Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: Effect of the plasma membrane-associated NEU3

Adenocarcinoma of Non-Small Cell Lung Cancer (NSCLC) is a severe disease. Patients carrying EGFR mutations may benefit from EGFR targeted therapies (e.g.: gefitinib). Recently, it has been shown that sialidase NEU3 directly interacts and regulates EGFR. In this work, we investigate the effect of sialidase NEU3 overexpression on EGFR pathways activation and EGFR targeted therapies sensitivity, in a series of lung cancer cell lines. NEU3 overexpression, forced after transfection, does not affect NSCLC cell viability. We demonstrate that NEU3 overexpression stimulates the ERK pathway but this activation is completely abolished by gefitinib treatment. The Akt pathway is also hyper-activated upon NEU3 overexpression, but gefitinib is able only to decrease, and not to abolish, such activa- tion. These findings indicate that NEU3 can act directly on the ERK pathway through EGFR and both directly and indirectly with respect to EGFR on the Akt pathway. Furthermore, we provide evidence that a healthy mucosa cell line (with EGFR wild-type gene sequence) is slightly sensitive to gefitinib, especially in the presence of NEU3 overexpression, thus hypothesizing that NEU3 overexpressing patients may benefit from EGFR targeted thera- pies also in absence of EGFR point mutations. Overall, the expression of NEU3 may be a novel diagnostic marker in NSCLC because, by its ability to stimulate EGFR downstream pathways with direct and indirect mechanisms, it may help in the identification of patients who can profit from EGFR targeted therapies in absence of EGFR activating mutations or from new combinations of EGFR and Akt inhibitors

Surgical treatment of pleural empyema in Coronavirus disease 19 patients: the Southern Switzerland experience

We report the first surgical series of patients developing pleural empyema after severe bilateral interstitial lung disease in confirmed severe acute respiratory syndrome coronavirus 2 infection. The empyema results in a complex medical challenge that requires combination of medical therapies, mechanical ventilation and surgery. The chest drainage approach was not successful to relieve the symptomatology and to drain the excess fluid. After multidisciplinary discussion, a surgical approach was recommended. Even though decortication and pleurectomy are high-risk procedures, they must be considered as an option for pleural effusion in Coronavirus disease-positive patients. This is a life-treating condition, which can worsen the coronavirus disease manifestation and should be treated immediately to improve patient's status and chance of recovery

Evaluation of sialidase NEU3 effect on cell viability with or without gefitinib.

<p>MTT test was performed on HSAEC1, HCC4006 and H1734 cell lines transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3 and then treated or not with 27 nM or 1 μM gefitinib for 36 h <i>post</i>-transfection. Data were normalized on control cells transfected with the empty vector (A). Cell viabilities of mock and NEU3 transfected cells are reported after treatment with either 27 nM (B) or 1 μM gefitinib (C). Data were normalized on control cells without drug. Values are presented as means ± standard error (SE).</p

Sensitivity to gefitinib.

<p>(A) Dose-response curves of human lung cell lines to gefitinib. Cell survival was determined by MTT assay in the absence or the presence of different gefitinib doses (0, 0.01, 0.1, 1 and 10 μM) for 72 h. Viabilities are expressed as a percentage of the untreated control ± standard error (SE). (B) Nonlinear Regression of experimental data for HSAEC1 and HCC4006 cells lines was obtained using a Four Parameter Logistic Curve f1 = min + (max-min)/(1 + (x/EC₅₀)^(-Hillslope)). (C) Cell death of HSAEC1, HCC4006 and H1734 cells lines was determined by flow cytometry through PI staining. The cells were treated with different gefitinib concentration ranging from 0.01 to 10 μM for 72 h and stained by PI dye. The zero concentration was defined as a control and cell death was expressed as a percentage of the control ± standard error (SE).</p

EGFR phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment.

<p>(A) Representative Western-blot analyses performed on a normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected either with the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-EGFR, anti-P-EGFR antibodies. GAPDH was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained in the absence of EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).</p

Endoscopic treatment of ewing sarcoma of the sinonasal tract

The extra-skeletal form is an unusual type of Ewing sarcoma (ES) arising from soft tissue and in the literature there are reports of less than 50 patients describing the tumor in the paranasal sinuses and skull base. The histological diagnosis is crucial to plan the correct treatment and the molecular confirmation is mandatory in equivocal patients. A multimodality treatment with chemotherapy, surgery and radiotherapy improved the outcomes of these diseases during the last decades and a free-margin resection with the endoscopic transnasal technique is one of the most recent ways to manage these pathologies in selected patients, reducing the morbidities of the external approaches and preserving the quality of life of the patient. Here, the authors present the first patient of primary sinonasal ES free from disease after 5 years of follow-up and treated with an endoscopic endonasal approach and a second patient of sinonasal metastases of ES treated with and endoscopic transnasal approach

Prognostic implication of PD-L1 in early-stage non-small cell lung cancer: a retrospective single-centre study

BACKGROUND: The prognostic role of programmed death-ligand 1 (PD-L1) expression in patients with localised and locally advanced non-small cell lung cancer has not been fully elucidated. This information could help to better interpret recent and upcoming results of phase III adjuvant or neoadjuvant anti-PD-1/PD-L1 immunotherapy studies. METHODS: In a cohort of 146 patients with early or locally advanced non-small cell lung cancer treated with curative intent (by surgery or radiotherapy), we investigated the prognostic value of PD-L1 expression and its correlation with other biological and clinical features. PD-L1 expression was stratified by quartiles. Primary endpoints were overall and disease-free survival. We also analysed the prognostic impact of the presence of actionable mutations, implemented treatment modality and completion of the treatment plan. Neither type of patient received neoadjuvant or adjuvant immunotherapy or target therapy. RESULTS: Of the 146 selected patients, 32 (21.9%) presented disease progression and 15 died (10.3%) at a median follow-up of 20 months. In a univariable analysis, PD-L1 expression ≥25% was associated with significantly lower disease-free survival (hazard ratio [HR]) 1.9, 95% confidence interval [CI] 1.0–3.9, p= 0.049). PD-L1 expression ≥50% did not lead to disease-free survival or overall survival benefits (HR 1.2 and 1.1, respectively; 95% CI 0.6–2.6 and 0.3–3.4, respectively; pnot significant). In a multivariate analysis, a stage >I (HR 2.7, 95% CI 1.2–6, p = 0.012) and having an inoperable tumour (HR 3.2, 95% CI 1.4–7.4, p = 0.005) were associated with lower disease-free survival. CONCLUSION: The population of patients with early-stage non-small cell lung cancer and PD-L1 expression ≥25% who were treated with curative intent during the pre-immunotherapy era exhibited a worse prognosis. This finding provides justification for the utilisation of adjuvant immunotherapy in this subgroup of patients, based on the current evidence derived from disease-free survival outcomes. However, for patients with PD-L1 expression <25%, opting to wait for the availability of the overall survival results may be a prudent choice

ERK phosphorylation levels after EGF stimulation, sialidase NEU3 overexpression and gefitinib treatment.

<p>(A) Representative Western-blot analyses performed on normal lung cell line (HSAEC1) and NSCLC cell lines (HCC4006 and H1734) transfected with either the empty vector (mock) or pcDNA3.1-HsNEU3. Cells were treated for 3 h with 1 μM gefitinib, followed by the addition of EGF (20 ng/mL) for 15 min. Protein extracts were separated on a 10% SDS-PAGE and probed with anti-ERK1/2, anti-P-ERK1/2 antibodies. Vinculin was used as a loading control. The experiments were performed in triplicate. (B)–(C)–(D) Densitometric analysis was performed with Scion Image Software. Values are expressed by comparing the data obtained after EGF stimulation with those obtained without EGF (B); by comparing the data obtained after transfection with NEU3 with those obtained after transfection with the empty vector (mock) (C); by comparing the data obtained after gefitinib treatment with those obtained without gefitinib administration (D). Statistical analyses were performed using Student’s t-test. Values are presented as means ± standard error (SE). *p<0.05, **p<0.01, and ***p<0.001 (Student’s t-test).</p

IC₅₀ values for gefitinib.

<p>IC₅₀ values for gefitinib.</p