Understanding mechanisms of resistance to FLT3 inhibitors in adult FLT3-mutated acute myeloid leukemia to guide treatment strategy.

The presence of FLT3 mutations, including the most common FLT3-ITD (internal tandem duplications) and FLT3-TKD (tyrosine kinase domain), is associated with an unfavorable prognosis in patients affected by acute myeloid leukemia (AML). In this setting, in recent years, new FLT3 inhibitors have demonstrated efficacy in improving survival and treatment response. Nevertheless, the development of primary and secondary mechanisms of resistance poses a significant obstacle to their efficacy. Understanding these mechanisms is crucial for developing novel therapeutic approaches to overcome resistance and improve the outcomes of patients. In this context, the use of novel FLT3 inhibitors and the combination of different targeted therapies have been studied. This review provides an update on the molecular alterations involved in the resistance to FLT3 inhibitors, and describes how the molecular monitoring may be used to guide treatment strategy in FLT3-mutated AML.

IDH1 mutation is detectable in plasma cell-free DNA and is associated with survival outcome in glioma patients.

BACKGROUND: Circulating cell-free DNA (cfDNA, liquid biopsy) is a powerful tool to detect molecular alterations. However, depending on tumor characteristics, biology and anatomic localization, cfDNA detection and analysis may be challenging. Gliomas are enclosed into an anatomic sanctuary, which obstacles the release of cfDNA into the peripheral blood. Therefore, the advantages of using liquid biopsy for brain tumors is still to be confirmed. The present study evaluates the ability of liquid biopsy to detect IDH1 mutations and its correlation with survival and clinical characteristics of glioma patients. METHODS: Blood samples obtained from glioma patients were collected after surgery prior to the adjuvant therapy. cfDNA was extracted from plasma and IDH1 p.R132H mutation analysis was performed on a digital droplet PCR. χ2-test and Cohen k were used to assess the correlation between plasma and tissue IDH1 status, while Kaplan Meier curve and Cox regression analysis were applied to survival analysis. Statistical calculations were performed by MedCalc and GraphPad Prism software. RESULTS: A total of 67 samples were collected. A concordance between IDH1 status in tissue and in plasma was found (p = 0.0024), and the presence of the IDH1 mutation both in tissue (138.8 months vs 24.4, p < 0.0001) and cfDNA (116.3 months vs 35.8, p = 0.016) was associated with longer median OS. A significant association between IDH1 mutation both in tissue and cfDNA, age, tumor grade and OS was demonstrated by univariate Cox regression analysis. No statistically significant association between IDH1 mutation and tumor grade was found (p = 0.10). CONCLUSIONS: The present study demonstrates that liquid biopsy may be used in brain tumors to detect IDH1 mutation which represents an important prognostic biomarker in patients with different types of gliomas, being associated to OS.

Clinical utility of Next Generation Sequencing of plasma cell-free DNA for the molecular profiling of patients with NSCLC at diagnosis and disease progression.

BACKGROUND: The present study evaluates the utility of NGS analysis of circulating free DNA (cfDNA), which incorporates small amounts of tumor DNA (ctDNA), at diagnosis or at disease progression (PD) in NSCLC patients. METHODS: Comprehensive genomic profiling on cfDNA by NGS were performed in NSCLC patients at diagnosis (if tissue was unavailable/insufficient) or at PD to investigate potential druggable molecular aberrations. Blood samples were collected as routinary diagnostic procedures, DNA was extracted, and the NextSeq 550 Illumina platform was used to run the Roche Avenio ctDNA Expanded Kit for molecular analyses. Gene variants were classified accordingly to the ESCAT score. RESULTS: A total of 106 patients were included in this study; 44 % of cases were requested because of tissue unavailability at the diagnosis and 56 % were requested at the PD. At least one driver alteration was observed in 62 % of cases at diagnosis. Driver druggable variants classified as ESCAT level I were detected in 34 % of patients, including ALK-EML4, ROS1-CD74, EGFR, BRAF, KRAS p.G12C, PI3KCA. In the PD group, most patients were EGFR-positive, progressing to a first line-therapy. Sixty-three percent of patients had at least one driver alteration detected in blood and 17 % of patients had a known biological mechanism of resistance allowing further therapeutic decisions. CONCLUSIONS: The present study confirms the potential of liquid biopsy to detect tumour molecular heterogeneity in NSCLC patients at the diagnosis and at PD, demonstrating that a significant number of druggable mutations and mechanisms of resistance can be detected by NGS analysis on ctDNA.

Expression of Circulating let-7e and miR-126 May Predict Clinical Remission in Patients With Crohn's Disease Treated With Anti-TNF-α Biologics.

BACKGROUND: The identification of new biomarkers predictive of response to antitumor necrosis factor alpha (anti-TNF-α) monoclonal antibodies remains an unmet medical need in Crohn's disease (CD) because a high percentage of patients show no clinical improvement after treatment or can lose response over time. MicroRNAs (miRNAs) can regulate inflammatory and immunological responses and were found to play a role in CD. METHODS: Baseline serum samples from 37 CD patients previously treated with infliximab or adalimumab, as per clinical practice, were obtained from the serum library at the Gastroenterology Unit of the University Hospital of Pisa, Italy. Patients were categorized as responders or nonresponders based on the following treatment outcomes: clinical remission at weeks 14 and 54 and endoscopic remission at week 54. The expression levels of a panel of selected miRNAs were analyzed by real-time polymerase chain reaction. Comparisons of miRNA expression between responders and nonresponders and statistical analyses were performed by MedCalc and GraphPad Prism software. Receiver operating characteristic curve analyses were calculated to evaluate the predictive performance of potential biomarkers. RESULTS: Patients in clinical remission at week 14 had a lower let-7e expression, whereas those in clinical remission at week 54 had lower levels of circulating miR-126 than nonresponders. The receiver operating characteristic curve analysis identified optimal cutoff values with assay sensitivity and specificity of 92.9% and 61.1%, for let-7e, and 62.5% and 83.3%, for miR-126, respectively. CONCLUSION: These results provide evidence that expression levels of circulating let-7e and miR-126 at baseline may predict clinical remission in CD patients treated with anti-TNF-α biologics.

We found that the baseline expression of 2 microRNAs (Let-7e and miR126) involved in inflammation and immune system regulation may predict short- and long-term clinical remission in CD patients treated with anti-TNF-α biologics, supporting clinicians in therapeutic decision-making.

Gene-network analysis predicts clinical response to immunotherapy in patients affected by NSCLC.

OBJECTIVES: Predictive biomarkers of response to immune checkpoint inhibitors (ICIs) have been extensively studied in non-small cell lung cancer (NSCLC) with controversial results. Recently, gene-network analysis emerged as a new tool to address tumor biology and behavior, representing a potential tool to evaluate response to therapies. METHODS: Clinical data and genetic profiles of 644 advanced NSCLCs were retrieved from cBioPortal and the Cancer Genome Atlas (TCGA); 243 ICI-treated NSCLCs were used to identify an immunotherapy response signatures via mutated gene network analysis and K-means unsupervised clustering. Signatures predictive values were tested in an external dataset of 242 cases and assessed versus a control group of 159 NSCLCs treated with standard chemotherapy. RESULTS: At least two mutations in the coding sequence of genes belonging to the chromatin remodelling pathway (A signature), and/or at least two mutations of genes involved in cell-to-cell signalling pathways (B signature), showed positive prediction in ICI-treated advanced NSCLC. Signatures performed best when combined for patients undergoing first-line immunotherapy, and for those receiving combined ICIs. CONCLUSIONS: Alterations in genes related to chromatin remodelling complexes and cell-to-cell crosstalk may force dysfunctional immune evasion, explaining susceptibility to immunotherapy. Therefore, exploring mutated gene networks could be valuable for determining essential biological interactions, contributing to treatment personalization.

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins.

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

Comparison of digital PCR systems for the analysis of liquid biopsy samples of patients affected by lung and colorectal cancer.

BACKGROUND AND AIMS: Highly sensitive technologies are available for the molecular characterization of solid tumors, including digital PCR (dPCR). Liquid biopsy, based on the analysis of cell-free DNA (cfDNA), is often used to assess EGFR or RAS alterations in lung and colorectal cancers. Our study aimed to compare the results of two different dPCR platforms for the detection of mutations in cfDNA. METHODS: Plasma samples from lung and colorectal cancer patients collected as per routine procedures have been tested. cfDNA Was extracted from plasma, and samples were screened on the droplet digital PCR (ddPCR, BioRad) and solid dPCR QIAcuity (Qiagen). RESULTS: A total of 42 samples were analyzed, obtained from 20 Non-Small Cell Lung Cancer (NSCLC) patients carrying an EGFR or a KRAS mutation on tissue at diagnosis, and from 22 samples of colorectal cancer (CRC) patients, 10 of which presenting a KRAS mutation. EGFR mutation detection was 58.8% for ddPCR and 100% for dPCR (κ = 0.54; 95% CI, 0.37-0.71), compared to tissue results. The detection rate for RAS mutations was 72.7% for ddPCR and 86.4% for dPCR (κ = 0.34; 95% CI, 0.01-0.68), compared to tissue results. CONCLUSIONS: This study showed moderate agreement between dPCR and ddPCR. Sampling effect or threshold settings may potentially explain the differences in the cfDNA data between the two different platforms.

CDK4/6 Inhibitors Overcome Endocrine ESR1 Mutation-Related Resistance in Metastatic Breast Cancer Patients.

ESR1 mutations contribute to endocrine resistance and occur in a high percentage of hormone-receptor-positive (HR+) metastatic breast cancer (mBC) cases. Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) changed the treatment landscape of HR+ mBC, as they are able to overcome estrogen resistance. The present retrospective study investigates the clinical benefit of CDK4/6i in ESR1 mutant HR+ mBC patients treated with a CDK4/6i as first- or second-line therapy. Plasma was collected at baseline prior to CDK4/6i plus hormone therapy as a first- or second-line treatment. Circulating free DNA (cfDNA) was extracted from plasma, and ESR1 mutation analysis was performed on a ddPCR. Statistical analyses were performed to investigate the predictive power of ESR1 mutations and any association with clinical factors. A total of 42 patients with mBC treated with CDK4/6i plus endocrine therapy as first- (n = 35) or second-line (n = 7) were enrolled. Twenty-eight patients received hormonal therapy (AI or tamoxifen) in the adjuvant setting. ESR1 mutation status in blood was associated with shorter median disease-free survival (DFS) (30 vs. 110 months; p = 0.006). Multivariate analysis confirmed ESR1 mutations as independent factors of resistance in adjuvant hormone therapy. On the contrary, no difference in progression-free survival (PFS) was observed in the presence or absence of an ESR1 mutation in patients treated with CDK4/6i as first-line treatment (p = 0.29). No statistically significant correlation between the best response to CDK4/6i and ESR1 mutation was found (p = 0.46). This study indicates that the ESR1 mutation detected in cfDNA is an independent predictive factor of clinical recurrence in the adjuvant setting and that CDK4/6i can overcome ESR1-dependent resistance.

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy.

In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.