Deep Learning-Based Objective and Reproducible Osteosarcoma Chemotherapy Response Assessment and Outcome Prediction.

Osteosarcoma is the most common primary bone cancer, whose standard treatment includes pre-operative chemotherapy followed by resection. Chemotherapy response is used for prognosis and management of patients. Necrosis is routinely assessed after chemotherapy from histology slides on resection specimens, where necrosis ratio is defined as the ratio of necrotic tumor/overall tumor. Patients with necrosis ratio ≥90% are known to have a better outcome. Manual microscopic review of necrosis ratio from multiple glass slides is semiquantitative and can have intraobserver and interobserver variability. In this study, an objective and reproducible deep learning-based approach was proposed to estimate necrosis ratio with outcome prediction from scanned hematoxylin and eosin whole slide images (WSIs). To conduct the study, 103 osteosarcoma cases with 3134 WSIs were collected. Deep Multi-Magnification Network was trained to segment multiple tissue subtypes, including viable tumor and necrotic tumor at a pixel level and to calculate case-level necrosis ratio from multiple WSIs. Necrosis ratio estimated by the segmentation model highly correlates with necrosis ratio from pathology reports manually assessed by experts. Furthermore, patients were successfully stratified to predict overall survival with P = 2.4 × 10-6 and progression-free survival with P = 0.016. This study indicates that deep learning can support pathologists as an objective tool to analyze osteosarcoma from histology for assessing treatment response and predicting patient outcome.

Autophagy in non-immune-mediated rhabdomyolysis: Assessment of p62 immunohistochemistry.

INTRODUCTION/AIMS: p62 immunochemistry (IHC) has been shown to aid diagnosis with distinct patterns of muscle fiber staining observed in some inflammatory, hereditary, and degenerative myopathies, such as immune-mediated necrotizing myopathy (IMNM). The pattern of p62 staining may help narrow the pathological differential diagnosis of rhabdomyolysis. However, there is a lack of information on the pattern of p62 IHC in non-immune-mediated rhabdomyolysis. In this study we aim to describe histopathological findings in non-immune-mediated rhabdomyolysis, with particular emphasis on the pattern of p62 IHC. METHODS: We retrospectively reviewed the histopathological features of patients with a confirmed diagnoses of non-immune-mediated rhabdomyolysis referred to our center. RESULTS: Five patients were identified. Rhabdomyolysis was determined to be due to statin-associated toxicity in three patients, alcohol overuse in one patient, and intensive exercise in one patient. All patients showed increased numbers of necrotic and regenerating muscle fibers. Diffuse and finely granular sarcoplasmic positive p62 staining was present in scattered non-necrotic muscle fibers in all patients. DISCUSSION: Disturbance of autophagy appears to be a common mechanism in non-immune-mediated rhabdomyolysis. Our results show p62 IHC is sensitive but lacks specificity. Therefore, the pattern of p62 staining does not distinguish non-immune-mediated rhabdomyolysis from histopathologically similar IMNM.

Assessment of the Immune Response to Tumor Cell Apoptosis and Efferocytosis.

Apoptotic cells are cleared from the body principally through recognition and engulfment by neighboring phagocytes, a process known as efferocytosis. During efferocytosis, phagocytes are recruited to the site/activated by "find me" signals released from apoptotic cells, precisely identify apoptotic cells by the recognition of "eat me" signals on the apoptotic cell surface, and engulf the apoptotic cells to prevent secondary necrosis and inflammation. Thus, efferocytosis is critical for tissue homeostasis in normal physiology. However, efferocytosis of apoptotic tumor cells-performed by tumor-associated macrophages-suppresses immunity within the tumor microenvironment and limits the antitumor response. This phenomenon is further exacerbated in tumor residual disease because of the high apoptotic cell burden generated by cytotoxic therapies. Blocking efferocytosis could be a powerful approach to boost tumor immunogenicity, particularly as a combination approach with cytotoxic therapies that produce many apoptotic cells, but little is currently known about the immune response to efferocytosis. Moreover, there is a dearth of in vivo models available to study the immunologic and therapeutic consequences of blocking efferocytosis in tumor residual disease.Here, we describe a model that enables in vivo studies of tumor immunology in the aftermath of cytotoxic therapy with an emphasis on the impact of efferocytosis. Orthotopic HER2+ mammary tumors are established in immune-competent mice, followed by a single administration of lapatinib, a receptor tyrosine kinase inhibitor of HER2, to the mice that induces widespread, transient apoptosis in the tumor microenvironment. In the days following lapatinib treatment, agents that block efferocytosis such as BMS-777607 are administered. Tissue is collected from cohorts of mice at day 2 (after lapatinib treatment only) to assess apoptosis, day 8 (after lapatinib treatment followed by blockade of efferocytosis) to assess the immune response to apoptosis and efferocytosis, and day 28 (after 4 consecutive weeks of treatment) to assess therapeutic efficacy. This model enables mechanistic studies of tumor immunology in residual disease as well as therapeutic efficacy studies of targeted agents that disrupt efferocytosis.

In vivo porcine characterization of atrial lesion safety and efficacy utilizing a circular pulsed-field ablation catheter including assessment of collateral damage to adjacent tissue in supratherapeutic ablation applications.

INTRODUCTION: Pulsed-field ablation (PFA), an ablative method that causes cell death by irreversible electroporation, has potential safety advantages over radiofrequency ablation and cryoablation. Pulmonary vein (PV) isolation was performed in a porcine model to characterize safety and performance of a novel, fully-integrated biphasic PFA system comprising a multi-channel generator, variable loop circular catheter, and integrated PFA mapping software module. METHODS: Eight healthy porcine subjects were included. To evaluate safety, multiple ablations were performed, including sites not generally targeted for therapeutic ablation, such as the right inferior PV lumen, right superior PV ostium, and adjacent to the esophagus and phrenic nerve. To evaluate the efficacy, animals were recovered, followed for 30(±3) days, then re-mapped. Gross pathological and histopathological examinations assessed procedural injuries, chronic thrombosis, tissue ablation, penetration depth, healing, and inflammatory response. RESULTS: All eight animals survived follow-up. PV narrowing was not observed acutely nor at follow-up, even when ablation was performed deep to the PV ostium. No injury was seen grossly or histologically in adjacent structures. All PVs were durably isolated, confirmed by bidirectional block at re-map procedure. Histological examination showed complete, transmural necrosis around the circumference of the ablated section of right PVs. CONCLUSION: This preclinical evaluation of a fully-integrated PFA system demonstrated effective and durable ablation of cardiac tissue and PV isolation without collateral damage to adjacent structures, even when ablation was performed in more extreme settings than those used therapeutically. Histological staining confirmed complete transmural cell necrosis around the circumference of the PV ostium at 30 days.

The centrally restricted diffusion sign on MRI for assessment of radiation necrosis in metastases treated with stereotactic radiosurgery.

PURPOSE: Differentiation of radiation necrosis from tumor progression in brain metastases treated with stereotactic radiosurgery (SRS) is challenging. For this, we assessed the performance of the centrally restricted diffusion sign. METHODS: Patients with brain metastases treated with SRS who underwent a subsequent intervention (biopsy/resection) for a ring-enhancing lesion on preoperative MRI between 2000 and 2020 were included. Excluded were lesions containing increased susceptibility limiting assessment of DWI. Two neuroradiologists classified the location of the diffusion restriction with respect to the post-contrast T1 images as centrally within the ring-enhancement (the centrally restricted diffusion sign), peripherally correlating to the rim of contrast enhancement, both locations, or none. Measures of diagnostic accuracy and 95% CI were calculated for the centrally restricted diffusion sign. Cohen's kappa was calculated to identify the interobserver agreement. RESULTS: Fifty-nine patients (36 female; mean age 59, range 40 to 80) were included, 36 with tumor progression and 23 with radiation necrosis based on histopathology. Primary tumors included 34 lung, 12 breast, 5 melanoma, 3 colorectal, 2 esophagus, 1 head and neck, 1 endometrium, and 1 thyroid. The centrally restricted diffusion sign was seen in 19/23 radiation necrosis cases (sensitivity 83% (95% CI 63 to 93%), specificity 64% (95% CI 48 to 78%), PPV 59% (95% CI 42 to 74%), NPV 85% (95% CI 68 to 94%)) and 13/36 tumor progression cases (difference p < 0.001). Interobserver agreement was substantial, at 0.61 (95% CI 0.45 to 70.8). CONCLUSION: We found a low probability of radiation necrosis in the absence of the centrally restricted diffusion sign.

Histology to 3D in vivo MR registration for volumetric evaluation of MRgFUS treatment assessment biomarkers.

Advances in imaging and early cancer detection have increased interest in magnetic resonance (MR) guided focused ultrasound (MRgFUS) technologies for cancer treatment. MRgFUS ablation treatments could reduce surgical risks, preserve organ tissue and function, and improve patient quality of life. However, surgical resection and histological analysis remain the gold standard to assess cancer treatment response. For non-invasive ablation therapies such as MRgFUS, the treatment response must be determined through MR imaging biomarkers. However, current MR biomarkers are inconclusive and have not been rigorously evaluated against histology via accurate registration. Existing registration methods rely on anatomical features to directly register in vivo MR and histology. For MRgFUS applications in anatomies such as liver, kidney, or breast, anatomical features that are not caused by the treatment are often insufficient to drive direct registration. We present a novel MR to histology registration workflow that utilizes intermediate imaging and does not rely on anatomical MR features being visible in histology. The presented workflow yields an overall registration accuracy of 1.00 ± 0.13 mm. The developed registration pipeline is used to evaluate a common MRgFUS treatment assessment biomarker against histology. Evaluating MR biomarkers against histology using this registration pipeline will facilitate validating novel MRgFUS biomarkers to improve treatment assessment without surgical intervention. While the presented registration technique has been evaluated in a MRgFUS ablation treatment model, this technique could be potentially applied in any tissue to evaluate a variety of therapeutic options.

Ki-67 immunoexpression and radiological assessment of necrosis improves accuracy of conventional and modified core biopsy systems in predicting the final grade assigned to adult-soft tissue sarcomas. An international collaborative study.

Based on the French Federation Nationale des Centers de Lutte Contre le Cancer (FNCLCC) grading system, this study assesses the accuracy of conventional and modified core biopsy (CB) systems in predicting the final grade (low vs high) assigned to the resected specimen. Substituting Ki-67 immunoexpression for mitotic count, and radiological for histological assessment of necrosis, we used two modified FNCLCC CB grading systems: (1) Ki-67 immunoexpression alone, and (2) Ki-67 plus radiological assessment of necrosis. We graded 199 soft tissue sarcomas (STS) from nine centers, and compared the results for the conventional (obtained from local histopathology reports) and modified CB systems with the final FNCLCC grading of the corresponding resected specimens. Due to insufficient sample quality or lack of available radiologic data, five cases were not evaluated for Ki67 or radiological assessment of necrosis. The conventional FNCLCC CB grading system accurately identified 109 of the 130 high-grade cases (83.8%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (87.9%) of the 199 resected tumors; overestimating the final grade in three cases and underestimating in 21 cases. Modified system 1 (Ki-67) accurately identified 117 of the 130 high-grade cases (90.0%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (89.7%) of the 195 evaluated cases; overestimating seven and underestimating 13 cases. Modified system 2 (Ki-67 plus radiological necrosis) accurately identified 120 of the 130 high-grade cases (92.3%). This last matched the final FNCLCC grading (low vs high) in 177 (91.2%) of the 194 evaluated cases; overestimating seven and underestimating 10 cases. Modified system 2 obtained highest area under ROC curves, although not statistically significant. Underestimated CB grades did not correlate with histological subtypes, although many of the discrepant cases were myxoid tumors (myxofibrosarcomas or myxoid liposarcomas), leiomyosarcomas or undifferentiated pleomorphic/spindle cell sarcomas. Using modified FNCLCC CB grading systems to replace conventional mitotic count and histologic assessment of necrosis may improve the distinction between low and high-grade STS on CB. Our study confirms that classifying grade 1 as low grade and grades 2 and 3 as high grade improves correlation between CB and final grade by up to 21%, irrespective of CB system used. A higher than expected Ki-67 score in a low-grade sarcoma diagnosed on CB should raise concern that a higher-grade component may not have been sampled. Furthermore, correlation of all clinicopathological and radiological findings at multidisciplinary meetings is essential to assess the histological grade on CB as accurately as possible.

Assessment of metals induced histopathological and gene expression changes in different organs of non-diabetic and diabetic rats.

Diabetes is a complex metabolic disorder and different environmental toxicants including heavy metals have been involved in diabetes induction. Therefore, assessment of the environmental risk factors and heavy metals induced toxicity have become critical for reducing the consequences of metals pollutants. Previously, we reported heavy metals induced nephrotoxicity in non-diabetic and diabetic rats. Here, we extended our analysis by examining the heavy metals induced organs (heart, kidney, liver, pancreas, and spleen) damage in diabetic and non-diabetic Wistar rats using histopathology and quantitative real-time PCR (qRT-PCR). Following the generation of the diabetic rat model, the animals were exposed to heavy metals including lead (Pb), arsenic (As), manganese (Mn) and cadmium (Cd). Both non-diabetic and diabetic rats were exposed to heavy metals for 30 days and subsequently, the heart, kidney, liver, pancreas and spleen tissues were examined. Heavy metal treatment resulted in irregularly arranged myofibrils and vacuolization in the heart tissue of metal treated groups as evident from hematoxylin and eosin (H & E) staining. The kidney tissue of rats treated with heavy metals showed tubular degeneration, fibrosis, hemorrhage, and vacuolation. The liver of the heavy metals treated rats exhibited cellular degeneration and necrosis. The pancreatic tissue of streptozotocin injected untreated and metal treated rats revealed severe degeneration, necrosis, degranulation, shrinkage, and depression in the islets of Langerhans. Increased red pulp area and congestion were observed in the spleen of the metal mixture treated non-diabetic and diabetic rats. In line with the histological data, the qRT-PCR analysis showed downregulated expression of Bcl2 and upregulation of Caspase-3 in non-diabetic and diabetic metal treated rats as compared to the non-diabetic untreated rats. In conclusion, the present study revealed, diabetic rats are more prone to metal alone as well as metal mixture induced organ damage as compared to non-diabetic rats.

Clinical Performance of the Idylla MSI Test for a Rapid Assessment of the DNA Microsatellite Status in Human Colorectal Cancer.

In this study, the clinical performance of the Idylla MSI test (investigational use only) was evaluated in 330 colorectal carcinoma samples (all stages). This test is fully automated, from formalin-fixed, paraffin-embedded slide to result, and gives a result in <2.5 hours. Compared with the Promega MSI Analysis System version 1.2, an overall agreement, sensitivity, and specificity of 99.7%, 98.7%, and 100%, respectively, was reached. Whereas seven samples were invalid with the Promega MSI Analysis System, only two were invalid with the Idylla MSI test. Compared with the historical immunohistochemistry (IHC) data, overall agreement, sensitivity, and specificity of 98.7%, 94.4%, and 100%, respectively, were observed. Tumor mutation burden analysis of the discordant IHC cases was in favor of the Idylla MSI test result in three of the four samples. Furthermore, for those cases where the IHC data were invalid or hard to interpret because sole loss of one DNA mismatch repair deficiency marker was observed, Idylla MSI test results were always valid and accurate. Herein, the Idylla MSI test has been shown to be an accurate, fast screening assay for the detection of microsatellite status in colorectal cancer patients, with a low number of invalid results.

Viable and necrotic tumor assessment from whole slide images of osteosarcoma using machine-learning and deep-learning models.

Pathological estimation of tumor necrosis after chemotherapy is essential for patients with osteosarcoma. This study reports the first fully automated tool to assess viable and necrotic tumor in osteosarcoma, employing advances in histopathology digitization and automated learning. We selected 40 digitized whole slide images representing the heterogeneity of osteosarcoma and chemotherapy response. With the goal of labeling the diverse regions of the digitized tissue into viable tumor, necrotic tumor, and non-tumor, we trained 13 machine-learning models and selected the top performing one (a Support Vector Machine) based on reported accuracy. We also developed a deep-learning architecture and trained it on the same data set. We computed the receiver-operator characteristic for discrimination of non-tumor from tumor followed by conditional discrimination of necrotic from viable tumor and found our models performing exceptionally well. We then used the trained models to identify regions of interest on image-tiles generated from test whole slide images. The classification output is visualized as a tumor-prediction map, displaying the extent of viable and necrotic tumor in the slide image. Thus, we lay the foundation for a complete tumor assessment pipeline from original histology images to tumor-prediction map generation. The proposed pipeline can also be adopted for other types of tumor.

Assessment of tumour-associated necrosis provides prognostic information additional to World Health Organization/International Society of Urological Pathology grading for clear cell renal cell carcinoma.

AIMS: The aims of this study were to evaluate the impact of tumour-associated necrosis (TAN) on metastasis-free survival for clear cell renal cell carcinoma (RCC), and to determine whether TAN provides survival information additional to World Health Organization (WHO)/International Society of Urological Pathology (ISUP) grading. METHODS AND RESULTS: The study consisted of 376 cases of clear cell RCC treated by nephrectomy, for which follow-up was available. WHO/ISUP grade was assigned, and sections were assessed for the presence of TAN. American Joint Committee on Cancer (AJCC) pT staging category and tumour size were also recorded. The development of metastatic disease was taken as the clinical endpoint, and survival analyses, utilising univariate and multivariate models, were performed. WHO/ISUP grades were: grade 1, 35 cases (9.3%); grade 2, 188 cases (50.0%); grade 3, 91 cases (24.2%); and grade 4, 62 cases (16.5%). Staging categories were pT1-pT2 [234 tumours (62.2%)] and pT3-pT4 [139 tumours (37.0%)]. TAN was seen in 128 cases (34.0%). Neither TAN nor metastases were seen in grade 1 tumours. Among grade 2-4 tumours, those with TAN had a significantly worse prognosis than those without TAN (P = 0.017, P = 0.04, and P = 0.006, respectively). Multivariate analysis (WHO/ISUP grade, pT staging category, and TAN) showed all three variables to be independently associated with outcome (P = 0.009, P = 0.005, and P = 0.001, respectively). For all tumour grades and pT staging categories, it was found that the presence of TAN was associated with a 2.91-fold greater risk of metastatic disease. CONCLUSION: Tumour-associated necrosis is an important prognostic factor for clear cell RCC, independently of WHO/ISUP grade. This supports the suggestion that TAN could be incorporated into tumour grading criteria.

Volume and Shape Assessment of Renal Radiofrequency Ablation Lesion.

OBJECTIVE: To evaluate the microscopic characteristics and quantify the volume of a radiofrequency ablation (RFA) lesion. METHODS: Ten male pigs were submitted to laparoscopic RFA. An RFA needle was introduced in the lower pole of the left kidney to create a lesion expected to be a sphere of 2.0 cm diameter. The animals were followed up for 21 days. Kidneys were weighed and the volume was assessed using Scherle's method. Cavalieri's principle was used to assess the RFA volume, and sphericity was calculated to assess RFA lesion. One sample t test was used to compare RFA volume with the volume of a sphere of 2.0 cm diameter, and sphericity to hypothetical values of a sphere and an icosahedron. Fragments of RFA region were histologically evaluated. RESULTS: Three animals developed postoperative complications and were excluded from the analyses. There was no difference in the mean weight and volume between right and left kidneys following RFA. The mean total volume of RFA injury was 3.44 cm3. There was no difference in the RFA volume in comparison with hypothetical volumes of a sphere and an icosahedron of 2.0 cm diameter. Sphericity of the RFA injury was not similar with a sphere; however, it was similar to an icosahedron. Histology revealed areas of coagulation necrosis, fibrosis, and inflammatory mononuclear infiltration. Areas with normal tissue were also observed. CONCLUSION: The volume of injury caused by RFA was as expected and its shape was comparable with an icosahedron. Microscopic evaluation revealed areas of normal-appearing tissue.

First Evaluation of Radioiodinated Flavonoids as Necrosis-Avid Agents and Application in Early Assessment of Tumor Necrosis.

A rapid and accurate identification of necrotic tissues is of great importance to define disease severity, predict prognosis, and monitor responses to therapies. To seek necrosis-avid agents with clinically translational potential, we first evaluated the necrosis avidity of flavonoids in rodent models of muscular, myocardial, and tumoral necrosis. In this study, the necrosis avidity of eight radioiodinated 5,7-dihydroxyflavones was tested by ex vivo gamma counting, histochemical staining, and autoradiography in mouse models of ethanol-induced muscular necrosis. The necrosis avidity of a lead tracer, 131I-5, was further assessed in rat models of myocardial infarction and reperfusion. Therapy response was evaluated by 131I-5 single photon emission computed tomography/computed tomography imaging 24 h after combretastatin A-4 disodium phosphate (CA4P) therapy on rats bearing W256 breast carcinomas. The necrosis avidity mechanism for the tracers was studied by in vitro DNA binding experiments of 12 5,7-dihydroxyflavones and in vivo blocking experiments of 131I-5. In the results, all 131I-5,7-dihydroxyflavones showed intense uptake to necrotic muscles, and 131I-5 emerged as the most potential tracer among them. 131I-5 obtained a necrotic-viable myocardium ratio of 5.0 ± 0.9 in post-mortem biodistribution on reperfused myocardial infarction models and achieved necrosis imaging on CA4P-treated W256 tumors 4 h after tracer injection. DNA binding studies suggested that necrosis avidity was related to DNA binding to a certain extent. The uptake of 131I-5 in necrotic muscle was markedly blocked by excessive ethidium bromide and cold 5 with a 51.95% and 64.29% decline at 1 h after coinjection, respectively. In conclusion, flavonoids are necrosis-avid agents. Furthermore, 131I-5 can serve as a promising necrosis-avid diagnostic tracer for the rapid imaging of necrotic tissues, supporting the further molecular design of radiotracer based on 5.

Assessment of histopathological features of maculopapular viral exanthem and drug-induced exanthem.

BACKGROUND: Viral infections and drug reactions are the commonest causes of exanthems in clinical practice. Clinically, their overlapping features may pose a diagnostic challenge. Hematologic, in vitro, and drug provocation tests are either unreliable or impractical. METHODS: This was a descriptive, prospective study to assess and compare histopathological features of maculopapular viral and drug exanthem. Subjects fulfilling case definition of exanthems were included. Serum C-reactive protein (CRP) and absolute eosinophil count (AEC) were also studied. RESULTS: Skin biopsy slides of 48 cases were evaluated and AEC and CRP were performed. Both median AEC and CRP were lower in viral exanthem compared with drug exanthem. On histopathological evaluation, features such as lymphocytic exocytosis, and dermal infiltrate of eosinophils, lymphocytes and histiocytes were seen in a significantly greater number of drug exanthems. Other findings such as focal spongiosis, acanthosis, keratinocyte necrosis, basal cell damage, papillary dermal edema and atypical lymphocytes in the dermis were also observed in higher though not statistically significant number of drug exanthem biopsies. CONCLUSIONS: Certain histopathological features can help to differentiate between the two exanthems and this modality may be used in situations when there is clinical overlap and when drug rechallenge cannot be undertaken.

Risk assessment in solitary fibrous tumors: validation and refinement of a risk stratification model.

Solitary fibrous tumors are an uncommon sarcoma type characterized by NAB2-STAT6 gene fusion. While solitary fibrous tumors metastasize in 5-25% of cases, it has historically been challenging to determine which specific tumor and patient characteristics predict aggressive behavior. We previously reported on a novel risk stratification scheme for solitary fibrous tumors incorporating patient age, tumor size, and mitotic activity to predict risk of metastasis. Herein we validate this risk stratification scheme in an independent, lower-risk population of 79 patients with primary non-meningeal solitary fibrous tumors, and propose incorporating tumor necrosis as a fourth variable to further improve the risk score. Fifty-seven percent of cases were considered low risk, 29% intermediate risk, and 14% high risk for metastasis. Of 50 patients with sufficient clinical follow-up data, no metastases developed in the low-risk patients (n=23), while there was a 7% 10-year metastatic risk in the intermediate risk group (n=17), and a 49% 5-year metastatic risk for the high-risk patients (n=10). When tumor necrosis was added as a fourth variable to the model, predictive power was enhanced. Under the revised stratification, the proportion of tumors identified as low risk increased to 66%, with no metastasis at 10 years, intermediate risk cases comprised 24% with 10% risk of metastasis at 10 years, and high risk comprised 10% of cases with 73% risk of metastasis at 5 years. In Kaplan-Meier analysis, this fourth-variable stratification provided significant discrimination between the risk groups (P=0.0005). These findings confirmed the clinical utility of our previously published risk stratification model and support the inclusion of necrosis as a fourth variable in the model.

Histologic Appearance After Preoperative Radiation Therapy for Soft Tissue Sarcoma: Assessment of the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group Response Score.

PURPOSE: To critically assess the prognostic value of the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group (EORTC-STBSG) response score and define histologic appearance after preoperative radiation therapy (RT) for soft tissue sarcoma (STS). METHODS AND MATERIALS: For a cohort of 100 patients with STS of the extremity/trunk treated at our institution with preoperative RT followed by resection, 2 expert sarcoma pathologists evaluated the resected specimens for percent residual viable cells, necrosis, hyalinization/fibrosis, and infarction. The EORTC response score and other predictors of recurrence-free survival (RFS) and overall survival (OS) were assessed by Kaplan-Meier and proportional hazard models. RESULTS: Median tumor size was 7.5 cm; 92% were intermediate or high grade. Most common histologies were unclassified sarcoma (34%) and myxofibrosarcoma (25%). Median follow-up was 60 months. The 5-year local recurrence rate was 5%, 5-year RFS was 68%, and 5-year OS was 75%. Distribution of cases according to EORTC response score tiers was as follows: no residual viable tumor for 9 cases (9% pathologic complete response); <1% viable tumor for 0, ≥1% to <10% for 9, ≥10% to <50% for 44, and ≥50% for 38. There was no association between EORTC-STBSG response score and RFS or OS. Conversely, hyalinization/fibrosis was a significant independent favorable predictor for RFS (hazard ratio 0.49, P=.007) and OS (hazard ratio 0.36, P=.02). CONCLUSION: Histologic evaluation after preoperative RT for STS showed a 9% pathologic complete response rate. The EORTC-STBSG response score and percent viable cells were not prognostic. Hyalinization/fibrosis was associated with favorable outcome, and if validated, may become a valid endpoint for neoadjuvant trials.

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.

BACKGROUND/AIMS: For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. METHODS: A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. RESULTS: Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. CONCLUSION: Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments.

Assessment of micronecrotic tumor tissue using dynamic contrast-enhanced magnetic resonance imaging.

Compartmental models for evaluation of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) datasets assume a homogeneous interstitital volume distribution and homogeneous contrast agent (CA) distribution within each compartment, neglecting effects of CA diffusion within the compartments. When necrotic or micronecrotic tumor tissue is present, these assumptions may no longer be valid. Therefore, the present study investigates the validity of three compartmental models in assessing tumors with necrotic components. The general diffusion equation for inhomogeneous tissue was used to simulate the extravasation of a low-molecular-weight contrast agent from a feeding vessel into the interstitial space. The simulated concentration-time curves were evaluated using the extended Tofts model, a parallel 3-compartment model, and a sequential 3-compartment model. The extended Tofts model overestimated the interstitial volume fraction by a median of 6.9% resp. 10.0% and the parallel 3-compartment model by 8.6% resp. 15.5%, while the sequential 3-compartment model overestimated it by 0.2% resp. underestimated it by 18.8% when simulating a mean vessel distance of 100µm resp. 150µm. Overall, the sequential 3-compartment model provided more reliable results both for the total fractional interstitial volume and for the interstitial subcompartments.

Epidemiologic aspects of necrotic enteritis in broiler chickens - disease occurrence and production performance.

Since future conventional broiler production can no longer rely upon in-feed antimicrobials (anticoccidials and antibiotic growth promoters), understanding the most important non-antimicrobial factors influencing occurrence of necrotic enteritis (NE) in poultry will become urgent. Solid population-based data on NE occurrence are scarce. Additionally, data on cholangiohepatitis (CPH) at slaughter is a useful indirect measurement of NE occurrence. Existing data suggest that coccidiosis and nutritional factors are among the most important determinants of NE occurrence. Dietary cereal contents and dietary level of animal proteins can both influence NE occurrence, but cereal composition may be more important because cereals constitute a larger portion of the diet. Losses associated with NE vary depending on the severity of the disease, but data indicate that the farmers' profit may be reduced on average with as much as one third during an epidemic of clinical disease.

Best practices for application of attachment cells to in vitro micronucleus assessment by flow cytometry.

This work seeks to provide users with guidance on cell culture, treatment, processing and analytical conditions for achieving optimal performance of the in vitro micronucleus assay using the In Vitro MicroFlow(®) method. Experimental data are provided to support the advice described. The information provided covers specific topics or issues that are identified as critical to the methodology and thus is meant to work with instruction manuals, published papers and other references, and not as a replacement for these documents. The content is divided into several sections. Cell culture and treatment describes conditions for routine maintenance of cells as well as treatment with test articles. Preparation and processing of samples details steps found to be critical in execution of the procedure. Instrument parameters and analysis covers set-up of the flow cytometer and evaluation of the samples. General assay considerations and interpretation of results describes examination of data in terms of assay validity, viability and genotoxicity assessment. The goal is to educate users and enable them to design, conduct and interpret flow cytometric in vitro micronucleus (MN) studies. Readers should obtain an understanding of specific cell culture practices, options for assay formatting and execution and the information required to successfully integrate and validate the in vitro MN assay into their existing safety program.