The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

Colorectal cancer (CRC) is one of the most common and fatal types of cancer. Inflammation promotes CRC development, however, the underlying etiological factors are unknown. Human cytomegalovirus (HCMV), a virus that induces inflammation and other cancer hallmarks, has been detected in several types of malignancy, including CRC. The present study investigated whether HCMV infection was associated with expression of the pro‑inflammatory enzymes 5‑lipoxygenase (5‑LO) and cyclooxygenase‑2 (COX‑2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146 individual paraffin‑embedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki‑67 index and EGFR by immunohistochemistry (IHC). The cores were further analyzed by IHC for the expression of two HCMV proteins (Immediate Early, IE and pp65) and the inflammatory markers 5‑LO and COX‑2. The CRC cell lines Caco‑2 and LS‑174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or anti‑inflammatory drug celecoxib (CCX) and analyzed by reverse transcription‑quantitative PCR and immunofluorescence for 5‑LO, COX‑2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in ~90% of the CRC cases tested; this was correlated with COX‑2, 5‑LO and KI‑67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. In vitro, HCMV infection upregulated 5‑LO and COX‑2 transcript and proteins in both Caco‑2 and LS‑174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX‑2, 5‑LO, HCMV IE and pp65 in infected cells. HCMV was widely expressed in CRC and may promote inflammation and serve as a potential new target for CRC therapy.

A considerable effort has been devoted in all countries to react to the COVID-19 pandemic by tracing infected individuals, containing the spread of the disease, identifying therapies, and producing and distributing vaccines. Currently, a significant concern is the appearance of variants of the virus that may frustrate these efforts by showing increased transmissibility, increased disease severity, reduced response to therapy or vaccines, and ability to escape diagnosis. All countries have therefore devoted a massive attempt to the identification and tracking of these variants, which requires a vast technological effort to sequence a large number of viral genomes. In this paper, we report our experience as one of the Italian laboratories involved in SARS-CoV-2 variant tracing. We summarize the different approaches used, and outline a potential model combining several techniques to increase tracing ability while at the same time minimizing costs.

Head and neck paragangliomas (HNPGLs), rare chemoresistant tumors curable only with surgery, are strongly influenced by genetic predisposition, hence patients and relatives require lifetime follow-up with MRI and/or PET-CT because of de novo disease risk. This entails exposure to electromagnetic/ionizing radiation, costs, and organizational challenges, because patients and relatives are scattered far from reference centers. Simplified first-line screening strategies are needed. We employed flow injection analysis tandem mass spectrometry, as used in newborn metabolic screening, to compare the plasma metabolic profile of HNPGL patients (59 samples, 56 cases) and healthy controls (24 samples, 24 cases). Principal Component Analysis (PCA) and Partial Least Discriminant Analysis (PLS-DA) highlighted a distinctive HNPGL signature, likely reflecting the anaplerotic conversion of the TCA cycle to glutaminolysis and catabolism of branched amino acids, DNA damage and deoxyadenosine (dAdo) accumulation, impairment of fatty acid oxidation, switch towards the Warburg effect and proinflammatory lysophosphatidylcholines (LPCs) signaling. Statistical analysis of the metabolites that most impacted on PLS-DA was extended to 10 acoustic neuroma and 2 cholesteatoma patients, confirming significant differences relative to the HNPGL plasma metabolomic profile. The best confusion matrix from the ROC curve built on 2 metabolites, dAdo and C26:0-LPC, provided specificity of 94.29% and sensitivity of 89.29%, with positive and negative predictive values of 96.2% and 84.6%, respectively. Analysis of dAdo and C26:0-LPC levels in dried venous and capillary blood confirmed that dAdo, likely deriving from 2'-deoxy-ATP accumulated in HNPGL cells following endogenous genotoxic damage, efficiently discriminated HNPGL patients from healthy controls and acoustic neuroma/cholesteatoma patients on easily manageable dried blood spots.