Activity of osimeRTInib in non-small-cell lung Cancer with UNcommon epidermal growth factor receptor mutations: retrospective Observational multicenter study (ARTICUNO).

BACKGROUND: Osimertinib represents the standard of care for the treatment of advanced non-small-cell lung cancer (NSCLC) harboring classical epidermal growth factor receptor (EGFR) mutations, constituting 80%-90% of all EGFR alterations. In the remaining cases, an assorted group of uncommon alterations of EGFR (uEGFR) can be detected, which confer variable sensitivity to previous generations of EGFR inhibitors, overall with lower therapeutic activity. Data on osimertinib in this setting are limited and strongly warranted. PATIENTS AND METHODS: The ARTICUNO study retrospectively evaluated data on osimertinib activity from patients with advanced NSCLC harboring uEGFR treated in 21 clinical centers between August 2017 and March 2023. Data analysis was carried out with a descriptive aim. Investigators collected response data according to RECIST version 1.1 criteria. The median duration of response, progression-free survival (mPFS), and overall survival were estimated by the Kaplan-Meier method. RESULTS: Eighty-six patients harboring uEGFR and treated with osimertinib were identified. Patients with 'major' uEGFR, that is, G719X, L861X, and S768I mutations (n = 51), had an overall response rate (ORR) and mPFS of 50% and 9 months, respectively. Variable outcomes were registered in cases with rarer 'minor' mutations (n = 27), with ORR and mPFS of 31% and 4 months, respectively. Among seven patients with exon 20 insertions, ORR was 14%, while the best outcome was registered among patients with compound mutations including at least one classical EGFR mutation (n = 13). Thirty patients presented brain metastases (BMs) and intracranial ORR and mPFS were 58% and 9 months, respectively. Amplification of EGFR or MET, TP53 mutations, and EGFR E709K emerged after osimertinib failure in a dataset of 18 patients with available rebiopsy. CONCLUSION: The ARTICUNO study confirms the activity of osimertinib in patients with uEGFR, especially in those with compound uncommon-common mutations, or major uEGFR, even in the presence of BMs. Alterations at the E709 residue of EGFR are associated with resistance to osimertinib.

The lung immuno-oncology prognostic score (LIPS-3): a prognostic classification of patients receiving first-line pembrolizumab for PD-L1 ≥ 50% advanced non-small-cell lung cancer.

BACKGROUND: To stratify the prognosis of patients with programmed cell death-ligand 1 (PD-L1) ≥ 50% advanced non-small-cell lung cancer (aNSCLC) treated with first-line immunotherapy. METHODS: Baseline clinical prognostic factors, the neutrophil-to-lymphocyte ratio (NLR), PD-L1 tumour cell expression level, lactate dehydrogenase (LDH) and their combination were investigated by a retrospective analysis of 784 patients divided between statistically powered training (n = 201) and validation (n = 583) cohorts. Cut-offs were explored by receiver operating characteristic (ROC) curves and a risk model built with validated independent factors by multivariate analysis. RESULTS: NLR < 4 was a significant prognostic factor in both cohorts (P < 0.001). It represented 53% of patients in the validation cohort, with 1-year overall survival (OS) of 76.6% versus 44.8% with NLR > 4, in the validation series. The addition of PD-L1 ≥ 80% (21% of patients) or LDH < 252 U/l (25%) to NLR < 4 did not result in better 1-year OS (of 72.6% and 74.1%, respectively, in the validation cohort). Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 2 [P < 0.001, hazard ratio (HR) 2.04], pretreatment steroids (P < 0.001, HR 1.67) and NLR < 4 (P < 0.001, HR 2.29) resulted in independent prognostic factors. A risk model with these three factors, namely, the lung immuno-oncology prognostic score (LIPS)-3, accurately stratified three OS risk-validated categories of patients: favourable (0 risk factors, 40%, 1-year OS of 78.2% in the whole series), intermediate (1 or 2 risk factors, 54%, 1-year OS 53.8%) and poor (>2 risk factors, 5%, 1-year OS 10.7%) prognosis. CONCLUSIONS: We advocate the use of LIPS-3 as an easy-to-assess and inexpensive adjuvant prognostic tool for patients with PD-L1 ≥ 50% aNSCLC.

Double-filtration plasmapheresis in the treatment of leg ulcers in cryoglobulinemia.

Hepatitis C virus (HCV) is the major cause of cryoglobulinemia. Skin lesions are frequent and can be cured from the removal of cryoglobulins by therapeutic apheresis. We describe a case of HCV-positive type I cryoglobulinemia with severe leg ulcers, not responsive to antiviral and immunosuppressive treatment. Thirty sessions of double filtration plasmapheresis were performed, over a period of 6 months, with no other associated treatment. Before and after each session an assessment of immunoglobulins, complement, cryocrit, and fibrinogen was made. HCV RNA levels were determined in serum cryoprecipitate, supernatant before and after each session, and in the collection bag. No differences in pre and postapheresis values were observed in the serum concentrations and the supernatant, whereas the postapheresis cryoprecipitate showed a significantly reduced viral load (P < 0.02) as compared with the preapheresis values. There was improvement in the condition of ulcers in the leg during apheresis and had completely regressed by the end of the cycle.

Hepatitis C virus productive infection in mononuclear cells from patients with cryoglobulinaemia.

The relationship between the occurrence of cryoglobulins and hepatitis C virus (HCV) productive infection in peripheral blood and bone marrow-derived lymphocytes was explored. HCV minus strand RNA, the viral replicative intermediate, was searched for by a polyA(+) tract strand-specific Tth-based reverse transcriptase-polymerase chain reaction (RT-PCR) in lymphoid cells of 46 patients with acute and chronic infection. The HCV minus strand was demonstrated in RNA extracted from six (13%) and five (11%) peripheral blood and bone marrow-derived lymphocytes, respectively. The HCV replicating form in lymphoid cells was associated strictly with mixed cryoglobulinaemia (MCG), in that it was found in six of 13 (46%) MCG patients, including two with B cell non-Hodgkin's lymphoma (NHL). No traces of HCV-negative strand RNA were found in four patients with acute hepatitis C, in 15 with chronic active hepatitis without extrahepatic disorders, in seven with monoclonal gammopathy of undetermined significance, and in seven with B-NHL without MCG. These results emphasize the direct role of the virus in the pathogenesis of MCG and support the contention that HCV is not specifically lymphotropic, its entry and replication in lymphoid cells being determined largely by selective interactions.

Virological analysis and phenotypic characterization of peripheral blood lymphocytes of hepatitis C virus-infected patients with and without mixed cryoglobulinaemia.

In clinical and pathological terms hepatitis C virus (HCV)-infected patients can be subdivided into two main groups with and without mixed cryoglobulinaemia (MC). Involvement of blood mononuclear cells by HCV has potentially important implications. To this end, HCV-RNA levels in peripheral blood lymphocytes (PBL) preparations of 20 chronically HCV-infected patients with MC were measured and compared with those found in a group of 20 patients without MC matched for age, serum HCV-RNA, infectious genotype, source and presumable duration of infection. Phenotypic abnormalities of PBL subsets in each group of patients were determined by cell surface marker expression and compared. Results showed a significant enrichment of HCV-RNA in PBL of MC patients compared with a non-MC group (P = 0.01). Different distribution of HCV-RNA was accompanied by evidence of an increased frequency of circulating B cells. These data indicate that MC patients are characterized distinctly by a higher quota of cell-associated viral load.

Hepatitis C virus RNA and core protein in kidney glomerular and tubular structures isolated with laser capture microdissection.

The role of hepatitis C virus (HCV) in the production of renal injury has been extensively investigated, though with conflicting results. Laser capture microdissection (LCM) was performed to isolate and collect glomeruli and tubules from 20 consecutive chronically HCV-infected patients, namely 6 with membranoproliferative glomerulonephritis, 4 with membranous glomerulonephritis, 7 with focal segmental glomerulosclerosis and 3 with IgA-nephropathy. RNA for amplification of specific viral sequences was provided by terminal continuation methodology and compared with the expression profile of HCV core protein. For each case two glomeruli and two tubular structures were microdissected and processed. HCV RNA sequences were demonstrated in 26 (65%) of 40 glomeruli, but in only 4 (10%) of the tubules (P < 0.05). HCV core protein was concomitant with viral sequences in the glomeruli and present in 31 of the 40 tubules. HCV RNA and/or HCV core protein was found in all four disease types. The immunohistochemical picture of HCV core protein was compared with the LCM-based immunoassays of the adjacent tissue sections. Immune deposits were detected in 7 (44%) of 16 biopsy samples shown to be positive by extraction methods. The present study indicates that LCM is a reliable method for measuring both HCV RNA genomic sequences and HCV core protein in kidney functional structures from chronically HCV-infected patients with different glomerulopathies and provides a useful baseline estimate to define the role of HCV in the production of renal injury. The different distribution of HCV RNA and HCV-related proteins may reflect a peculiar 'affinity' of kidney microenvironments for HCV and point to distinct pathways of HCV-related damage in glomeruli and tubules.

Evaluation of the BDProbeTec strand displacement amplification assay in comparison with the AMTD II direct test for rapid diagnosis of tuberculosis.

The BDProbeTec MTB assay for direct detection of Mycobacterium tuberculosis was evaluated in comparison with the AMTD-II assay on 94 samples from different patients with clinical suspicion of tuberculosis. Using a combination of culture on Lowenstein-Jensen medium (with or without preculture in BACTEC 9000) and clinical diagnosis as the standard, BDProbeTec MTB showed high sensitivity and specificity (96.1% and 100%, respectively), similar to AMTD-II (96.1% and 97.1%, respectively), with significantly higher sensitivity than the Ziehl-Neelsen stain for acid-fast bacilli (73%, p < 0.05).

Regulation of switching frequency and bias of the bacterial flagellar motor by CheY and fumarate.

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of Switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counter-clockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.

Phosphorylation-independent bacterial chemoresponses correlate with changes in the cytoplasmic level of fumarate.

Bacterial chemotaxis is based on modulation of the probability to switch the direction of flagellar rotation. Responses to many stimuli are transduced by a two-component system via reversible phosphorylation of CheY, a small cytoplasmic protein that directly interacts with the switch complex at the flagellar motor. We found that the chemorepellents indole and benzoate induce motor switching in Escherichia coli cells with a disabled phosphorylation cascade. This phosphorylation-independent chemoresponse is explained by reversible inhibition of fumarase by indole or benzoate which leads to an increased level of cellular fumarate, a compound involved in motor switching for bacteria and archaea. Genetic deletion of fumarase increased the intracellular concentration of fumarate and enhanced the switching frequency of the flagellar motors irrespective of the presence or absence of the phosphorylation cascade. These correlations provide evidence for fumarate-dependent metabolic signal transduction in bacterial chemosensing.

Mechanism of photosensory adaptation in Halobacterium salinarium.

Phototaxis in Halobacterium salinarium is the result of an interplay of sensory rhodopsin excitation and adaptation to the stimulus background. Adaptation to orange light, received by sensory rhodopsin I was probed by measuring the behavioral response of cells to a step-like decrease in intensity. Cells were able to adapt to an intensity range of more than four orders of magnitude. The data were analysed on the basis of theoretical fluence rate response relationships calculated from the photocycle kinetics of the complex of sensory rhodopsin I with its transducer HtrI. Independent of the stimulus background, the cellular response was shown to be a function of the absolute number of photoreceptor complex molecules turned over by the light stimulus. Receptor deactivation was identified as the underlying mechanism of adaptation and was sufficient to account for the experimental results. We suggest that reversible methylation of the transducer protein HtrI provides the chemical mechanism of sensory adaptation in H. salinarium and also explains the different sensitivity of the cells to orange and UV light.

Sensory rhodopsin-controlled release of the switch factor fumarate in Halobacterium salinarium.

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low-molecular-mass fraction (< 5 kDa) of the lysate was assayed enzymatically and a light-induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light-induced release was not detected in a mutant defective in sensory rhodopsin-I and -II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin-II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light-induced fumarate release is at least 10-times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.