER: consulting fees (eg, advisory boards): Novartis; other, travel: Genesis Pharmaceuticals, Pfizer, Roche, Bristol-Myers Squibb, Genekor; honoraria: Novartis. PP: advisory role: Roche, Merck, Genesis Pharmaceuticals; honoraria: Roche, Merck. GA: advisory boards: Novartis, BMS, Roche Hellas, AstraZeneca, Sanofi, Amgen, Genesis Pharma, Merck, Pfizer. GF: advisory board: Pfizer, Sanofi and Roche; honoraria from AstraZeneca.

Background/aim: PTEN-loss and PIK3CA mutations have been addressed as markers of PI3K activation in breast cancer. We evaluated these markers in early high-risk breast cancer (EBC) focusing on PTEN immunohistochemistry (IHC) issues, particularly in HER2-positive disease., Materials and Methods: We examined PTEN-loss and PIK3CA mutations in 1265 EBC patients treated with adjuvant chemotherapy within two clinical trials. Two different methods for the evaluation of PTEN IHC were used, one upfront binary (loss; no-loss) and the other initially multi-scale allowing for the classification of "grey zone" tumors with low and very low PTEN protein expression., Results: PTEN-loss (33.4% and 22.1%, depending on the IHC method) and PIK3CA mutations (29.6%) were associated with ER/PgR/HER2-negative and ER/PgR-positive disease, respectively. Concordance of the two IHC methods was moderate (Cohen's kappa 0.624). PTEN-loss discrepancy and intra-tumor heterogeneity concerned "grey zone" tumors that were prevalent among HER2-positive cancers. PTEN-loss independently conferred higher risk for relapse and death. Compared to single PIK3CA mutations,single PTEN-loss was independently associated with increased risk for relapse and death. Depending on the evaluation method, in HER2-positive cancer, PTEN-loss was without- or of marginal unfavorable prognostic significance., Conclusion: In EBC, PTEN-loss is an independent predictor of poor outcome. When occurring singly, PTEN-loss and PIK3CA mutations have opposite prognostic impact. In HER2-positive disease, assessment of PTEN-loss by IHC appears unreliable and the marker is without clear prognostic significance., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Src, CDKN1B, and JAK2 play a crucial role in the coordination of cell signaling pathways. In the present study, we aim to investigate the prognostic significance of these biomarkers in HER2-positive metastatic breast cancer (MBC) patients treated with trastuzumab (T)., Methods: Formalin-fixed paraffin-embedded tumor tissue samples from 197 patients with HER2-positive MBC treated with T were retrospectively collected. All tissue samples were centrally assessed for ER, PgR, Ki67, HER2, and PTEN protein expression; EGFR gene amplification; PI3KCA mutational status; and tumor-infiltrating lympocytes density. Src, CDKN1B, and JAK2 mRNA expression was evaluated using quantitative reverse transcription-polymerase chain reaction., Results: Only 133 of the 197 patients (67.5%) were found to be HER2-positive by central assessment. CDKN1B mRNA expression was strongly correlated with Src (rho = 0.71) and JAK2 (rho = 0.54). In HER2-positive patients, low CDKN1B conferred higher risk for progression [hazard ratio (HR) = 1.58, 95% confidence interval (CI) 1.08-2.32, P = .018]. In HER2-negative patients, low Src was associated with longer survival (HR = 0.56, 95% CI 0.32-0.99, P = .045). Upon multivariate analyses, only low CDKN1B and JAK2 mRNA expression remained unfavorable factors for PFS in de novo and relapsed (R)-MBC patients, respectively (HR = 2.36, 95% CI 1.01-5.48, P = .046 and HR = 1.76, 95% CI 1.01-3.06, P = .047, respectively)., Conclusions: Low CDKN1B and JAK2 mRNA expressions were unfavorable prognosticators in a cohort of T-treated MBC patients. Our results suggest that CDKN1B and JAK2, if validated, may serve as prognostic factors potentially implicated in T resistance, which seems to be associated with distinct pathways in de novo and R-MBC., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: We examined tumor genotype characteristics of human epidermal growth factor receptor 2 (HER2)-positive relapsed (R-) and de novo (dn-) metastatic breast cancer (MBC) in trastuzumab-treated patients who were previously not exposed to this agent., Materials and Methods: We analyzed genotypes obtained upon deep sequencing from 113 HER2-positive primary tumors from 69 patients with R-MBC and 44 patients with dn-MBC., Results: Mutations were observed in 90 (79.6%) tumors, 56 R-MBC and 34 dn-MBC (median number per tumor: 2; mean: 11.2; range: 0-150). The top mutated gene was TP53 (63.7%) followed by PIK3CA (24.8%) and others that were mostly co-mutated with TP53 (eg, 22 of 28 PIK3CA mutated tumors were co-mutated in TP53, 17 of these were R-MBC [P = .041]). dn-MBC had higher CEN17 average copies (P = .048). Tumor mutational burden inversely correlated with average HER2 copies (rho -0.32; P < .001). In all patients, PIK3CA mutations and higher proliferation rate were independent unfavorable prognosticators. In R-MBC, longer disease-free interval between initial diagnosis and relapse conferred lower risk for time-to-progression (P < .001) and death (P = .009); PIK3CA mutations conferred higher risk for death (P = .035). In dn-MBC, surgical removal of the primary tumor before any other therapy was favorable for time-to-progression (P = .002); higher tumor mutational burden was unfavorable for survival (P = .026)., Conclusions: Except for the overall unfavorable prognostic effect of PIK3CA mutations in trastuzumab-treated MBC, our exploratory findings indicate that the outcome of patients with R-MBC is related to patient benefit from the preceding adjuvant chemotherapy and provide initial evidence that tumor mutational burden may be related to prognosis in dn-MBC, which is of potential clinical relevance and merits further investigation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Tissue genomic heterogeneity (t-HET) in patients with epithelial ovarian cancer (OVCA) is related to tissue plasticity, i.e., flexibility to adapt to adverse molecular environments. Here, we interrogated the presence and clinical relevance of OVCA t-HET., Methods: We applied high-depth (>2000×) sequencing on 297 paraffin tissue samples (fallopian tubes, ovaries, intra-abdominal metastases) from 71 treatment-naïve patients who subsequently received first-line platinum-based chemotherapy. Based on tissue mutation patterns, we distinguished tissue genotypes into: no mutation (33/297 samples; 11.1%), stable (173; 58.2%) and unstable (91; 30.7%). We profiled genotypes per patient and assessed t-HET in 69 patients. Predicted pathogenic mutations refer to germline and/or tissues., Results: Among all 71 patients, 46 (64.8%) had pathogenic BRCA1 mutations and 15 (21.7%) had BRCA1/2 disruption (i.e., pathogenic mutations with position-LOH). We classified 29 patients with t-HET (42%), all with pathogenic BRCA1; t-HET was observed in 64% with such mutations (p < 0.001). As opposed to non-t-HET, matched tissues in t-HET shared pathogenic BRCA1 (p < 0.001) but not BRCA2 and TP53. Germline BRCA1 mutations in tissues exhibited position-LOH; heterozygous status; or, partial loss of the inherited allele accompanied by additional clonal mutations. Patients with t-HET had worse outcome (log-rank p = 0.048 [progression-free]; p = 0.037 [overall survival]), including 12/15 patients with disrupted BRCA1/2 and 3 BRCA1 carriers with partial germline loss in tissues., Conclusions: Pathogenic BRCA1 mutations appear necessary but may not be sufficient for the establishment of t-HET. t-HET may be associated with worse outcome, including in patients with disrupted BRCA1/2, which is usually considered as a favourable marker. OVCA t-HET may need to be addressed for treatment decisions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the current study, we performed a complete analysis, with four different methods, of all four HER family receptors, in a series of patients with metastatic breast cancer treated with trastuzumab-based regimens and evaluated their prognostic value. Formalin-fixed paraffin-embedded tumor tissue samples were collected from 227 patients, considered to be HER2-positive when assessed at the local laboratories. We evaluated gene amplification, copy number variations (CNVs), mRNA and protein expression of all four HER family members. In addition, our analysis included the evaluation of several other factors by immunohistochemistry (IHC), such as pHER2Tyr1221/1222, pHER2Tyr877 and PTEN. Central review of HER2 status by IHC and fluorescence in situ hybridization revealed that of the 227 patients, only 139 (61.2%) were truly HER2-positive. Regarding the 191 patients treated with trastuzumab as first-line therapy, median time to progression (TTP) was 15.3 and 10.4 months for HER2-positive and HER2-negative participants, respectively, whereas median survival was 50.4 and 38.1 months, respectively. In HER2-positive patients, high HER3 mRNA expression was of favorable prognostic significance for TTP and survival (HR = 0.43, 95% CI 0.21-0.88, Wald's p = 0.022 and HR = 0.43, 95% CI 0.21-0.88, p = 0.021, respectively), while EGFR copy gain and EGFR protein expression were associated with higher risk for disease progression in HER2-negative patients (HR = 3.53, 95% CI 1.19-10.50, p = 0.023 and HR = 3.37, 95% CI 1.12-10.17, p = 0.031, respectively). Positive HER3 protein expression was a favorable factor for TTP in HER2-negative patients (HR = 0.43, 95% CI 0.22-0.84, p = 0.014). In the multivariate analysis, only EGFR copy gain retained its prognostic significance for TTP in the HER2-negative population (HR = 3.96, 95% CI 1.29-12.16, p = 0.016), while high HER3 mRNA expression retained its favorable prognostic significance for TTP in the HER2-positive subgroup (HR = 0.47, 95% CI 0.23-0.99, p = 0.048). The present study suggests that EGFR copy gain represents a negative prognostic factor for TTP in HER2-negative patients with metastatic breast cancer treated with trastuzumab. In addition, high HER3 mRNA expression appears to be of favorable prognostic significance for TTP in HER2-positive patients. Given the small number of patients included in the current analysis and the retrospective nature of the study, our findings should be validated in larger cohorts., Competing Interests: The authors declare the following competing interests: Koutras A: Advisory Role: Roche. Christodoulou C: Advisory Role: Roche, Honoraria: Roche. Pentheroudakis G: Advisory Role: Roche, Honoraria: Roche, Speaker bureau: Roche. Papakostas P: Advisory Role: Roche, Honoraria: Roche. Razis E: Advisory Role: Roche, Travel: Roche, Honoraria: Roche. Psyrri A: Consultation Fees: Roche, Honoraria Roche, Research Funding: Roche/Genentech. Fountzilas G: Advisory Role: Roche. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Tumor-infiltrating lymphocytes (TILs) have been shown to be of prognostic value in several cancer types. In early breast cancer, TILs have a prognostic utility, as well, especially in HER2-positive and triple-negative breast cancer. TILs presence is broadly associated with improved survival; however, there is controversy regarding TILs subpopulations., Patients and Methods: Early-stage breast cancer patients treated with anthracycline-based chemotherapy within two randomized trials were included in the study. We evaluated, by qRT-PCR, 826 tumor tissue samples for mRNA expression of CD3, CD8, and FOXP3 for potential prognostic significance in terms of disease-free survival (DFS) and overall survival (OS)., Results: After a median follow-up of 133.0 months, 255 patients (30.9%) had died and 314 (38.0%) had disease progression. In the univariate analysis, high CD3 and CD8 mRNA expression was found to be of favorable prognostic value for DFS (P = 0.007 and P = 0.016, respectively). In multivariate analyses, the association of high CD8 mRNA expression with increased DFS was retained (HR = 0.77, 95% CI 0.60-0.998, Wald's P = 0.048), whereas that of high CD3 mRNA expression was of marginal statistical significance (HR = 0.77, 95% CI 0.59-1.01, P = 0.059). Moreover, a significant interaction was observed between HER2 status and CD3 mRNA expression with respect to DFS (interaction P = 0.032). In the HER2-positive subgroup, the hazard ratio associated with high CD3 mRNA expression was of greater magnitude (HR = 0.48, 95% CI 0.30-0.76, P = 0.002) compared with the hazard ratio presented above, for the entire cohort. No significant findings were observed for FOXP3 in terms of DFS, while none of the studied markers were of prognostic value for OS., Conclusions: High CD3 and CD8 mRNA expression in early-stage breast cancer patients is of prognostic value for decreased risk of relapse and, in the future, could potentially be of importance in deciding the most appropriate therapeutic strategy in light of the recent immune-related treatment developments., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

New material of the Mio-Pliocene colobine Mesopithecus from the Turolian locality of Kryopigi (Greece) is described here. It includes a complete skull with the atlas attached and other dental and postcranial elements representing at least five individuals (four males and one female). The material is compared with Mesopithecus delsoni, Mesopithecus pentelicus, Mesopithecus monspessulanus and intermediate forms from more than a dozen Turolian localities of the Greco-Iranian province. These comparisons support the attribution of the Kryopigi material to M. pentelicus. The chronostratigraphic distribution of Mesopithecus species and intermediate forms suggests that the Kryopigi fauna could be dated as younger than the Perivolaki locality with M. delsoni/pentelicus (7.1-7.3 Ma, MN12) and older than the Dytiko localities with M. aff. pentelicus, M. cf. pentelicus and M. cf. monspessulanus (?middle MN13). The dimensions of the atlas are within the distribution of extant colobines. The skull shows bite-marks, probably caused by the hyaena Adcrocuta eximia., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Several studies support an important role of angiogenesis in breast cancer growth and metastasis. The main objectives of the study were to investigate the immunohistochemical expression of vascular endothelial growth factor (VEGF) family ligands (VEGF-A and VEGF-C) and receptors (VEGFR1, VEGFR2 and VEGFR3) in breast cancer and their associations with clinicopathological parameters, cancer subtypes/subgroups and patient outcome. Formalin-fixed paraffin-embedded tumor tissue samples were collected from early-stage breast cancer patients treated with anthracycline-based chemotherapy within a randomized trial. Immunohistochemistry was performed on serial 2.5 μm thick tissue sections from tissue microarray blocks. High VEGF-A, VEGF-C, VEGFR1, VEGFR2 and VEGFR3 protein expression was observed in 11.8% (N = 87), 80.8% (N = 585), 28.1% (N = 202), 64.6% (N = 359) and 71.8% (N = 517) of the cases, respectively. Significant associations were observed among all proteins (all p-values <0.05), with the exception of the one between VEGF-C and VEGFR1 (chi-square test, p = 0.15). Tumors with high VEGF-A protein expression, as compared to tumors with low expression were more frequently ER/PgR-negative (33.3% vs. 20.8%, chi-square test, p = 0.009) and HER2-positive (44.8% vs. 20.6%, p<0.001). In addition, tumors with high VEGFR1 expression, were more frequently HER2-positive (32.8% vs. 19.6%, p<0.001), while tumors with high VEGFR3 expression were more frequently ER/PgR-negative (24.9% vs. 17.0%, p = 0.024) and HER2-positive (26.9% vs. 14.8%, p = 0.001). High VEGF-A and VEGF-C protein expression was associated with increased DFS in the entire cohort (HR = 0.57, 95% CI 0.36-0.92, Wald's p = 0.020 and HR = 0.71, 95% CI 0.52-0.96, p = 0.025, respectively), as well as in specific subtypes/subgroups, such as HER2-positive (VEGF-A, HR = 0.32, 95% CI 0.14-0.74, p = 0.008) and triple-negative (VEGF-C, HR = 0.44, 95% CI 0.21-0.91, p = 0.027) patients. High vs. low VEGFR1 expression was an unfavorable factor for DFS in triple-negative patients (HR = 2.74, 95% CI 1.26-5.98, p = 0.011), whereas the opposite was observed among the ER/PgR-positive patients (HR = 0.69, 95% CI 0.48-0.98, p = 0.041). Regarding OS, high VEGF-C protein expression was associated with increased OS in the entire cohort (HR = 0.64, 95% CI 0.46-0.89, Wald's p = 0.008), as well as in in specific subtypes/subgroups, such as ER/PgR-negative (HR = 0.37, 95% CI 0.20-0.71, p = 0.003) and triple-negative (HR = 0.42, 95% CI 0.19-0.90, p = 0.026) patients. In conclusion, high expression of angiogenesis-related proteins is associated with adverse clinicopathological parameters in early-stage breast cancer patients and may be surrogate markers of biologically distinct subgroups of ER/PgR-negative or triple-negative tumors with superior outcome. Further validation of our findings in independent cohorts is needed., Competing Interests: As per Journal requirements, and on behalf of all authors, the authors confirm that they have the following interests: HG: Honoraria from BMS, MSD, Roche, Novartis, AMGEN. Research grants from BMS, Roche, Novartis. ES: Advisory Board of Merck, MSD, AstaZeneca, Roche, Amgen and Genesis. GF: Advisory Board of Pfizer, Sanofi and Roche. Honoraria from AstraZeneca. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. The rest of the authors have declared that no competing interests exist.

Biliary tract cancer (BTC) represents a heterogeneous disease with dismal outcome. Herein, we examined genotype and angiogenesis features in BTC. We applied genotyping (Sanger, qPCR, 101-gene panel NGS), mRNA relative quantification methods, and β-catenin immunohistochemistry in 84 FFPE BTC (55 gallbladder [GBC], 14 intrahepatic [ICC], 15 extrahepatic [ECC] carcinomas). We identified 541 mutations in 68 (81%) tumors. Top mutated genes were CTNNB1 (36%); PTEN (33%); TP53 (31%); PIK3R1 (29%); PIK3CA (13%); BRCA2 and KRAS (12%); BRCA1 (11%). Six GBCs were hypermutated [hm] displaying a distinct mutational pattern. Mutations in TP53 and PI3K, Wnt and RAS components were prevalent among non-hypermutated tumors. All hmGBCs carried mutations in BRCA2 and other homologous recombination repair (HRR) genes, in PD1, but not in CTNNB1 and KRAS. None of the pathogenic BRCA2 p.D2723G and BRCA1 p.Q563* and c.5266dupC was present at frequencies expected for germline mutations. We observed copy gains (>6 copies) in EGFR (9% of informative tumors), PRKAR1A (7%), PIK3CA (6%), ERBB2 (5%) and MET (4%). TP53 mutations were prevalent in GBC (P<0.001) and PRKAR1A copy gains in ICC (P=0.007). PTEN was frequently co-mutated with CTNNB1 (P<0.001). Unrelated to CTNNB1 mutations, nuclear β-catenin was detected in 45% of tumors, among them in 5/6 hmGBC. We observed strong mRNA expression correlation of the two neuropilins (NRP1 and NRP2) with each other (Spearman's rho 0.59) and with the endothelin receptor (NRP2 rho 0.66; NRP2 rho 0.51), and between VEGFA and its receptors (FLT1 rho 0.49; KDR rho 0.45). All PIK3CA mutated tumors expressed endothelin 1 mRNA (P=0.010). Most tumors expressing nuclear β-catenin were negative for VEGFC (P=0.009) and FLT4 (P=0.002) mRNA expression. In conclusion, we confirmed the presence of known genomic aberrations in BTC and different genotypes between BTC subsets. Novel findings are the coexistence of PI3K and WNT pathway gene alterations in BTC, their association with angiogenesis, and the hypermutated GBCs with HRR gene mutations, all of which may be considered for new treatment options in this difficult to treat disease., Competing Interests: None.

Background-Aim: To evaluate the prognostic role of elaborate molecular clusters encompassing cyclin D1, cyclin E1, p21, p27 and p53 in the context of various breast cancer subtypes., Methods: Cyclin E1, cyclin D1, p53, p21 and p27 were evaluated with immunohistochemistry in 1077 formalin-fixed paraffin-embedded tissues from breast cancer patients who had been treated within clinical trials. Jaccard distances were computed for the markers and the resulted matrix was used for conducting unsupervised hierarchical clustering, in order to identify distinct groups correlating with prognosis., Results: Luminal B and triple-negative (TNBC) tumors presented with the highest and lowest levels of cyclin D1 expression, respectively. By contrast, TNBC frequently expressed Cyclin E1, whereas ER-positive tumors did not. Absence of Cyclin D1 predicted for worse OS, while absence of Cyclin E1 for poorer DFS. The expression patterns of all examined proteins yielded 3 distinct clusters; (1) Cyclin D1 and/or E1 positive with moderate p21 expression; (2) Cyclin D1 and/or E1, and p27 positive, p53 protein negative; and, (3) Cyclin D1 or E1 positive, p53 positive, p21 and p27 negative or moderately positive. The 5-year DFS rates for clusters 1, 2 and 3 were 70.0%, 79.1%, 67.4% and OS 88.4%, 90.4%, 78.9%, respectively., Conclusions: It seems that the expression of cell cycle regulators in the absence of p53 protein is associated with favorable prognosis in operable breast cancer.

Background: The retinoblastoma (RB) gene is a tumor-suppressor gene that plays a central role in regulating the cell cycle. Inactivation of this gene is involved in breast cancer., Patients and Methods: A total of 827 patients with breast cancer treated with taxane-based adjuvant chemotherapy were included in the study. Protein expression of RB, phosphorylated RB (pRB), p16, cyclin D1 and p53 was evaluated by immunohistochemistry., Results: Neither of the retinoblastoma markers (RB and pRB) reached statistical significance in terms of their association with disease-free or overall survival. Nevertheless, when clustering analysis was performed, patients with tumors featuring low levels of p16, cyclin D1 and p53 with concomitantly high levels of pRB had reduced risk for relapse (Wald's p=0.015)., Conclusion: The p53-mediated sensitivity of breast cancer cells to chemotherapeutic agents appears to be driven mostly by pRB. Using agents that enhance RB phosphorylation might possibly increase the chemosensitivity of breast cancer cells., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The Notch pathway has been implicated in triple-negative breast cancer (TNBC). Herein, we studied the subcellular localization of the less investigated Notch2 and Notch3 and that of the Jagged1 (Jag1) ligand in patients with operable TNBC., Patients and Methods: We applied immunohistochemistry for Notch2, Notch3 and Jag1 in 333 tumors from TNBC patients treated with adjuvant anthracycline-based chemotherapy. We evaluated cytoplasmic (c), membranous (m) and nuclear (n) protein localization., Results: c-Notch2 (35% positive tumors), c-Notch3 (63%), c-Jag1 (43%), m-Notch3 (23%) and n-Jag1 (17%) were analyzed individually and by using hierarchical clustering for prognostic evaluation. Upon multivariate analysis, compared to high m-Notch3 in the absence of n-Jag1 (cluster 4), all other marker combinations (clusters 1, 2, 3) conferred significantly higher risk for relapse (p<0.05)., Conclusion: Specific Notch3 and Jag1 subcellular localization patterns may provide clues for the behavior of the tumors and potentially for Jag1 targeting in TNBC patients., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The shift towards an earlier diagnosis of breast cancer (BC) highlights the need for biomarkers that would identify patients at risk for relapse and metastatic spread and indicate the potential value of additional treatment strategies. Osteopontin (OPN) is a matricellular protein that has been suggested to be a potential biomarker in BC. In the present study, we used archived BC patient samples to assess the clinical utility of OPN., Methods: Formalin-fixed paraffin-embedded tumor tissue samples from 975 patients were collected from two large phase III randomized adjuvant chemotherapy trials (HE10/97 and HE10/00) that included patients with high risk BC. All tissue samples were assessed for ER, PgR, Ki67 and HER2 protein expression. OPN protein and mRNA expression was evaluated using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction, respectively., Results: OPN mRNA expression data were available for 814 patients, whereas OPN protein expression data were available for 546 patients. The majority of patients were ER/PgR-positive (78.3%), HER2-negative (76.5%) and Ki67-positive (55.2%) and had received adjuvant radiation therapy (76.8%) and hormonal therapy (81.1%). OPN mRNA expression was significantly associated with age (60.9% in high OPN tumors vs. 54.1% in low OPN tumors, p = 0.047), ER/PgR-negative status (25.7 vs. 17.2%, p = 0.004) and BC subtypes (p = 0.021). In addition, high OPN mRNA expression was significantly associated with reduced DFS (HR 1.26, 95% CI 1.00-1.59, Wald's p = 0.050) and OS (HR 1.37, 95% CI 1.05-1.78, p = 0.019), while it retained its prognostic significance for both DFS (HR 1.39, 95% CI 1.10-1.77, p = 0.007) and OS (HR 1.54, 95% CI 1.61-2.05, p = 0.003) in the multivariate analysis., Conclusions: We showed that high OPN mRNA expression is associated with decreased DFS and OS in a large cohort of BC patients treated with adjuvant chemotherapy in a clinical trial setting. Our results suggest that OPN may serve as a prognostic factor and a potential target for therapy. Trial registration Australian New Zealand Clinical Trials Registry; HE10/97 ACTRN12611000506998; HE10/00 ACTRN12609001036202.

The preservation of pathogenic BRCA1/2 germline mutations in tumor tissues is usually not questioned, while it remains unknown whether these interact with somatic genotypes for patient outcome. Herein we compared germline and tumor genotypes in operable triple-negative breast cancer (TNBC) and evaluated their combined effects on prognosis. We analyzed baseline germline and primary tumor genotype data obtained by Sanger and Next Generation Sequencing in 194 TNBC patients. We also performed multiple tests interrogating the preservation of germline mutations in matched tumors and breast tissue from carriers with available material. Patients had been treated within clinical trials with adjuvant anthracyclines-taxanes based chemotherapy. We identified 50 (26%) germline mutation carriers (78% in BRCA1 ) and 136 (71%) tumors with somatic mutations (83% in TP53 ). Tumor mutation patterns differed between carriers and non-carriers (P<0.001); PIK3CA mutations were exclusively present in non-carriers (P=0.007). Germline BRCA1/2 mutations were not detected in matched tumors and breast tissues from 14 out of 33 (42%) evaluable carriers. Microsatellite markers revealed tumor loss of the germline mutant allele in one case only. Tumors that had lost the germline mutation demonstrated a higher incidence of somatic TP53 mutations as compared to tumors with preserved germline mutations (P=0.036). Germline mutation status significantly interacted with tumor TP53 mutations for patient disease-free survival (interaction P=0.026): In non-carriers, tumor TP53 mutations did not affect outcome; In carriers, those with mutated TP53 tumors experienced more relapses compared to those with wild-type TP53 tumors (36% vs. 9% relapse rate, respectively). In conclusion, we show that loss of germline BRCA1/2 mutations is not a rare event in TNBC. This finding, the observed differences in tumor genotypes with respect to germline status and the prognostic interaction between germline BRCA1-related and tumor TP53 mutation status prompt for combined germline and tumor genotyping for the classification of TNBC, particularly in the context of clinical trials evaluating synthetic lethality drugs.

Background-Aim: Early breast cancer is a heterogeneous disease, and, therefore, prognostic tools have been developed to evaluate the risk for distant recurrence. In the present study, we sought to develop a risk for recurrence score (RRS) based on mRNA expression of three proliferation markers in high-risk early breast cancer patients and evaluate its ability to predict risk for relapse and death. In addition the Adjuvant! Online score (AOS) was also determined for each patient, providing a 10-year estimate of relapse and mortality risk. We then evaluated whether RRS or AOS might possibly improve the prognostic information of the clinical treatment score (CTS), a model derived from clinicopathological variables., Methods: A total of 1,681 patients, enrolled in two prospective phase III trials, were treated with anthracycline-based adjuvant chemotherapy. Sufficient RNA was extracted from 875 samples followed by multiplex quantitative reverse transcription-polymerase chain reaction for assessing RACGAP1, TOP2A and Ki67 mRNA expression. The CTS, slightly modified to fit our cohort, integrated the prognostic information from age, nodal status, tumor size, histological grade and treatment. Patients were also classified to breast cancer subtypes defined by immunohistochemistry. Likelihood ratio (LR) tests and concordance indices were used to estimate the relative increase in the amount of information provided when either RRS or AOS is added to CTS., Results: The optimal RRS, in terms of disease-free survival (DFS) and overall survival (OS), was based on the co-expression of two of the three evaluated genes (RACGAP1 and TOP2A). CTS was prognostic for DFS (p<0.001), while CTS, AOS and RRS were all prognostic for OS (p<0.001, p<0.001 and p = 0.036, respectively). The use of AOS in addition to CTS added prognostic information regarding DFS (LR-Δχ2 8.7, p = 0.003), however the use of RRS in addition to CTS did not. For estimating OS, the use of either AOS or RRS in addition to CTS added significant prognostic information. Specifically, the use of both CTS and AOS had significantly better prognostic value vs. CTS alone (LR-Δχ2 20.8, p<0.001), as well as the use of CTS and RRS vs. CTS alone (LR-Δχ2 4.8, p = 0.028). Additionally, more patients were scored as high-risk by AOS than CTS. According to immunohistochemical subtypes, prognosis was improved in the Luminal A (LR-Δχ2 7.2, p = 0.007) and Luminal B (LR-Δχ2 8.3, p = 0.004) subtypes, in HER2-negative patients (LR-Δχ2 23.4, p<0.001) and in patients with >3 positive nodes (LR-Δχ2 23.9, p<0.001) when AOS was added to CTS., Conclusions: The current study has shown a clear benefit in predicting overall survival of high-risk early breast cancer patients when combining CTS with either AOS or RRS. The combination of CTS and AOS adds significant prognostic information compared to CTS alone for DFS, while the combination of CTS with either AOS or RRS has better prognostic value than CTS alone for OS. These findings could possibly add on the information needed for the best risk prediction strategy in high-risk early breast cancer patients in a rather simple and inexpensive way, especially in Luminal A and B subtypes, HER2-negative patients and those with >3 positive nodes., Competing Interests: Zoi Alexopoulou is employed by Health Data Specialists Ltd. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials. The rest of the authors have declared that no competing interests exist. There are no patents, products in development or marketed products to declare.

The purpose of this study is to investigate whether the outcome of breast cancer (BC) patients treated with adjuvant chemotherapy is affected by co-mutated TP53 and PIK3CA according to stromal tumor-infiltrating lymphocytes (TILs). Paraffin tumors of all clinical subtypes from 1661 patients with operable breast cancer who were treated within 4 adjuvant trials with anthracycline-taxanes chemotherapy were informative for TP53 and PIK3CA mutation status (semiconductor sequencing genotyping) and for stromal TILs density. Disease-free survival (DFS) was examined. TP53 mutations were associated with higher (p < 0.001) and PIK3CA with lower (p = 0.004) TILs in an ER /PgR-specific manner (p < 0.001). Mutations did not affect the favorable DFS of patients with lymphocyte-predominant (LP) BC. Within non-LPBC, PIK3CA-only mutations conferred best, while TP53-PIK3CA co-mutations (6 % of all tumors) conferred worst DFS (HR 0.59; 95 % CI 0.44-0.79; p = 0.001 for PIK3CA-only). TP53-only mutations were unfavorable in patients with lower TILs, while patients with lower TILs performed worse if their tumors carried TP53-only mutations (interaction p = 0.046). Multivariate analysis revealed favorable PIK3CA-only mutations in non-LPBC (HR 0.64; 95 % CI 0.47-0.88; p = 0.007), and unfavorable TP53 mutations in ER/PgRpos/HER2neg (HR 1.55; 95 % CI 1.07-2.24; p = 0.021). Mutations did not interact with TILs in non-LP triple-negative and HER2-positive patients. TP53 and PIK3CA mutations appear to have diverse effects on the outcome of early BC patients, according to whether these genes are co-mutated or not, and for TP53 according to TILs density and ER/PgR-status. These findings need to be considered when evaluating the effect of these two most frequently mutated genes in the context of large clinical trials.

Background: Although host immune response is an emerging prognostic factor for colorectal cancer, there is no consensus on the optimal methodology, surrogate markers or tissue for study., Patients and Methods: Tumour blocks were prospectively collected from 344 patients with stage II/III colorectal cancer (CRC) treated with adjuvant chemotherapy. Whole section lymphocytic infiltration was studied along with mRNA expression of CD3Z, CD8, CD4, CXCL9, CXCL13, IGHM, FOXP3, SNAI2 and ESR1 by qRT-qPCR in tissue microarray (TMA) cores from the centre of tumour, invasive margin and adjacent normal mucosa., Results: Lymphocytic infiltration, deficient MMR (10.9%), KRAS (40.7%) and BRAF (4.9%) mutations or single mRNA gene expression were not prognostic. Tumour ESR1 gene expression (Hazard Ratio [HR] for relapse 2.33, 95% CI 1.35-4.02; HR for death 1.74, 95% CI 1.02-2.97) and absence of necrosis (HR for relapse 1.71, 95% CI 1.05-2.71; HR for death 1.98, 95% CI 1.14-3.43) were adverse prognostic features. We used CD3Z and CD8 expression in order to devise the mRNA-based Immune Score (mIS) and proceeded to partitioning analysis in 267 patients, with age, stage, tumour site (Right vs Left CRC), KRAS mutation and tumour mIS as input factors. Only in patients with stage III right-sided colon cancer, a low immune response was associated with inferior disease-free survival (mIS-low, HR for relapse 2.28, 95% CI 1.05-8.02). No prognostic significance was seen for tumour mIS in any other stage or site of CRC, or for a similar mIS score derived from adjacent normal mucosa. Independent adverse prognostic significance was retained in multivariable analysis for absence of necrosis, tumour ESR1 expression in all patients and low tumour mIS in stage III right-sided CRC., Conclusions: In localised CRC, mRNA-based CD3Z/CD8 profiling of tumour immune response may have stage, site and tissue-specific prognostic significance, along with ESR1 expression., Trial Registration: ANZCTR.org.au ACTRN12610000509066.

Background and Purpose: There are scarce data available on the prognostic/predictive value of p-Akt and p-mTOR protein expression in patients with high-risk early breast cancer., Patients and Methods: Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 997 patients participating in two adjuvant phase III trials were assessed for EGFR, PTEN, p-Akt, p-mTOR protein expression, and PIK3CA mutational status. These markers were evaluated for associations with each other and with selected patient and tumor characteristics, immunohistochemical subtypes, disease-free survival (DFS), and overall survival (OS)., Results: p-mTOR protein expression was negatively associated with EGFR and positively associated with PTEN, with p-Akt473, and with the presence of PIK3CA mutations. EGFR expression was positively associated with p-Akt473, p-Akt308, and PIK3CA wild-type tumors. Finally, p-Akt308 was positively associated with p-Akt473 expression. In univariate analysis, EGFR (p = 0.016) and the coexpression of EGFR and p-mTOR (p = 0.015) were associated with poor OS. Among patients with p-Akt308-negative or low-expressing tumors, those treated with hormonal therapy were associated with decreased risk for both relapse and death (p = 0.013 and p < 0.001, respectively). In the subgroup of patients with locoregional relapse, positive EGFR and mTOR protein expression was found to be associated with increased (p = 0.034) and decreased (p < 0.001) risk for earlier relapse, respectively. In multivariate analysis, low levels of p-Akt308 and the coexpression of EGFR and p-mTOR retained their prognostic value., Conclusion: Low protein expression of p-Akt308 was associated with improved DFS and OS among patients treated with hormonal therapy following adjuvant chemotherapy. Coexpression of EGFR and p-mTOR was associated with worse OS.