Improving the Accuracy and Speed of Visual Field Testing in Glaucoma With Structural Information and Deep Learning.

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.

The Impact of the COVID-19 Pandemic on Antimicrobial Resistance and Management of Bloodstream Infections.

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.

Predicting Visual Fields From Optical Coherence Tomography via an Ensemble of Deep Representation Learners.

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 R2 = 0.78 and R2 = 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.

Comparison of a Rapid Light-Induced and Forced Test to Study the Oxidative Stability of White Wines.

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 (R2 > 0.89) between optical density at 420 nm and b* parameter was highlighted. The synergic effect of light and H2O2 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.

Improving statistical power of glaucoma clinical trials using an ensemble of cyclical generative adversarial networks.

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.

OCT Signal Enhancement with Deep Learning.

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.

Merging Information From Infrared and Autofluorescence Fundus Images for Monitoring of Chorioretinal Atrophic Lesions.

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.

Phase 2 study of cabazitaxel as second-line treatment in patients with HER2-negative metastatic breast cancer previously treated with taxanes-a Hellenic Cooperative Oncology Group (HeCOG) Trial.

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.

Artificial Neural Network-Based Automatic Detection of Food Intake for Neuromodulation in Treating Obesity and Diabetes.

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.

Cyclin D1 differential activation and its prognostic impact in patients with advanced breast cancer treated with trastuzumab.

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.

Opposite Prognostic Impact of Single PTEN-loss and PIK3CA Mutations in Early High-risk Breast Cancer.

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.

Prognostic Impact of Src, CDKN1B, and JAK2 Expression in Metastatic Breast Cancer Patients Treated with Trastuzumab.

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.

Pathogenic BRCA1 mutations may be necessary but not sufficient for tissue genomic heterogeneity: Deep sequencing data from ovarian cancer patients.

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.

Relapsed and De Novo Metastatic HER2-positive Breast Cancer Treated With Trastuzumab: Tumor Genotypes and Clinical Measures Associated With Patient Outcome.

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.

Evaluation of the prognostic value of all four HER family receptors in patients with metastatic breast cancer treated with trastuzumab: A Hellenic Cooperative Oncology Group (HeCOG) study.

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.

Evaluation of the prognostic value of CD3, CD8, and FOXP3 mRNA expression in early-stage breast cancer patients treated with anthracycline-based adjuvant chemotherapy.

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.

Associations of angiogenesis-related proteins with specific prognostic factors, breast cancer subtypes and survival outcome in early-stage breast cancer patients. A Hellenic Cooperative Oncology Group (HeCOG) trial.

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.

Mesopithecus pentelicus from the Turolian locality of Kryopigi (Kassandra, Chalkidiki, Greece).

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.

Genotyping and mRNA profiling reveal actionable molecular targets in biliary tract cancers.

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 ß-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 ß-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 ß-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.