Non-Small Cell Lung Cancer: Targetable Variants in Concurrent Tissue and Liquid Biopsy Testing in a North Indian Cohort.

OBJECTIVES: Testing for EGFR, ALK, ROS1 and MET alterations in paired tissue and plasma samples of treatment-naïve patients of NSCLC and correlating their status with overall survival. MATERIALS AND METHODS: One hundred treatment-naïve patients were recruited after obtaining informed consent. Ten ml of blood was collected within a period of two weeks from histological diagnosis, prior to the start of any treatment. DNA & RNA extraction was done from formalin-fixed paraffin embedded (FFPE) tissue and total cell-free nucleic acid extraction was done from plasma samples. EGFR mutation, ALK, ROS1 and MET rearrangements were tested by ARMS (Amplification Refractory Mutation System) PCR. All statistical analyses were conducted in R version 4.1.1. RESULTS: A total of 61 cases showed molecular alterations in tissue samples which included EGFR mutations (47), ALK rearrangements (12), ROS1 fusion (2). MET alteration was not detected. Forty-three cases showed EGFR mutations in plasma, 26 of which were concurrently positive in tissue. Concordance observed was 62%. ALK-EML4 rearrangement, ROS1 fusion and MET were not detected in plasma samples. Sensitivity and specificity for detection of EGFR mutation in plasma were 55.3% and 67.9% respectively. Univariate Cox regression analysis showed a positive association between EGFR mutation in tissue and overall survival (HR = 0.4; 95% CI: 0.2-0.7; p = 0.003) and improved overall survival in those who received targeted therapy (HR = 0.29; 95% CI: 0.1-0.8; p = 0.02). CONCLUSION: Concurrent testing in tissue and liquid biopsy in NSCLC increased the detection of EGFR mutations (47% to 64%). This has substantial implications in deciding treatment and administration targeted therapy and the consequent overall survival.

A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

Objectives Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX. Materials and Methods Serum protein precipitation was performed using acetonitrile-water containing 250 µM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy. Results The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 µM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 µM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel. Conclusion We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.

Outcome Measures and Inclusion/Exclusion Criteria in Benign Prostatic Hyperplasia Trials on ClinicalTrials.gov.

INTRODUCTION: For benign prostatic hyperplasia, clinical trials help assess new medical and surgical treatment options. The U.S. National Library of Medicine maintains ClinicalTrials.gov to provide access to prospective trials on diseases. This study investigates registered benign prostatic hyperplasia trials to determine if there are widespread differences in outcome measures and study criteria. METHODS: Interventional research with known study status on ClinicalTrials.gov identified by the keywords "benign prostatic hyperplasia" was examined. Inclusion/exclusion criteria, primary outcomes, secondary outcomes, study status, study enrollment, country of origin, and intervention category were studied. RESULTS: Of the 411 studies identified, International Prostate Symptom Score was the most common study outcome and was the primary or secondary study outcome in 65% of trials. Maximum urinary flow was the second most common study outcome (40.1% of studies). No other outcomes were measured as the primary or secondary outcome for more than 30% of studies. The most common inclusion criteria were a minimum International Prostate Symptom Score (48.9%), maximum urinary flow (34.8%), and minimum prostate volume (25.8%). Among studies using a minimum International Prostate Symptom Score, 13 was the most common minimum (35.3%) and a range of 7-21 was noted. The most common maximum urinary flow for inclusion was 15 mL/s (78 trials). CONCLUSIONS: Among clinicals trials on benign prostatic hyperplasia registered on ClinicalTrials.gov, a majority of studies utilized International Prostate Symptom Score as a primary or secondary outcome. Unfortunately, there were major differences in the inclusion criteria; these dissimilarities between trials may limit comparability of results across trials.

Electrostatic Surface Potential of Macrophages Correlates with Their Functional Phenotype.

Macrophages exist in various functional phenotypes, which could be identified by specific surface molecules. Previous studies have shown that modulation of surface charges could alter the phagocytic function of macrophages. In this study, we show that activation of both human peripheral blood monocyte and THP-1-derived macrophages with lipopolysaccharide (LPS) or IL-1ß resulted in a significant decrease in the zeta potential compared to freshly isolated monocytes and unstimulated macrophages. Interestingly, interaction with bacteria significantly increased the zeta potential of such cells irrespective of activation conditions. Similarly, IFNγ-treated pro-inflammatory macrophages showed lesser negative zeta potential compared to untreated control. A moderate reduction was also seen in IL-4-treated anti-inflammatory subtype. Additionally, in an LPS-induced systemic inflammation model, bone marrow cells isolated after 2 h of LPS injection showed significant reduction in zeta potential compared to naïve cells. Furthermore, electrostatic potential measurement of surface proteins associated with pro-inflammatory and anti-inflammatory macrophages, using in silico modeling under physiological and protonation conditions, showed that the average electrostatic potential of pro-inflammatory type surface proteins was less negative than anti-inflammatory subtype. These data suggest that the expression of different protein molecules on macrophages under different environments may contribute to the zeta potential and that this quick and low-cost technique could be used in monitoring macrophage functional phenotypes.

Clinical evaluation of 4D MRI in the delineation of gross and internal tumor volumes in comparison with 4DCT.

PURPOSE: To evaluate clinical utility of respiratory-correlated (RC) four-dimensional magnetic resonance imaging (4DMRI) for lung tumor delineation and motion assessment, in comparison with the current clinical standard of 4D computed tomography (4DCT). METHODS AND MATERIALS: A prospective T2-weighted (T2w) RC-4DMRI technique was applied to acquire coronal 4DMRI images for 14 lung cancer patients (16 lesions) during free breathing (FB) under an IRB-approved protocol, together with a breath-hold (BH) T1w 3DMRI and axial 4DMRI. Clinical simulation CT and 4DCT were acquired within 2 h. An internal navigator was applied to trigger amplitude-binned 4DMRI acquisition whereas a bellows or real-time position management (RPM) was used in the 4DCT reconstruction. Six radiation oncologists manually delineated the gross and internal tumor volumes (GTV and ITV) in 399 3D images using programmed clinical workflows under a tumor delineation guideline. The ITV was the union of GTVs within the breathing cycle without margin. Average GTV and motion range were assessed and ITV variation between 4DMRI and 4DCT was evaluated using the Dice similarity index, mean distance agreement (MDA), and volume difference. RESULTS: The mean tumor volume is similar between 4DCT (GTV4DCT  = 1.0, as the reference) and T2w-4DMRI (GTVT2wMR  = 0.97), but smaller in T1w MRI (GTVT1wMR  = 0.76), suggesting possible peripheral edema around the tumor. Average GTV variation within the breathing cycle (22%) in 4DMRI is slightly greater than 4DCT (17%). GTV motion variation (-4 to 12 mm) and ITV variation (∆VITV =-25 to 95%) between 4DCT and 4DMRI are large, confirmed by relatively low ITV similarity (Dice = 0.72 ± 0.11) and large MDA = 2.9 ± 1.5 mm. CONCLUSION: Average GTVs are similar between T2w-4DMRI and 4DCT, but smaller by 25% in T1w BH MRI. Physician training and breathing coaching may be necessary to reduce ITV variability between 4DMRI and 4DCT. Four-dimensional magnetic resonance imaging is a promising and viable technique for clinical lung tumor delineation and motion assessment.

In Silico Mining of Conserved miRNAs of Indian Catfish Clarias batrachus (Linnaeus, 1758) from the Contigs, ESTs, and BAC End Sequences.

MicroRNAs are small non-coding RNAs which play significant role in RNA interference. The present work deals with mining of the conserved miRNA and their target genes from the contigs, ESTs, and BAC end sequences of commercially important catfish, Clarias batrachus, from India. A total of 138, 1 and 1 conserved pre-miRNA sequences, were mined from the contigs, ESTs, and BAC end sequences, respectively. The analysis of families of the conserved pre-miRNA revealed conservation of the fish-specific family mir-430 and other important families, such as mir-455, let-7, mir-133, and mir-137. The mir-455 is involved in hypoxia signaling, let-7 family represents potential anti-tumor molecules involved in human cancer therapy, whereas mir-133 and mir-137 have high therapeutic potentials. Using an alternate computational in silico approach, mining of mature miRNAs resulted in identification of 210 mature miRNAs from contigs, 1 from EST, and 2 each from forward as well as reverse BAC end sequences. Target prediction of these putative miRNAs resulted in the identification of 66,758 and 18,747 target genes in C. batrachus and Danio rerio, respectively. Functional annotation of these miRNAs indicated their involvement in diverse biological functions. The findings of the present study can serve as a valuable resource for further functional genomics studies in C. batrachus.

Nodal Langerhans cell histiocytosis with prominent eosinophilic emperipolesis.

Multinucleated giant cells of osteoclastic type are known to occur in nodal and extranodal lesions of Langerhans cell histiocytosis (LCH). These giant cells are thought to be derived from phagocytic histiocytes, which correlate with the degree of necrosis in LCH lesions. Emperipolesis commonly seen in Rosai-Dorfman disease is a distinct phenomenon characterized by intact phagocytosed cells in an intracytoplasmic vacuole protected from proteolytic digestion. We present a case of emperipolesis of inflammatory cells especially of eosinophils by multinucleated giant cells of Langhans type in a lymph node involved by LCH--a finding that has not been described previously in the literature.