Prostate cancer-derived extracellular vesicles metabolic biomarkers: Emerging roles for diagnosis and prognosis.

Prostate cancer (PCa) is a global health concern, ranking as the most common cancer among men in Western countries. Traditional diagnostic methods are invasive with adverse effects on patients. Due to the heterogeneous nature of PCa and their multifocality, tissue biopsies often yield false-negative results. To address these challenges, researchers are exploring innovative approaches, particularly in the realms of proteomics and metabolomics, to identify more reliable biomarkers and improve PCa diagnosis. Liquid biopsy (LB) has emerged as a promising non-invasive strategy for PCa early detection, biopsy selection, active surveillance for low-risk cases, and post-treatment and progression monitoring. Extracellular vesicles (EVs) are lipid-bilayer nanovesicles released by all cell types and play an important role in intercellular communication. EVs have garnered attention as a valuable biomarker resource in LB for PCa-specific biomarkers, enhancing diagnosis, prognostication, and treatment guidance. Metabolomics provides insight into the body's metabolic response to both internal and external stimuli, offering quantitative measurements of biochemical alterations. It excels at detecting non-genetic influences, aiding in the discovery of more accurate cancer biomarkers for early detection and disease progression monitoring. This review delves into the potential of EVs as a resource for LB in PCa across various clinical applications. It also explores cancer-related metabolic biomarkers, both within and outside EVs in PCa, and summarises previous metabolomic findings in PCa diagnosis and risk assessment. Finally, the article addresses the challenges and future directions in the evolving field of EV-based metabolomic analysis, offering a comprehensive overview of its potential in advancing PCa management.

Sharing reference intervals and monitoring patients across laboratories - findings from a likely commutable external quality assurance program.

OBJECTIVES: Laboratory results are increasingly interpreted against common reference intervals (CRIs), published clinical decision limits, or previous results for the same patient performed at different laboratories. However, there are no established systems to determine whether current analytical performance justifies these interpretations. We analysed data from a likely commutable external quality assurance program (EQA) to assess these interpretations. METHODS: The use of CRIs was assessed by evaluating instrument group medians against minimum specifications for bias. The use of clinical decision limits was assessed using specifications from professional bodies, and the monitoring of patients by testing at different laboratories was assessed by comparing all-laboratory imprecision to within-subject biological variation. RESULTS: Five of the 18 analytes with Australasian CRIs did not meet specification for all instrument groups. Among these, calcium and magnesium failed for one instrument group out of seven, while bicarbonate, chloride, and lipase failed for two instrument groups. Of the 18 analytes reviewed currently without CRIs in Australasia, 10 candidates were identified. Among analytes with clinical decision limits, i.e. lipids, glucose, and vitamin D, only triglycerides met both bias and imprecision specifications, while vitamin D met the imprecision specification. Monitoring patients by testing at different laboratories was supported for 15 of the 46 (33 %) analyte-method principles groups that met minimum imprecision specifications. CONCLUSIONS: Analysis of data from commutable EQA programs can provide a mechanism for monitoring whether analytical performance justifies the interpretations made in contemporary laboratory practice. EQA providers should establish systems for routinely providing this information to the laboratory community.

Ensuring quality in 17OHP mass spectrometry measurement: an international study assessing isomeric steroid interference.

OBJECTIVES: Interference from isomeric steroids is a potential cause of disparity between mass spectrometry-based 17-hydroxyprogesterone (17OHP) results. We aimed to assess the proficiency of mass spectrometry laboratories to report 17OHP in the presence of known isomeric steroids. METHODS: A series of five samples were prepared using a previously demonstrated commutable approach. These samples included a control (spiked to 15.0 nmol/L 17OHP) and four challenge samples further enriched with equimolar concentrations of 17OHP isomers (11α-hydroxyprogesterone, 11ß-hydroxyprogesterone, 16α-hydroxyprogesterone or 21-hydroxyprogesterone). These samples were distributed to 38 participating laboratories that reported serum 17OHP results using mass spectrometry in two external quality assurance programs. The result for each challenge sample was compared to the control sample submitted by each participant. RESULTS: Twenty-six laboratories (68 % of distribution) across three continents returned results. Twenty-five laboratories used liquid chromatography-tandem mass spectrometry (LC-MS/MS), and one used gas chromatography-tandem mass spectrometry to measure 17OHP. The all-method median of the control sample was 14.3 nmol/L, ranging from 12.4 to 17.6 nmol/L. One laboratory had results that approached the lower limit of tolerance (minus 17.7 % of the control sample), suggesting the isomeric steroid caused an irregular result. CONCLUSIONS: Most participating laboratories demonstrated their ability to reliably measure 17OHP in the presence of the four clinically relevant isomeric steroids. The performance of the 12 (32 %) laboratories that did not engage in this activity remains unclear. We recommend that all laboratories offering LC-MS/MS analysis of 17OHP in serum, plasma, or dried bloodspots determine that the isomeric steroids are appropriately separated.

Repeat patient testing-quality control with canine samples shows promise as an alternative to commercial quality control material for a network of four Sysmex XT-2000iV hematology analyzers.

BACKGROUND: Repeat patient testing-quality control (RPT-QC) uses retained patient samples as an alternative to commercial quality control material (QCM). We elected to calculate and validate RPT-QC limits for red blood cell count (RBC), hemoglobin (HBG), hematocrit (HCT), and white blood cell count (WBC). OBJECTIVES: (1) To validate RPT-QC across a network of four harmonized Sysmex XT-2000iV hematology analyzers and determine the total error that can be controlled with RPT-QC. (2) To generate quality control (QC) limits using the standard deviation (SD) of the duplicate measurement differences and determine a suitable simple QC rule with a probability of error detection >0.85 and probability of false rejection <0.05. (3) Monitor RPT-QC using sigma metrics as a performance indicator and (4) to challenge RPT-QC to ensure acceptable sensitivity. METHODS: Fresh adult canine EDTA samples with results within reference intervals were selected and run again on days 2, 3, and 4. QC limits were generated from the SD of the duplicate measurement differences. The QC limits were challenged using interventions designed to promote unstable system performance. The total error detectable by RPT-QC was determined using EZRULES 3 software. RESULTS: In all, 20-40 data points were needed for RPT-QC calculations and validated using 20 additional data points. The calculated limits differed among the network of analyzers. The total error that could be controlled was the same or better than that of the manufacturer's commercially available quality control material using the same analyzer for all measurands except hematocrit, which required a higher total error goal than that proposed by ASVCP guidelines to achieve an acceptable probability of error detection. The challenges designed to mimic unstable system performance were successfully detected as out-of-control QC. CONCLUSIONS: The challenges for RPT-QC resulted in acceptable detection of potential unstable system performance. This initial study demonstrates that RPT-QC limits differ among the network of Sysmex XT-2000iV analyzers, indicating a requirement to customize for the individual analyzer and laboratory conditions. RPT-QC could achieve ASVCP total allowable error goals for RBC, HGB, and WBC, but not for HCT. Sigma metrics were consistently >5.5 for RBC, HGB, and WBC, but not for HCT.

Quality assurance programs for vitamin A and E in serum: are we doing enough to assess laboratory performance?

OBJECTIVES: Monitoring serum vitamin A (retinol) and vitamin E (α-tocopherol) concentrations is common practice for assessing nutritional status. Measurement of these vitamins can be challenging due to several factors. Whilst the RCPAQAP Vitamins: Serum Program assists participating laboratories in harmonisation, the materials provided do not contain the analogues of retinol and α-tocopherol that may be present in real patient samples. We aimed to assess participants' capacity to accurately report retinol and α-tocopherol in the presence of the vitamin E analogues tocopherol acetate and γ-tocopherol. METHODS: A supplementary series of a control sample and three matched spiked samples were distributed to each laboratory participating in the Program. Retinol and α-tocopherol results for each spiked sample were compared to the results of the control sample submitted by each participant. Acceptability of retinol and α-tocopherol results was determined based on the RCPAQAP allowable performance specifications (APS). RESULTS: Thirteen participants returned results for the supplementary sample series. Interference from α-tocopherol acetate was observed with results below the APS in 30 % (n=4) of laboratories for retinol quantification and in 23 % (n=3) for α-tocopherol quantification. One laboratory returned results above the APS for α-tocopherol when γ-tocopherol was present. CONCLUSIONS: This supplementary sample series has shown that the presence of vitamin E analogues can lead to the over or under estimation of nutritional status by some participants. Affected laboratories are encouraged to review their analytical procedures. To further assess laboratory competence, EQA providers should consider using patient samples or spiked challenge samples.

Comparison Study of Small Extracellular Vesicle Isolation Methods for Profiling Protein Biomarkers in Breast Cancer Liquid Biopsies.

Small extracellular vesicles (sEVs) are an important intercellular communicator, participating in all stages of cancer metastasis, immunity, and therapeutic resistance. Therefore, protein cargoes within sEVs are considered as a superior source for breast cancer (BC) biomarker discovery. Our study aimed to optimise the approach for sEV isolation and sEV proteomic analysis to identify potential sEV protein biomarkers for BC diagnosis. sEVs derived from BC cell lines, BC patients' plasma, and non-cancer controls were isolated using ultracentrifugation (UC), a Total Exosome Isolation kit (TEI), and a combined approach named UCT. In BC cell lines, the UC isolates showed a higher sEV purity and marker expression, as well as a higher number of sEV proteins. In BC plasma samples, the UCT isolates showed the highest proportion of sEV-related proteins and the lowest percentage of lipoprotein-related proteins. Our data suggest that the assessment of both the quantity and quality of sEV isolation methods is important in selecting the optimal approach for the specific sEV research purpose, depending on the sample types and downstream analysis.

SABR for Early Non-Small Cell Lung Cancer: Changes in Pulmonary Function, Dyspnea, and Quality of Life.

PURPOSE: The aim of this study was to report pulmonary function tests (PFTs) and clinician-reported and patient-reported quality-of-life (QoL) outcomes on a cohort of patients with non-small cell lung cancer (NSCLC) treated with SABR. METHODS AND MATERIALS: A total of 119 patients with NSCLC were treated with SABR in the prospective cohort SSBROC study of patients with T1-T2N0M0 NSCLC. PFTs and QoL measures were obtained at baseline pretreatment and at 6-month intervals. Here we report on the 6- to 18-month time points. Analysis of covariance (ANCOVA) methods adjusting for baseline analyzed potential predictors on outcomes of PFTs and patient-reported dyspnea at 18 months. RESULTS: The only statistically significant decline in PFTs was seen in forced expiratory volume in 1 second (FEV1) at 18 months post-SABR, with a decline of -0.11 L (P = .0087; 95% CI, -0.18 to -0.02). Of potential predictors of decline, only a 1-unit increase in smoking pack-years resulted in a -0.12 change in diffusing capacity for carbon monoxide (P = .026; 95% CI, -0.02 to -0.23) and a 0.003 decrease in FEV1 (P = .026; 95% CI, -0.006 to -0.0004). For patient-reported outcomes, statistically significant worsening in both the European Organisation for Research and Treatment of Cancer Quality of Life Core Questionnaire (QLQ-C30 Version 3) and the lung module (QLQ-LC13) dyspnea scores occurred at the 18-month time point, but not earlier. No potential predictors of worsening dyspnea were statistically significant. There was no statistically significant decline in clinician-reported outcomes or global QoL scores. CONCLUSIONS: We found a statistically significant decline in FEV1 at 18 months posttreatment. Smoking pack-years was a predictor for decline in diffusing capacity for carbon monoxide and FEV1 at 18 months. Worsening of patient-reported dyspnea scores was observed, consistent with the expected progression of lung comorbid disease.

hist2RNA: An Efficient Deep Learning Architecture to Predict Gene Expression from Breast Cancer Histopathology Images.

Gene expression can be used to subtype breast cancer with improved prediction of risk of recurrence and treatment responsiveness over that obtained using routine immunohistochemistry (IHC). However, in the clinic, molecular profiling is primarily used for ER+ breast cancer, which is costly, tissue destructive, requires specialised platforms, and takes several weeks to obtain a result. Deep learning algorithms can effectively extract morphological patterns in digital histopathology images to predict molecular phenotypes quickly and cost-effectively. We propose a new, computationally efficient approach called hist2RNA inspired by bulk RNA sequencing techniques to predict the expression of 138 genes (incorporated from 6 commercially available molecular profiling tests), including luminal PAM50 subtype, from hematoxylin and eosin (H&E)-stained whole slide images (WSIs). The training phase involves the aggregation of extracted features for each patient from a pretrained model to predict gene expression at the patient level using annotated H&E images from The Cancer Genome Atlas (TCGA, n = 335). We demonstrate successful gene prediction on a held-out test set (n = 160, corr = 0.82 across patients, corr = 0.29 across genes) and perform exploratory analysis on an external tissue microarray (TMA) dataset (n = 498) with known IHC and survival information. Our model is able to predict gene expression and luminal PAM50 subtype (Luminal A versus Luminal B) on the TMA dataset with prognostic significance for overall survival in univariate analysis (c-index = 0.56, hazard ratio = 2.16 (95% CI 1.12-3.06), p < 5 × 10-3), and independent significance in multivariate analysis incorporating standard clinicopathological variables (c-index = 0.65, hazard ratio = 1.87 (95% CI 1.30-2.68), p < 5 × 10-3). The proposed strategy achieves superior performance while requiring less training time, resulting in less energy consumption and computational cost compared to patch-based models. Additionally, hist2RNA predicts gene expression that has potential to determine luminal molecular subtypes which correlates with overall survival, without the need for expensive molecular testing.

Outcomes of Rural Men with Breast Cancer: A Multicenter Population Based Retrospective Cohort Study.

BACKGROUND: Breast cancer is rare in men. This population-based study aimed to determine outcomes of male breast cancer in relation to residence and other variables. METHODS: In this retrospective cohort study, men diagnosed with breast cancer in Saskatchewan during 2000-2019 were evaluated. Cox proportional multivariable regression analyses were performed to determine the correlation between survival and clinicopathological and contextual factors. RESULTS: One hundred-eight eligible patients with a median age of 69 years were identified. Of them, 16% had WHO performance status ≥ 2 and 61% were rural residents. The stage at diagnosis was as follows: stage 0, 7%; I, 31%; II, 42%; III, 11%; IV, 8%. Ninety-eight percent had hormone receptor-positive breast cancer. The median disease-free survival of urban patients was 97 (95% CI: 50-143) vs. 64 (46-82) months of rural patients (p = 0.29). The median OS of urban patients was 127 (94-159) vs. 93 (32-153) months for rural patients (p = 0.27). On multivariable analysis, performance status ≥ 2, hazard ratio (HR) 2.82 (1.14-6.94), lack of adjuvant systemic therapy, HR 2.47 (1.03-5.92), and node-positive disease, HR 2.32 (1.22-4.40) were significantly correlated with inferior disease-free survival in early-stage invasive breast cancer. Whereas stage IV disease, HR 7.8 (3.1-19.5), performance status ≥ 2, HR 3.25 (1.57-6.71), and age ≥ 65 years, HR 2.37 (1.13-5.0) were correlated with inferior overall survival in all stages. CONCLUSIONS: Although residence was not significantly correlated with outcomes, rural men had numerically inferior survival. Poor performance status, node-positive disease, and lack of adjuvant systemic therapy were correlated with inferior disease-free survival.

Recent advances of small extracellular vesicle biomarkers in breast cancer diagnosis and prognosis.

Current clinical tools for breast cancer (BC) diagnosis are insufficient but liquid biopsy of different bodily fluids has recently emerged as a minimally invasive strategy that provides a real-time snapshot of tumour biomarkers for early diagnosis, active surveillance of progression, and post-treatment recurrence. Extracellular vesicles (EVs) are nano-sized membranous structures 50-1000 nm in diameter that are released by cells into biological fluids. EVs contain proteins, nucleic acids, and lipids which play pivotal roles in tumourigenesis and metastasis through cell-to-cell communication. Proteins and miRNAs from small EVs (sEV), which range in size from 50-150 nm, are being investigated as a potential source for novel BC biomarkers using mass spectrometry-based proteomics and next-generation sequencing. This review covers recent developments in sEV isolation and single sEV analysis technologies and summarises the sEV protein and miRNA biomarkers identified for BC diagnosis, prognosis, and chemoresistance. The limitations of current sEV biomarker research are discussed along with future perspective applications.

Extracellular vesicles as a novel approach for breast cancer therapeutics.

Breast cancer (BC) still lacks effective management approaches to control metastatic and therapy-resistant disease. Extracellular vesicles (EVs), with a diameter of 50-1000 nm, are secreted by all types of living cells, are protected by a lipid bilayer and encapsulate biological cargos including RNAs, proteins and lipids. They play an important role in intercellular communications and are significantly associated with pathological conditions. Accumulating evidence indicates that cancer cells secrete EVs and communicate with neighboring cells within the tumor microenvironment (TME), which plays an important role in BC metastasis, immune escape and chemoresistance, thus providing a new therapeutic window. EVs can stimulate angiogenesis and extracellular matrix remodeling, establish premetastatic niches, inhibit immune response and promote cancer metastasis. Recent advances have demonstrated that EVs are a potential therapeutic target or carrier and have emerged as promising strategies for BC treatment. In this review, we summarize the role of EVs in BC metastasis, chemoresistance and immune escape, which provides the foundation for developing novel therapeutic approaches. We also focus on current EV-based drug delivery strategies in BC and EV cargo-targeted BC therapy and discuss the limitations and future perspectives of EV-based drug delivery in BC.

Dysbiosis in Head and Neck Cancer: Determining Optimal Sampling Site for Oral Microbiome Collection.

Recent research suggests that dysbiosis of the oral microbial community is associated with head and neck cancer (HNC). It remains unclear whether this dysbiosis causes chemo-radiotherapy (CRT)-related complications. However, to address this question, it is essential to determine the most representative oral site for microbiome sampling. In this study, our purpose was to determine the optimal site for oral sample collection and whether the presence of HNC is associated with altered oral microbiome from this site. In 21 newly diagnosed HNC patients and 27 healthy controls, microbiome samples were collected from saliva, swabs from buccal mucosa, tongue, hard palate, faucial pillars and all mucosal sites combined. Microbial DNA was extracted and underwent 16S rRNA amplicon gene sequencing. In healthy controls, analysis of observed taxonomic units detected differences in alpha- and beta-diversity between sampling sites. Saliva was found to have the highest intra-community microbial diversity and lowest within-subject (temporal) and between-subject variance. Feature intersection showed that most species were shared between all sites, with saliva demonstrating the most unique species as well as highest overlap with other sites. In HNC patients, saliva was found to have the highest diversity but differences between sites were not statistically significant. Across all sites, HNC patients had lower alpha diversity than healthy controls. Beta-diversity analysis showed HNC patients' microbiome to be compositionally distinct from healthy controls. This pattern was confirmed when the salivary microbiome was considered alone. HNC patients exhibited reduced diversity of the oral microbiome. Salivary samples demonstrate temporal stability, have the richest diversity and are sufficient to detect perturbation due to presence of HNC. Hence, they can be used as representative oral samples for microbiome studies in HNC patients.

The STING agonist, DMXAA, reduces tumor vessels and enhances mesothelioma tumor antigen presentation yet blunts cytotoxic T cell function in a murine model.

We assessed the murine Stimulator of Interferon Genes (STING) agonist, DMXAA, for anti-mesothelioma potential using the AE17-sOVA model that expresses ovalbumin (OVA) as a neo tumor antigen. Dose response experiments alongside testing different routes of administration identified a safe effective treatment regimen that induced 100% cures in mice with small or large tumors. Three doses of 25mg/kg DMXAA given intra-tumorally every 9 days induced tumor regression and long-term survival (>5 months). Re-challenge experiments showed that tumor-free mice developed protective memory. MTT and propidium-iodide assays showed that DMXAA exerted direct cytotoxic effects at doses >1mg/ml on the murine AE17 and AB1 mesothelioma cell lines. In-vivo studies using a CFSE-based in-vivo proliferation assay showed that DMXAA improved tumor-antigen presentation in tumor-draining lymph nodes, evidenced by OVA-specific OT-1 T cells undergoing more divisions. An in-vivo cytotoxic T lymphocyte (CTL) assay showed that DMXAA blunted the lytic quality of CTLs recognizing the dominant (SIINFEKL) and a subdominant (KVVRFDKL) OVA epitopes. DMXAA reduced tumor vessel size in-vivo and although the proportion of T cells infiltrating tumors reduced, the proportion of tumor-specific T cells increased. These data show careful dosing and treatment protocols reduce mesothelioma cell viability and modulate tumor vessels such that tumor-antigen specific CTLs access the tumor site. However, attempts to enhance DMXAA-induced anti-tumor responses by combination with an agonist anti-CD40 antibody or IL-2 reduced efficacy. These proof-of-concept data suggest that mesothelioma patients could benefit from treatment with a STING agonist, but combination with immunotherapy should be cautiously undertaken.

Urine exosomes as biomarkers in bladder cancer diagnosis and prognosis: From functional roles to clinical significance.

Bladder cancer is one of the top ten most common cancers and top ten causes of cancer death globally. 5-year survival rates have decreased in Australia from 66% to 55% in the past three decades. The current gold standard for diagnosis is cystoscopy. However, cystoscopies are an invasive and health-resource intensive procedure which has sub-optimal sensitivity for flat lesions such as CIS (carcinoma in situ) and low specificity for differentiating inflammation from cancer - hence requiring biopsies under anesthesia. Frequent and life-long surveillance cystoscopy is required for most patients since there are high rates of progression and local recurrence in high-risk non-muscle invasive cancer (NMIBC) as well as poor outcomes associated with delayed detection of muscle-invasive bladder cancer (MIBC). There is an unmet need for a non-invasive test to provide better discrimination and risk-stratification of bladder cancer which could aid clinicians by improving patient selection for cystoscopy; enhanced risk stratification methods may guide the frequency of surveillance cystoscopies and inform treatment choices. Exosomes, which are nano-sized extracellular vesicles containing genetic material and proteins, have been shown to have functional roles in the development and progression of bladder cancer. Exosomes have also been demonstrated to be a robust source of potential biomarkers for bladder cancer diagnosis and prognosis and may also have roles as therapeutic agents. In this review, we summarize the latest evidence of biological roles of exosomes in bladder cancer and highlight their clinical significance in bladder cancer diagnosis, surveillance and treatment.

Successful use of a benchtop fluorescent enzyme immunoassay analyzer to measure serum cortisol concentration as a screening test for hypoadrenocorticism in dogs.

OBJECTIVE: To assess the diagnostic performance of a benchtop fluorescent enzyme immunoassay analyzer (AIA-360; Tosoh Bioscience Inc) for the measurement of serum cortisol concentration as a screening test for hypoadrenocorticism in dogs. ANIMALS: 173 client-owned dogs (20 with hypoadrenocorticism and 153 with nonadrenal illness). PROCEDURES: Medical records of all dogs that underwent an ACTH stimulation test between June 2015 and October 2019 were reviewed retrospectively. Dogs were excluded if the ACTH stimulation test was performed on the basis of a suspicion of hypercortisolism, serum cortisol concentrations were measured using an analyzer other than the one assessed in the present study, or dogs had received medication known to affect the pituitary-adrenal axis in the 4 weeks1,2 preceding ACTH stimulation testing. The diagnostic performance of the benchtop analyzer was evaluated by calculating sensitivity, specificity, and likelihood ratios at various cutoff points. RESULTS: Serum resting cortisol cutoff point concentrations of 0.8 µg/dL (22 nmol/L), 1 µg/dL (28 nmol/L), and 2 µg/dL (55 nmol/L) had a sensitivity of 100%. An optimal serum resting cortisol cutoff point of 0.58 µg/dL (16 nmol/L) had a sensitivity, specificity, and positive and negative likelihood ratios of 100%, 97%, and 30.6 and 0.0, respectively. CLINICAL RELEVANCE: Findings indicated that previously derived cutoff points could be used with excellent sensitivity to exclude hypoadrenocorticism in this population of dogs when serum cortisol concentration was measured with the evaluated benchtop analyzer. An ACTH stimulation test may need to only be performed to diagnose hypoadrenocorticism if resting serum cortisol concentration is ≤ 0.58 µg/dL when measured with the evaluated benchtop analyzer.

Malignant inflammatory myofibroblastic tumor: a rare case presentation.

Malignant inflammatory myofibroblastic tumors (IMT) are extremely rare, aggressive tumors with variable presentation. This is a case of a 29-year-old female presented with severe anemia and a large abdominal mass presumed to be a gastrointestinal stromal tumor (GIST). Severe anemia, leukocytosis and thrombocytosis accompanied the presentation. Final pathological assessment yielded a diagnosis of malignant IMT. Given the rarity of these tumors, no established diagnostic criteria exist aside from histological analysis of the tissue, which may result in delays or inappropriate treatment. As these tumors are aggressive in nature, a high index of suspicion is critical to improve outcomes. Further reports on the presentation, diagnosis and treatment of such rare tumors are important to develop clinical diagnostic guidelines to improve diagnosis and treatment and improve outcomes.

Survival prediction in triple negative breast cancer using multiple instance learning of histopathological images.

Computational pathology is a rapidly expanding area for research due to the current global transformation of histopathology through the adoption of digital workflows. Survival prediction of breast cancer patients is an important task that currently depends on histopathology assessment of cancer morphological features, immunohistochemical biomarker expression and patient clinical findings. To facilitate the manual process of survival risk prediction, we developed a computational pathology framework for survival prediction using digitally scanned haematoxylin and eosin-stained tissue microarray images of clinically aggressive triple negative breast cancer. Our results show that the model can produce an average concordance index of 0.616. Our model predictions are analysed for independent prognostic significance in univariate analysis (hazard ratio = 3.12, 95% confidence interval [1.69,5.75], p < 0.005) and multivariate analysis using clinicopathological data (hazard ratio = 2.68, 95% confidence interval [1.44,4.99], p < 0.005). Through qualitative analysis of heatmaps generated from our model, an expert pathologist is able to associate tissue features highlighted in the attention heatmaps of high-risk predictions with morphological features associated with more aggressive behaviour such as low levels of tumour infiltrating lymphocytes, stroma rich tissues and high-grade invasive carcinoma, providing explainability of our method for triple negative breast cancer.

Quality control validation for a veterinary laboratory network of six Sysmex XT-2000iV hematology analyzers.

BACKGROUND: Quality control (QC) validation is an important step in the laboratory harmonization process. This includes the application of statistical QC requirements, procedures, and control rules to identify and maintain ongoing stable analytical performance. This provides confidence in the production of patient results that are suitable for clinical interpretation across a network of veterinary laboratories. OBJECTIVES: To determine that a higher probability of error detection (Ped ) and lower probability of false rejection (Pfr ) using a simple control rule and one level of quality control material (QCM) could be achieved using observed analytical performance than by using the manufacturer's acceptable ranges for QCM on the Sysmex XT-2000iV hematology analyzers for veterinary use. We also determined whether Westgard Sigma Rules could be sufficient to monitor and maintain a sufficiently high level of analytical performance to support harmonization. METHODS: EZRules3 was used to investigate candidate QC rules and determine the Ped and Pfr of manufacturer's acceptable limits and also analyzer-specific observed analytical performance for each of the six Sysmex analyzers within our laboratory system using the American Society of Veterinary Clinical Pathology (ASVCP)-recommended or internal expert opinion quality goals (expressed as total allowable error, TEa ) as the quality requirement. The internal expert quality goals were generated by consensus of the Quality, Education, Planning, and Implementation (QEPI) group comprised of five clinical pathologists and seven laboratory technicians and managers. Sigma metrics, which are a useful monitoring tool and can be used in conjunction with Westgard Sigma Rules, were also calculated. RESULTS: The QC validation using the manufacturer's acceptable limits for analyzer 1 showed only 3/10 measurands reached acceptable Ped for veterinary laboratories (>0.85). For QC validation based on observed analyzer performance, the Ped was >0.94 using a 1-2.5s QC rule for the majority of observations (57/60) across the group of analyzers at the recommended TEa . We found little variation in Pfr between manufacturer acceptable limits and individual analyzer observed performance as this is a characteristic of the rule used, not the analyzer performance. CONCLUSIONS: An improved probability of error detection and probability of false rejection using a 1-2.5s QC rule for individual analyzer QC was achieved compared with the use of the manufacturers' acceptable limits for hematology in veterinary laboratories. A validated QC rule (1-2.5s) in conjunction with sigma metrics (>5.5), desirable bias, and desirable CV based on biologic variation was successful to evaluate stable analytical performance supporting continued harmonization across the network of analyzers.

Radiation doses and fractionation schedules in non-low-risk ductal carcinoma in situ in the breast (BIG 3-07/TROG 07.01): a randomised, factorial, multicentre, open-label, phase 3 study.

BACKGROUND: Whole breast irradiation (WBI) after conservative surgery for ductal carcinoma in situ (DCIS) reduces local recurrence. We investigated whether a tumour bed boost after WBI improved outcomes, and examined radiation dose fractionation sensitivity for non-low-risk DCIS. METHODS: The study was an international, randomised, unmasked, phase 3 trial involving 136 participating centres of six clinical trials organisations in 11 countries (Australia, New Zealand, Singapore, Canada, the Netherlands, Belgium, France, Switzerland, Italy, Ireland, and the UK). Eligible patients were women aged 18 years or older with unilateral, histologically proven, non-low-risk DCIS treated by breast-conserving surgery with at least 1 mm of clear radial resection margins. They were assigned to one of four groups (1:1:1:1) of no tumour bed boost versus boost after conventional versus hypofractionated WBI, or randomly assigned to one of two groups (1:1) of no boost versus boost after each centre prespecified conventional or hypofractionated WBI. The conventional WBI used was 50 Gy in 25 fractions, and hypofractionated WBI was 42·5 Gy in 16 fractions. A boost dose of 16 Gy in eight fractions, if allocated, was delivered after WBI. Patients and clinicians were not masked to treatment allocation. The primary endpoint was time to local recurrence. This trial is registered with ClinicalTrials.gov (NCT00470236). FINDINGS: Between June 25, 2007, and June 30, 2014, 1608 patients were randomly assigned to have no boost (805 patients) or boost (803 patients). Conventional WBI was given to 831 patients, and hypofractionated WBI was given to 777 patients. Median follow-up was 6·6 years. The 5-year free-from-local-recurrence rates were 92·7% (95% CI 90·6-94·4%) in the no-boost group and 97·1% (95·6-98·1%) in the boost group (hazard ratio 0·47; 0·31-0·72; p<0·001). The boost group had higher rates of grade 2 or higher breast pain (10% [8-12%] vs 14% [12-17%], p=0·003) and induration (6% [5-8%] vs 14% [11-16%], p<0·001). INTERPRETATION: In patients with resected non-low-risk DCIS, a tumour bed boost after WBI reduced local recurrence with an increase in grade 2 or greater toxicity. The results provide the first randomised trial data to support the use of boost radiation after postoperative WBI in these patients to improve local control. The international scale of the study supports the generalisability of the results. FUNDING: National Health and Medical Research Council of Australia, Susan G Komen for the Cure, Breast Cancer Now, OncoSuisse, Dutch Cancer Society, Canadian Cancer Trials Group.

Moving towards the optimization of diagnosis for patients with sarcoma: A 10-year review of externally consulted sarcoma cases in a general anatomical pathology service.

INTRODUCTION: Soft tissue sarcomas (STS) are rare, diagnostically challenging, malignant tumors with diverse histomorphologic, immunohistochemical and molecular features. In our practice, STS are reported in a general anatomical pathology practice with no formal subspecialized training in reporting these complex specimens. Our study was performed to look at the rate of external consultation (EC), along with other parameters including discordance rate, associated diagnostic delay with EC and extent of secondary work-up performed by the consultant for correct diagnosis. METHODS: The reports from 880 soft tissue sarcomas cases in the province of Saskatchewan between January 1, 2010, and December 31, 2020, were analyzed descriptively. RESULTS: Of the 880 cases reviewed in our database, 51.9% (n = 457) cases were sent to 35 different North American institutions for expert opinion. The initial diagnosis and expert opinion were in full agreement for 182 cases (39.8%), while 194 cases (42.5%) had partial agreement and 66 cases (14.4%) had zero agreement. Of the cases that had zero agreement, 20 cases (4.4%) were initially diagnosed as malignant, with a benign opinion given by the expert; and 10 cases (2.2%) were initially diagnosed as benign, which were malignant upon expert review. CONCLUSION: Soft tissue sarcomas are complex tumors that frequently require expert opinion and integration of ancillary techniques with histomorphologic features for definitive classification. A multidisciplinary, subspecialized approach to STS and availability of necessary ancillary tests would improve diagnostic accuracy. In centers where the case load would not support the full-time expertise of an STS multidisciplinary team, criteria should be developed to effectively utilize EC practices.