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Second harmonic generation (SHG) microscopy is acknowledged as an established imaging technique capable to provide information on the collagen architecture in tissues that is highly valuable for the diagnostics of various pathologies. The polarization-resolved extension of SHG (PSHG) microscopy, together with associated image processing methods, retrieves extensive image sets under different input polarization settings, which are not fully exploited in clinical settings. To facilitate this, we introduce PSHG-TISS, a collection of PSHG images, accompanied by additional computationally generated images which can be used to complement the subjective qualitative analysis of SHG images. These latter have been calculated using the single-axis molecule model for collagen and provide 2D representations of different specific PSHG parameters known to account for the collagen structure and distribution. PSHG-TISS can aid refining existing PSHG image analysis methods, while also supporting the development of novel image processing and analysis methods capable to extract meaningful quantitative data from the raw PSHG image sets. PSHG-TISS can facilitate the breadth and widespread of PSHG applications in tissue analysis and diagnostics., (© 2022. The Author(s).)

Background: Systemic insulin resistance is generally postulated as an independent risk factor of cardiovascular events in type 2 diabetes (T2D). However, the role of myocardial insulin resistance (mIR) remains to be clarified., Methods: Two 18 F-FDG PET/CT scans were performed on forty-three T2D patients at baseline and after hyperinsulinemic-euglycemic clamp (HEC). Myocardial insulin sensitivity (mIS) was determined by measuring the increment in myocardial 18 F-FDG uptake after HEC. Coronary artery calcium scoring (CACs) and myocardial radiodensity (mRD) were assessed by CT., Results: After HEC, seventeen patients exhibited a strikingly enhancement of myocardial 18 F-FDG uptake and twenty-six a marginal increase, thus revealing mIS and mIR, respectively. Patients with mIR showed higher mRD (HU: 38.95 [33.81-44.06] vs. 30.82 [21.48-38.02]; p = 0.03) and CACs > 400 (AU: 52% vs. 29%; p = 0.002) than patients with mIS. In addition, HOMA-IR and mIS only showed a correlation in those patients with mIR., Conclusions: 18 F-FDG PET combined with HEC is a reliable method for identifying patients with mIR. This subgroup of patients was found to be specifically at high risk of developing cardiovascular events and showed myocardial structural changes. Moreover, the gold-standard HOMA-IR index was only associated with mIR in this subgroup of patients. Our results open up a new avenue for stratifying patients with cardiovascular risk in T2D.

Second harmonic generation (SHG) microscopy has emerged over the past two decades as a powerful tool for tissue characterization and diagnostics. Its main applications in medicine are related to mapping the collagen architecture of in-vivo, ex-vivo and fixed tissues based on endogenous contrast. In this work we present how H&E staining of excised and fixed tissues influences the extraction and use of image parameters specific to polarization-resolved SHG (PSHG) microscopy, which are known to provide quantitative information on the collagen structure and organization. We employ a theoretical collagen model for fitting the experimental PSHG datasets to obtain the second order susceptibility tensor elements ratios and the fitting efficiency. Furthermore, the second harmonic intensity acquired under circular polarization is investigated. The evolution of these parameters in both forward- and backward-collected SHG are computed for both H&E-stained and unstained tissue sections. Consistent modifications are observed between the two cases in terms of the fitting efficiency and the second harmonic intensity. This suggests that similar quantitative analysis workflows applied to PSHG images collected on stained and unstained tissues could yield different results, and hence affect the diagnostic accuracy., Competing Interests: The authors declare no competing interests., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Subsets of non-acute promyelocytic leukemia (APL) acute myelogenous leukemia (AML) exhibit aberrant retinoid signaling and demonstrate sensitivity to retinoids in vitro . We present the results of a phase 1 dose-escalation study that evaluated the safety, pharmacodynamics, and efficacy of IRX195183, a novel retinoic acid receptor α agonist, in patients with relapsed or refractory myelodysplastic syndrome (MDS) or AML. In this single center, single arm study, eleven patients with relapsed or refractory MDS/AML were enrolled and treated. Oral IRX195183 was administered at two dose levels: 50 mg daily or 75 mg daily for a total of two 28-day cycles. Patients with stable disease or better were allowed to continue on the drug for four additional 28-day cycles. Common adverse events included hypertriglyceridemia, fatigue, dyspnea, and edema. Three patients at the first dose level developed asymptomatic Grade 3 hypertriglyceridemia. The maximally tolerated dose was not reached. Four of the eleven patients had (36%) stable disease or better. One had a morphological complete remission with incomplete hematologic recovery while on the study drug. Two patients had evidence of in vivo leukemic blast maturation, as reflected by increased CD38 expression. In a pharmacodynamics study, plasma samples from four patients treated at the lowest dose level demonstrated the capacity to differentiate leukemic cells from the NB4 cell line in vitro . These results suggest that IRX195183 is safe, achieves biologically meaningful plasma concentrations and may be efficacious in a subset of patients with MDS/AML. Clinical Trial Registration: clinicaltrials.gov, identifier NCT02749708., (Copyright © 2020 Ambinder, Norsworthy, Hernandez, Palau, Paun, Duffield, Chandraratna, Sanders, Varadhan, Jones, Douglas Smith and Ghiaur.)

Background: Skeletal muscle injury characterisation during healing supports trauma prognosis. Given the potential interest of computed tomography (CT) in muscle diseases and lack of in vivo CT methodology to image skeletal muscle wound healing, we tracked skeletal muscle injury recovery using in vivo micro-CT in a rat model to obtain a predictive model., Methods: Skeletal muscle injury was performed in 23 rats. Twenty animals were sorted into five groups to image lesion recovery at 2, 4, 7, 10, or 14 days after injury using contrast-enhanced micro-CT. Injury volumes were quantified using a semiautomatic image processing, and these values were used to build a prediction model. The remaining 3 rats were imaged at all monitoring time points as validation. Predictions were compared with Bland-Altman analysis., Results: Optimal contrast agent dose was found to be 20 mL/kg injected at 400 μL/min. Injury volumes showed a decreasing tendency from day 0 (32.3 ± 12.0mm 3 , mean ± standard deviation) to day 2, 4, 7, 10, and 14 after injury (19.6 ± 12.6, 11.0 ± 6.7, 8.2 ± 7.7, 5.7 ± 3.9, and 4.5 ± 4.8 mm 3 , respectively). Groups with single monitoring time point did not yield significant differences with the validation group lesions. Further exponential model training with single follow-up data (R 2 = 0.968) to predict injury recovery in the validation cohort gave a predictions root mean squared error of 6.8 ± 5.4 mm 3 . Further prediction analysis yielded a bias of 2.327., Conclusion: Contrast-enhanced CT allowed in vivo tracking of skeletal muscle injury recovery in rat.

Quantitative second harmonic generation microscopy was used to investigate collagen organization in the fibrillar capsules of human benign and malignant thyroid nodules. We demonstrate that the combination of texture analysis and second harmonic generation images of collagen can be used to differentiate between capsules surrounding the thyroid follicular adenoma and papillary carcinoma nodules. Our findings indicate that second harmonic generation microscopy can provide quantitative information about the collagenous capsule surrounding both the thyroid and thyroid nodules, which may complement traditional histopathological examination., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

The aim of the present study is to characterize the hemodynamics of left ventricular (LV) geometries to examine the impact of trabeculae and papillary muscles (PMs) on blood flow using high performance computing (HPC). Five pairs of detailed and smoothed LV endocardium models were reconstructed from high-resolution magnetic resonance images (MRI) of ex-vivo human hearts. The detailed model of one LV pair is characterized only by the PMs and few big trabeculae, to represent state of art level of endocardial detail. The other four detailed models obtained include instead endocardial structures measuring ≥1 mm 2 in cross-sectional area. The geometrical characterizations were done using computational fluid dynamics (CFD) simulations with rigid walls and both constant and transient flow inputs on the detailed and smoothed models for comparison. These simulations do not represent a clinical or physiological scenario, but a characterization of the interaction of endocardial structures with blood flow. Steady flow simulations were employed to quantify the pressure drop between the inlet and the outlet of the LVs and the wall shear stress (WSS). Coherent structures were analyzed using the Q-criterion for both constant and transient flow inputs. Our results show that trabeculae and PMs increase the intra-ventricular pressure drop, reduce the WSS and disrupt the dominant single vortex, usually present in the smoothed-endocardium models, generating secondary small vortices. Given that obtaining high resolution anatomical detail is challenging in-vivo , we propose that the effect of trabeculations can be incorporated into smoothed ventricular geometries by adding a porous layer along the LV endocardial wall. Results show that a porous layer of a thickness of 1.2·10 -2 m with a porosity of 20 kg/m 2 on the smoothed-endocardium ventricle models approximates the pressure drops, vorticities and WSS observed in the detailed models.

Cancers have been revealed to be extremely heterogenous in terms of the frequency and types of mutations present in cells from different malignant tumors. Thus, it is likely that uniform clinical treatment is not optimal for all patients, and that the development of individualized therapeutic regimens may be beneficial. We describe the generation of multiple, unique small peptides nine to thirty-four amino acids in length which, when labeled with the radioisotope (32)P, bind with vastly differing efficiencies to cell lines derived from different colon adenocarcinomas. In addition, the most effective of these peptides permanently transfers the (32)P radioisotope to colorectal cancer cellular proteins within two hours at a rate that is more than 150 times higher than in cell lines derived from other cancers or from the normal tissues tested. Currently, the only two FDA-approved radioimmunotherapeutic agents in use both employ antibodies directed against the B cell marker CD20 for the treatment of non-Hodgkin's lymphoma. By using the method described herein, large numbers of different (32)P-labeled peptides can be readily produced and assayed against a broad spectrum of cancer types. This report proposes the development and use of (32)P-labeled peptides as potential individualized peptide-binding therapies for the treatment of colon adenocarcinoma patients.

Human pituitary tumor-transforming 1 (PTTG1)/securin is a putative oncoprotein that is overexpressed in various tumor types. However, the involvement of PTTG1 in gastrointestinal cancer development and progression remains unclear. In this study, we investigated the clinical significance and biological effects of PTTG1 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical studies performed on 113 primary ESCC specimens revealed a high prevalence of PTTG1 overexpression (60.2%), which was significantly associated with lymph node metastasis (regional, P = 0.042; distant, P = 0.005), advanced tumor stage (P = 0.028), and poorer overall survival (P = 0.017, log-rank test; P = 0.044, Cox proportional hazard model). Eleven ESCC cell lines expressed PTTG1 protein at levels 2.4 to 6.6 times higher than those in normal esophageal epithelial cells (HEEpiC). PTTG1 protein expression was confined to the nucleus in HEEpiC cells but present in both the cytoplasm and nucleus in ESCC cells. Two small interfering RNAs (siRNA) inhibited PTTG1 mRNA and protein expression in three ESCC cell lines by 77% to 97%. In addition, PTTG1 down-regulation by these siRNAs significantly reduced cell motility in all three ESCC cell lines (P < 0.01) in vitro, as well as popliteal lymph node metastases of ESCC cells in nude mice (P = 0.020). Global gene expression profiling suggested that several members of the Ras and Rho gene families, including RRAS, RHOG, ARHGAP1, and ARHGADIA, represented potential downstream genes in the PTTG1 pathway. Taken together, these findings suggest that PTTG1 overexpression promotes cell motility and lymph node metastasis in ESCC patients, leading to poorer survival. Thus, PTTG1 constitutes a potential biomarker and therapeutic target in ESCCs with lymph node metastases.

Background: Barrett's esophagus predisposes to esophageal adenocarcinoma. However, the value of endoscopic surveillance in Barrett's esophagus has been debated because of the low incidence of esophageal adenocarcinoma in Barrett's esophagus. Moreover, high inter-observer and sampling-dependent variation in the histologic staging of dysplasia make clinical risk assessment problematic. In this study, we developed a 3-tiered risk stratification strategy, based on systematically selected epigenetic and clinical parameters, to improve Barrett's esophagus surveillance efficiency., Methods and Findings: We defined high-grade dysplasia as endpoint of progression, and Barrett's esophagus progressor patients as Barrett's esophagus patients with either no dysplasia or low-grade dysplasia who later developed high-grade dysplasia or esophageal adenocarcinoma. We analyzed 4 epigenetic and 3 clinical parameters in 118 Barrett's esophagus tissues obtained from 35 progressor and 27 non-progressor Barrett's esophagus patients from Baltimore Veterans Affairs Maryland Health Care Systems and Mayo Clinic. Based on 2-year and 4-year prediction models using linear discriminant analysis (area under the receiver-operator characteristic (ROC) curve: 0.8386 and 0.7910, respectively), Barrett's esophagus specimens were stratified into high-risk (HR), intermediate-risk (IR), or low-risk (LR) groups. This 3-tiered stratification method retained both the high specificity of the 2-year model and the high sensitivity of the 4-year model. Progression-free survivals differed significantly among the 3 risk groups, with p = 0.0022 (HR vs. IR) and p<0.0001 (HR or IR vs. LR). Incremental value analyses demonstrated that the number of methylated genes contributed most influentially to prediction accuracy., Conclusions: This 3-tiered risk stratification strategy has the potential to exert a profound impact on Barrett's esophagus surveillance accuracy and efficiency.

The A-kinase anchoring protein 12 (AKAP12) is a kinase scaffold protein with known tumor suppressor activity. Recently, AKAP12 promoter hypermethylation was reported in gastric and colorectal cancers. We examined AKAP12 promoter hypermethylation using real-time methylation-specific PCR in 259 human esophageal tissues. AKAP12 hypermethylation showed highly discriminative receiver-operator characteristic (ROC) curve profiles, clearly distinguishing esophageal adenocarcinoma (EAC) from esophageal squamous cell carcinoma and normal esophagus (P < 0.0001). AKAP12-normalized methylation values were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's, and EAC than in normal esophagus (P < 0.0000001). AKAP12 hypermethylation frequency was zero in normal esophagus but increased early during neoplastic progression, to 38.9% in BE from patients with Barrett's alone, 52.5% in dysplastic Barrett's metaplasia, and 52.2% in EAC. AKAP12 hypermethylation levels were significantly higher in normal esophageal epithelia from patients with EAC (mean = 0.00082) than in normal esophagi from patients without Barrett's or esophageal cancer (mean = 0.00007; P = 0.006). There was a significant correlation between AKAP12 hypermethylation and BE segment length, a known clinical neoplastic progression risk factor. In contrast, only 2 (7.7%) of 26 esophageal squamous cell carcinomas exhibited AKAP12 hypermethylation. Treatment of BIC and OE33 EAC cells with 5-aza-2'-deoxycytidine reduced AKAP12 methylation and increased AKAP12 mRNA expression. AKAP12 mRNA levels in EACs with unmethylated AKAP12 (mean = 0.1663) were higher than in EACs with methylated AKAP12 (mean = 0.0668). We conclude that promoter hypermethylation of AKAP12 is a common, tissue-specific event in human EAC, occurs early during Barrett's-associated esophageal neoplastic progression, and is a potential biomarker for the early detection of EAC.

Purpose: Our aim was to investigate whether and at what stage hypermethylation of the tachykinin-1 (TAC1) gene is associated with human esophageal neoplastic transformation., Experimental Design: TAC1 promoter hypermethylation was examined by real-time methylation-specific PCR in 258 human esophageal specimens and 126 plasma samples from patients or tissues at various stages of neoplastic evolution., Results: TAC1 hypermethylation in tissue samples showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < 0.0001). Both frequencies and normalized methylation values of TAC1 tissue methylation were significantly higher in Barrett's metaplasia (BE), dysplastic Barrett's esophagus, EAC, and ESCC than in normal esophagus (P < 0.01). The frequency of TAC1 hypermethylation increased dramatically and early during neoplastic progression, from 7.5% in normal esophagus to 55.6% in BE from patients with Barrett's metaplasia alone, 57.5% in dysplastic Barrett's esophagus, and 61.2% in EAC. There was a significant relationship between TAC1 hypermethylation and BE segment length, a known clinical risk factor for neoplastic progression. Twelve (50%) of 24 ESCC exhibited TAC1 hypermethylation. Overall patient survival correlated significantly with TAC1 methylation status in ESCC patients (mean survival, 22 versus 110 months; P = 0.0102, log-rank test), but not in EAC patients. Both mean normalized methylation values and frequency of TAC1 hypermethylation in plasma samples were significantly higher in EAC patients than in control subjects. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2'-deoxycytidine reduced TAC1 methylation and increased TAC1 mRNA expression., Conclusions: TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of a poor prognosis in ESCC. Circulating methylated TAC1 promoter DNA also offers potential as a biomarker for the diagnosis of EAC.

Background: The rapidly growing field of targeted tumor therapy often utilizes an antibody, sometimes tagged with a tumor-ablating material such as radioisotope, directed against a specific molecule., Methodology/principal Findings: This report describes the discovery of nine novel decapeptides which can be radioactively labeled, bind to, and deliver (32)P to colon cancer cells. The decapeptides vary from one another by one to three amino acids and demonstrate vastly different binding abilities. The most avidly binding decapeptide can permanently deliver very high levels of radioisotope to the adenocarcinoma cancer cell lines at an efficiency 35 to 150 times greater than to a variety of other cell types, including cell lines derived from other types of cancer or from normal tissue., Conclusions/significance: This experimental approach represents a new example of a strategy, termed peptide binding therapy, for the potential treatment of colorectal and other adenocarcinomas.

Background & Aims: Gene silencing via promoter hypermethylation is a central event in the pathogenesis of cancers. To identify novel methylation targets in colon cancer, we conducted a genome-wide, microarray-based, in silico, and epigenetic search., Methods: Complementary DNA microarray experiments were first performed to identify genes down-regulated in primary colon cancers and up-regulated in colon cancer cell lines after global DNA demethylation by 5-aza-2'-deoxycitidine. Candidate methylation targets were then identified by combining these microarray data with in silico genetic and functional searches. Candidate genes recognized by these searches were further investigated for promoter hypermethylation in colon cancer using methylation-specific polymerase chain reaction., Results: We identified 51 novel and 3 known candidate methylation targets. Subsequent epigenetic analysis revealed that primary colon cancers demonstrated frequent methylation of somatostatin (SST, 30 of 34 cases, 88%) and the substance P precursor gene tachykinin-1 (TAC1; 16 of 34 cases, 47%). TAC1 methylation intensity was significantly higher in Dukes A/B than in Dukes C/D cancers (P = .01). SST methylation intensity was significantly higher in low-level microsatellite instability (MSI-L) than in non-MSI-L cancers (P = .02). Methylation was associated with messenger RNA down-regulation for both SST and TAC1. Furthermore, we isolated 5 additional novel promoter methylation targets: NELL1, AKAP12, caveolin-1, endoglin, and MAL., Conclusions: These data strongly suggest that SST and TAC1 are involved in colon carcinogenesis. Further studies are now indicated to elucidate mechanisms underlying their involvement in colon cancer and their values as clinical biomarkers. NELL1, AKAP12, caveolin-1, endoglin, and MAL are also promising tumor suppressor gene candidates deserving of further study.

Background & Aims: Multiple studies have shown that promoter methylation of tumor suppressor genes underlies esophageal carcinogenesis. Hypothetically, methylation resulting in tumor suppressor gene inactivation might result in tumors that are unresponsive to chemotherapy and radiation. Accordingly, our aim was to find methylation markers that could be used to predict response to chemoradiation., Methods: Tumor specimens were obtained before treatment from 35 patients enrolled in a uniform chemoradiation treatment protocol. Methylation-specific quantitative polymerase chain reaction was performed on all samples. Pathology reports from esophagectomy specimens were used to define response to treatment., Results: Thirteen (37%) of 35 patients were responders, and 22 (63%) of 35 patients were nonresponders. The number of methylated genes per patient was significantly lower in responders than in nonresponders (1.4 vs 2.4 genes per patient; Student t test, P = .026). The combined mean level of promoter methylation of p16, Reprimo, p57, p73, RUNX-3, CHFR, MGMT, TIMP-3, and HPP1 was also lower in responders than in nonresponders (Student t test, P = .003; Mann-Whitney test, P = .001). The frequency (15% of responders vs 64% of nonresponders; Fisher exact test, P = .01) and level (0.078 in responders vs 0.313 in nonresponders; Mann-Whitney test, P = .037) of Reprimo methylation was significantly lower in responders than in nonresponders., Conclusions: Reprimo methylation occurred at significantly lower levels and less frequently in chemoradioresponsive than in nonresponsive esophageal cancer patients, suggesting potential clinical application of this single-gene biomarker in defining prognosis and management. In addition, increased methylation of a 9-gene panel correlated significantly with poor responsiveness to chemoradiation.