Comprehensive Approach to Genomic and Immune Profiling: Insights of a Real-World Experience in Gynecological Tumors.

Gynecological cancer accounts for an elevated incidence worldwide requiring responsiveness regarding its care. The comprehensive genomic approach agrees with the classification of certain tumor types. We evaluated 49 patients with gynecological tumors undergoing high-throughput sequencing to explore whether identifying alterations in cancer-associated genes could characterize concrete histological subtypes. We performed immune examination and analyzed subsequent clinical impact. We found 220 genomic aberrations mostly distributed as single nucleotide variants (SNV, 77%). Only 3% were classified as variants of strong clinical significance in BRCA1 and BRCA2 of ovarian high-grade serous (HGSC) and uterine endometrioid carcinoma. TP53 and BRCA1 occurred in 72% and 28% of HGSC. Cervical squamous cell carcinoma was entirely HPV-associated and mutations occurred in PIK3CA (60%), as well as in uterine serous carcinoma (80%). Alterations were seen in PTEN (71%) and PIK3CA (60%) of uterine endometrioid carcinoma. Elevated programmed death-ligand 1 (PD-L1) was associated with high TILs. Either PD-L1 augmented in deficient mis-matched repair (MMR) proteins or POLE mutated cases when compared to a proficient MMR state. An 18% received genotype-guided therapy and a 4% immunotherapy. The description of tumor subtypes is plausible through high-throughput sequencing by recognizing clinically relevant alterations. Additional concomitant assessment of immune biomarkers identifies candidates for immunotherapy.

Validation and clinical application of a targeted next-generation sequencing gene panel for solid and hematologic malignancies.

BACKGROUND: Next-generation sequencing (NGS) is a high-throughput technology that has become widely integrated in molecular diagnostics laboratories. Among the large diversity of NGS-based panels, the Trusight Tumor 26 (TsT26) enables the detection of low-frequency variants across 26 genes using the MiSeq platform. METHODS: We describe the inter-laboratory validation and subsequent clinical application of the panel in 399 patients presenting a range of tumor types, including gastrointestinal (GI, 29%), hematologic (18%), lung (13%), gynecological and breast (8% each), among others. RESULTS: The panel is highly accurate with a test sensitivity of 92%, and demonstrated high specificity and positive predictive values (95% and 96%, respectively). Sequencing testing was successful in two-thirds of patients, while the remaining third failed due to unsuccessful quality-control filtering. Most detected variants were observed in the TP53 (28%), KRAS (16%), APC (10%) and PIK3CA (8%) genes. Overall, 372 variants were identified, primarily distributed as missense (81%), stop gain (9%) and frameshift (7%) altered sequences and mostly reported as pathogenic (78%) and variants of uncertain significance (19%). Only 14% of patients received targeted treatment based on the variant determined by the panel. The variants most frequently observed in GI and lung tumors were: KRAS c.35G > A (p.G12D), c.35G > T (p.G12V) and c.34G > T (p.G12C). CONCLUSIONS: Prior panel validation allowed its use in the laboratory daily practice by providing several relevant and potentially targetable variants across multiple tumors. However, this study is limited by high sample inadequacy rate, raising doubts as to continuity in the clinical setting.

Prophylaxis with nebulized liposomal amphotericin B for Aspergillus infection in lung transplant patients does not cause changes in the lipid content of pulmonary surfactant.

BACKGROUND: Prophylaxis with inhaled liposomal amphotericin B has proven to be safe and effective for preventing infection due to Aspergillus spp in lung transplant recipients. However, the liposome contains a large quantity of phospholipids, and inhalation of these substances could potentially change the composition of pulmonary surfactant. The aim of this study was to determine the lipid composition of pulmonary surfactant in patients receiving inhaled liposomal amphotericin B prophylaxis. METHODS: A prospective, open, controlled multicenter study was conducted in 2 groups: 19 lung transplant recipients who received regular prophylaxis with inhaled amphotericin B (study group) and 19 recipients who did not receive inhaled prophylaxis (control group). From both groups, 15 ml of the third aliquot of bronchoalveolar lavage fluid was obtained and phospholipid content determined in the active fraction of surfactant (large aggregates) and in the inactive fraction (small aggregates). Large aggregate cholesterol content was also determined. RESULTS: Patient demographic data and characteristics were similar in the 2 groups. No between-group differences in median phospholipid content were found for large aggregates (study group, 0.4 [range, 0.18-1.9] µmol vs controls, 0.36 [range 2.15-0.12] µmol; p = 0.69) or small aggregates (study group, 0.23 [range, 0.1-0.58] µmol vs controls, 0.29 [range, 0.18-0.65] µmol; p = 0.33). The small aggregate-to-large aggregate phospholipid ratio, commonly used as a marker of alveolar injury, showed no differences between the groups (study group, 0.56 vs controls, 0.69; p = 0.28). Nor were there differences in the cholesterol content of large aggregates (study group, 0.04 µmol [range 0.01-0.1] vs controls, 0.04 µmol [range 0.02-0.27); p = 0.13). CONCLUSIONS: These results seem to indicate that prophylaxis with nebulized liposomal amphotericin B does not cause changes in the lipid content of pulmonary surfactant.

Conjugation of quinones with natural polyamines: toward an expanded antitrypanosomatid profile.

A combinatorial library of quinone-polyamine conjugates designed to optimize the antitrypanosomatid profile of hit compounds 1 and 2 has been prepared by a solid-phase approach. The conjugates were evaluated against the three most important human trypanosomatid pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani), and several showed promising activity. A subset also inhibited trypanothione reductase in vitro and induced oxidase activity of the enzyme. A highly potent analogue (7) was identified with activity against T. brucei as low as 70 nM and a selectivity index of 72. Interestingly, the presence of a cadaverine tail confers to 7 the ability to target mitochondrial function in Leishmania. In fact, in L. donovani promastigotes, we verified for 7 a decrease of cytoplasmic ATP and mitochondrial potential. Therefore, the current results support the suitability of the conjugation approach for the development of novel polyamine conjugates with enhanced therapeutic potential.

Surfactant protein A (SP-A)-tacrolimus complexes have a greater anti-inflammatory effect than either SP-A or tacrolimus alone on human macrophage-like U937 cells.

Intratracheal administration of immunosuppressive agents to the lung is a novel treatment after lung transplantation. Nanoparticles of tacrolimus (FK506) might interact with human SP-A, which is the most abundant lipoprotein in the alveolar fluid. This study was undertaken to determine whether the formation of FK506/SP-A complexes interferes with FK506 immunosuppressive actions on stimulated human macrophage-like U937 cells. We found that SP-A was avidly bound to FK506 (K(d) = 35 ± 4nM), as determined by solid phase-binding assays and dynamic light scattering. Free FK506, at concentrations ≤ 1 µM, had no effect on the inflammatory response of LPS-stimulated U937 macrophages. However, coincubation of FK506 and SP-A, at concentrations where each component alone did not affect LPS-stimulated macrophage response, significantly inhibited LPS-induced NF-κB activation and TNF-alpha secretion. Free FK506, but not FK506/SP-A, functioned as substrate for the efflux transporter P-glycoprotein. FK506 bound to SP-A was delivered to macrophages by endocytosis, since several endocytosis inhibitors blocked FK506/SP-A anti-inflammatory effects. This process depended partly on SP-A binding to its receptor, SP-R210. These results indicate that FK506/SP-A complexes have a greater anti-inflammatory effect than either FK506 or SP-A alone and suggest that SP-A strengthened FK506 anti-inflammatory activity by facilitating FK506 entrance into the cell, overcoming P-glycoprotein.

Fluidizing effects of C-reactive protein on lung surfactant membranes: protective role of surfactant protein A.

The purpose of this study was to investigate how surfactant membranes can be perturbed by C-reactive protein (CRP) and whether surfactant protein A (SP-A) might overcome CRP-induced surfactant membrane alterations. The effect of CRP on surfactant surface adsorption was evaluated in vivo after intratracheal instillation of CRP into rat lungs. Insertion of CRP into surfactant membranes was investigated through monolayer techniques. The effect of CRP on membrane structure was studied through differential scanning calorimetry and fluorescence spectroscopy and microscopy using large and giant unilamellar vesicles. Our results indicate that CRP inserts into surfactant membranes and drastically increases membrane fluidity, resulting in surfactant inactivation. At 10% CRP/phospholipid weight ratio, CRP causes disappearance of liquid-ordered/liquid-disordered phase coexistence distinctive of surfactant membranes. SP-A, the most abundant surfactant lipoprotein structurally similar to C1q, binds to CRP (K(d)=56±8 nM), as determined by solid-phase binding assays and dynamic light scattering. This novel SP-A/CRP interaction reduces CRP insertion and blocks CRP effects on surfactant membranes. In addition, intratracheal coinstillation of SP-A+CRP into rat lungs prevents surfactant inhibition induced by CRP, indicating that SP-A/CRP interactions might be an important factor in vivo in controlling harmful CRP effects in the alveolus.