The expression level of Programmed Death-Ligand 1 (PD-L1) determined by the immunohistochemical method is currently approved to test the potential efficacy of immune-checkpoint inhibitors and to candidate patients with Non-Small Cell Lung Cancer (NSCLC) for treatment with immunotherapeutic drugs. As part of the CORELAB (New prediCtivebiOmaRkers of activity and Efficacy of immune checkpoint inhibitors in advanced non-small cell Lung cArcinoma) project, aimed at identifying new predictive and prognostic biomarkers in NSCLC patients receiving immunotherapeutic drugs, we investigated the role of circulating tumor DNA (ctDNA) molecular characterization as an additional predictive biomarker. We analyzed plasma ctDNA by targeted Next Generation Sequencing in a subset of 50 patients at different time points. ctDNA content was inversely correlated with the clinical outcome both at a baseline and after 2 months of treatment. OS was significantly higher in patients with ≥50% ctDNA reduction. TP53 and KRAS were the most frequently mutated genes, and patients with KRAS and/or TP53 mutations showed worse outcomes than patients without detectable variants or with mutations in other genes. Fewer common variants were found in BRAF, EGFR, MAP2K1, MET, NRAS, and PIK3CA genes. Our data demonstrated that molecular characterization of ctDNA and also its quantitative evaluation could serve as a dynamic, real-time prognostic, and predictive biomarker, enabling regular molecular monitoring of therapy efficacy in support of other medical examinations.

Despite recent therapeutic advances, the adjuvant treatment of non-small cell lung cancer (NSCLC) remains a challenge. Reducing the risk of recurrence is still a concern, especially in the KRAS G12C population, for which platinum-based adjuvant chemotherapy (CT) remains the gold standard. In this study, we evaluated the efficacy, in terms of cell viability and volumetric reduction, of adding KRAS inhibitors (KRASi) sequentially or concurrently to CT in both parental (PR) and gemcitabine-resistant (GR) KRAS mutated NSCLC cell lines (SW1573 and H23). We demonstrated that KRASi added to CT (both sequential and concurrent treatment strategies) reduced cell viability in SW1573-PR and H23-PR and this effect is less evident in GR cell lines. Interestingly, in the 3D model, the concomitant use of KRASi+CT reduced spheroid volume in both PR and GR spheroids. Our results indicate that KRASi enhances the efficacy of CT in both NSCLC PR and GR cells, suggesting a potential therapeutic strategy to overcome chemoresistance in the adjuvant setting of NSCLC.

BACKGROUND:Lung cancer (LC) is the leading cause of cancer death worldwide. Non-small cell lung cancer is the most frequent and includes adenocarcinoma and squamous cell carcinoma. Currently, LC treatment is based on tumor molecular profiling. LC may display Epidermal Growth Factor Receptor (EGFR) gene mutation. Detecting mutations in the EGFR gene is crucial for the tyrosine kinase inhibitory therapy. METHODS:This study used a computer-based methodology with two Convolutional Neural Networks (CNNs) based on InceptionResNet-V2, applied to Whole Slide Images, to distinguish healthy from cancerous tissue and then EGFR mutated tumor tissue samples. We also integrated an Explainable AI technique (Grad-CAM) to clearly visualize insights into the model's decision-making process. The analysis was conducted on 259 LC cases collected from three different centers (Florence, Rome, and Sassari). RESULTS:This methodology achieved an accuracy of 96.17% in distinguishing healthy from cancerous tissue, with specificity of 87.89%, sensitivity of 98.43%, an F1 score of 97.59% and an AUC of 0.99. Additionally, Cohen's Kappa indicated a consistency of 0.7982, and the confusion matrix showed a correct classification rate of 96.2%. For EGFR mutation detection in cancer tissue, slide-level performance after aggregation reached an accuracy of 76.67% with specificity of 80.77%, sensitivity of 73.53%, an F1 score of 78.12%, a consistency of 0.5583 of Cohen's Kappa and an AUC of 0.77. The confusion matrix showed 76.7% as a correct classification rate. CONCLUSION:The two tested CNNs showed potential for assisting LC diagnosis, especially in distinguishing healthy from tumor tissue. While the direct detection of EGFR mutational status remains challenging, the results suggest that relevant predictive signals can still be extracted from routine H&E slides.

Background:Platinum chemotherapy (CT) remains the backbone of systemic therapy for patients with small-cell lung cancer (SCLC). The nucleotide excision repair (NER) pathway plays a central role in the repair of the DNA damage exerted by platinum agents. Alteration in this repair mechanism may affect patients' survival. Materials and Methods:We conducted a retrospective analysis of data from 38 patients with extensive disease (ED)-SCLC who underwent platinum-CT at the Clinical Oncology Unit, Careggi University Hospital, Florence (Italy), from 2015 to 2020. mRNA expression analysis and single nucleotide polymorphism (SNP) characterization of three NER pathway genes-namely ERCC1, ERCC2, and ERCC5-were performed on patient tumor samples. Results:Overall, elevated expression of ERCC genes was observed in SCLC patients compared to healthy controls. Patients with low ERCC1 and ERCC5 expression levels exhibited a better median progression-free survival (mPFS = 7.1 vs. 4.9 months, p = 0.39 for ERCC1 and mPFS = 6.9 vs. 4.8 months, p = 0.093 for ERCC5) and overall survival (mOS = 8.7 vs. 6.0 months, p = 0.4 for ERCC1 and mOS = 7.2 vs. 6.2 months, p = 0.13 for ERCC5). Genotyping analysis of five SNPs of ERCC genes showed a longer survival in patients harboring the wild-type genotype or the heterozygous variant of the ERCC1 rs11615 SNP (p = 0.24 for PFS and p = 0.14 for OS) and of the rs13181 and rs1799793 ERCC2 SNPs (p = 0.43 and p = 0.26 for PFS and p = 0.21 and p = 0.16 for OS, respectively) compared to patients with homozygous mutant genotypes. Conclusions:The comprehensive analysis of ERCC gene expression and SNP variants appears to identify patients who derive greater survival benefits from platinum-CT.

Introduction Hemophagocytic Lymphohistiocytosis (HLH) is a rare, aggressive, and life-threatening disorder characterized by sustained but ineffective immune system activation that leads to severe and systemic hyperinflammation. It may occur as a genetic or sporadic condition, often triggered by an infection. The multifaceted pathogenesis results in a wide range of non-specific symptoms, signs, and laboratory findings that challenge its recognition. The pulmonary involvement is underdiagnosed and may manifest as pneumonia, which can lead to respiratory failure. Despite the great improvement achieved in terms of survival, a considerable proportion of patients with HLH still die from progressive disease. Case Presentation We discuss the case of a unique form of respiratory distress and multiorgan failure with inconclusive radiological and lung biopsy investigations. The patient was finally diagnosed, by genetic analysis, with HLH, and promptly treated as per HLH-94 treatment protocol. Conclusion This case report aims to emphasize that clinical, laboratory, instrumental, and even pathological findings in HLH might not be unequivocal; nonetheless, a rapid diagnosis and treatment are mandatory, given the high mortality of the disease.

BACKGROUND:Emerging evidence identified sex as a variable regulating immune system functions and modulating response to immunotherapy in cancer patients. OBJECTIVE:This retrospective study analysed sex-related differences in immunotherapy outcomes in a real-world population of non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICIs). METHODS:We retrospectively investigated clinical data of 99 patients with advanced NSCLC and treated with single-agent nivolumab and pembrolizumab at Medical Oncology Unit, Careggi University Hospital, Florence (Italy), between April 2014 to August 2019. Main clinical characteristics and clinical outcomes were analysed. RESULTS:Our study showed that the efficacy of ICI treatment differed according to gender. A trend for better median progression-free survival (mPFS) was reported in males (mPFS 5.0 months, 95% Confidence Interval [CI] 4.0-11.0) than females (mPFS 4.5 months, 95% CI 2.0-9.0) (p=0.133), while no significant difference for overall survival (OS) between the two sex groups was observed (p=0.622). In the nivolumab cohort, we showed a statistically significant difference for a longer PFS in men compared to women (log-rank p=0.054), HR for PFS in females versus males was 1.81 (95% CI 0.97- 3.37, p=0.062). Disease control rate (DCR) was achieved in 55.7% and 45.7% of men and women, respectively, while disease progression was registered in 44.3% of males and 54.3% of females (p=0.386). CONCLUSION:Gender is a variable that should be taken into account in the choice of immunotherapy. Future prospective randomized trials testing tailored sex-based immunotherapy strategies are required to validate our findings before integrating into clinical practice.

Lung cancer is the malignancy with the highest morbidity and mortality worldwide. Approximately 60% of non-small cell lung cancer (NSCLC) presents driver alterations most of which are targetable. Nowadays, limited clinical data are available regarding the efficacy of epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors in patients with NSCLC harboring uncommon EGFR mutations, considering their heterogeneity. Herein, we report a rare case of EGFR-mutated lung adenocarcinoma which has developed into squamous cell carcinoma with uncommon EGFR (Ex18) compound mutations and phosphatidylinositol 3-kinase mutation receiving afatinib at the forefront.

Purpose: To investigate the early radiological features and survival of Large Cell Carcinoma (LCC) cases diagnosed in low-dose computed tomography (LDCT) screening trials. Methods: Two radiologists jointly reviewed the radiological features of screen-detected LCCs observed in NLST, ITALUNG, and LUSI trials between 2002 and 2016, comprising a total of 29,744 subjects who underwent 3-5 annual screening LDCT examinations. Survival or causes of death were established according to the mortality registries extending more than 12 years since randomization. Results: LCC was diagnosed in 30 (4 %) of 750 subjects with screen-detected lung cancer (LC), including 15 prevalent and 15 incident cases. Three additional LCCs occurred as interval cancers during the screening period. LDCT images were available for 29 cases of screen-detected LCCs, and 28 showed a single, peripheral, and welldefined solid nodule or mass with regularly smooth (39 %), lobulated (43 %), or spiculated (18 %) margins. One case presented as hilar mass. In 9 incident LCCs, smaller solid nodules were identified in prior LDCT examinations, allowing us to calculate a mean Volume Doubling Time (VDT) of 98.7 +/- 47.8 days. The overall five-year survival rate was 50 %, with a significant (p = 0.0001) difference between stages I-II (75 % alive) and stages IIIIV (10 % alive). Conclusions: LCC is a fast-growing neoplasm that can escape detection by annual LDCT screening. LCC typically presents as a single solid peripheral nodule or mass, often with lobulated margins, and exhibits a short VDT. The 5-year survival reflects the stage at diagnosis.

Diagnosing COVID-19 and treating its complications remains a challenge. This review reflects the perspective of some of the Dragon (IMI 2-call 21, #101005122) research consortium collaborators on the utility of bronchoalveolar lavage (BAL) in COVID-19. BAL has been proposed as a potentially useful diagnostic tool to increase COVID-19 diagnosis sensitivity. In both critically ill and non-critically ill COVID-19 patients, BAL has a relevant role in detecting other infections or supporting alternative diagnoses and can change management decisions in up to two-thirds of patients. BAL is used to guide steroid and immunosuppressive treatment and to narrow or discontinue antibiotic treatment, reducing the use of unnecessary broad antibiotics. Moreover, cellular analysis and novel multi-omics techniques on BAL are of critical importance for understanding the microenvironment and interaction between epithelial cells and immunity, revealing novel potential prognostic and therapeutic targets. The BAL technique has been described as safe for both patients and healthcare workers in more than a thousand procedures reported to date in the literature. Based on these preliminary studies, we recognize that BAL is a feasible procedure in COVID-19 known or suspected cases, useful to properly guide patient management, and has great potential for research.

Abstract Background: Platinum chemotherapy (CT) remains the backbone of systemic therapy for patients with small-cell lung cancer (SCLC). The nucleotide excision repair (NER) pathway plays a central role in the repair of the DNA damages exerted by platinum agents. Alteration in this repair mechanism may affect patients survival. Methods: We retrospectively collected data from 38 patients with extended disease (ED)-SCLC treated with platinum-CT at Clinical Oncology Unit, Careggi University Hospital, Florence (Italy), between 2015 to 2020. On patient tumor samples, we performed mRNA expression analysis and characterization of single nucleotide polymorphisms (SNPs) of three NER pathway genes, namely ERCC1, ERCC2 and ERCC5. Results: Overall, we found a higher expression of ERCC genes in SCLC patients compared to the healthy controls. Patients with low ERCC1 and ERCC5 expression levels had a better median progression free survival (mPFS=7.1 vs 4.9 months, p=0.39 for ERCC1 and mPFS=6.9 vs 4.8 months, p=0.093 for ERCC5) and overall survival (mOS=8.7 vs 6.0 months, p=0.4 for ERCC1 and mOS=7.2 vs 6.2 months, p=0.13 for ERCC5). Genotyping analysis of five SNPs of ERCC genes showed a longer survival in patients harboring the wild-type genotype or the heterozygous variant of the ERCC1 rs11615 SNP (p=0.24 for PFS and p=0.14 for OS) and of the rs13181 and rs1799793 ERCC2 SNPs (p=0.43 and p=0.26 for PFS and p=0.21 and p=0.16 for OS, respectively) compared to patients with homozygous mutant genotypes. Conclusions: The integrated analysis of ERCC genes expression and their SNPs variants seems to identify patients with better survival benefits to platinum-CT.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy of the pleural surface that includes three major histologic subtypes, epitheliod, sarcomatoid and biphasic. Epithelioid mesothelioma is usually associated with better prognosis. The genetic mechanisms driving MPM, the possible target mutations and the correlation with overall survival remain largely unsettled. We performed target exome sequencing in 29 cases of MPM aimed at identifying somatic mutations and, eventually, their correlation with phenotypic traits and prognostic significance. We found that KRAS mutations, occurring in 13.7% of cases, were associated with shortened median survival (7.6 versus 32.6 months in KRAS wild-type; p = 0.005), as it was the occurrence of any ≥3 mutations (7.6 versus 37.6 months; p = 0.049). Conversely, the presence of KDR single nucleotide polymorphism p.V297I (rs2305948) resulted in a favorable variable for survival (NR versus 23.4 months; p = 0.026). With the intrinsic limitations of a small number of cases and patient heterogeneity, results of this study contribute to the characterization of the mutation profile of MPM and the impact of selected somatic mutations, and possibly KDR polymorphism, on prognosis.

In immunocompromised hosts Coronavirus disease 2019 (COVID-19) can range from prolonged asymptomatic viral shedding to persistent/relapsing symptoms. In these conditions off-label use of remdesivir and Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) specific monoclonal antibodies (mAbs) has been endeavored. Nirmatrelvir-ritonavir (NM/r) is an oral antiviral, which has proven to reduce by 89% the risk of progression to severe COVID-19 among high-risk patients with early-stage, symptomatic COVID-19. To date, there are no data about NM/r or mAbs efficacy in patients with prolonged and/or relapsing SARS-CoV-2 infection. We describe 5 cases of persistent and/or relapsing COVID-19 in immunocompromised patients observed at the Careggi University Hospital, Florence, Italy successfully treated with oral NM/r or mAbs infusion. The clinical characteristics are summarized in Fig.1. Therapy course of symptoms was collegially discussed. In all cases relapse rather than reinfection was documented by genomic analysis. All patients showed a rapid and full clinical, virological and radiologic response to NM/r or mAbs treatment. In our cases, COVID-19 relapse occurred despite initial treatment with remdesivir and/or sotrovimab. At the time of writing, 8 months after NM/r or mABs administration, we have no evidence of further COVID-19 relapse in these patients.

Introduction: Given the current challenges in sampling in not directly visibile lesions (eg, sample size and quality) we propose an alternative to standard practice: a novel technique using the "ultrathin" 3mm bronchoscope with the 1.1mm cryoprobe as sampling tool. Objective: The main aim was to prove the safety and feasability of the proposed technique, measured as occurence of Adverse Events. Secondary aim included: Diagnostic Yeld, Operative time duration, adequacy and quality of both ROSE and histology evaluation. Methods: The study monocentric, retrospective study between two groups. The "ultrathin" in which sampling was conducted with the ultrathin instrument and the 1.1mm cryoprobe (UT). The "conventional" in which sampling is obtained using the "standard" 4 or 6mm bronchoscope with forceps (FBS-Tbbx) Results: We enrolled 9 pts or the UT group and 10 for the conventional. We registered similar occurence of AEs between the two groups (4/9 UT vs 5/10 FBS-Tbbx ); mainly intraoperative bleeding which was controlled endoscopically. None of the registered AEs required further invasive procedures. The DYs were also comparable (77 vs 80%). Notably in all cases ROSE showed a richer cellularity than conventional biopsy. Operative time were similar in both groups (mean duration 39,4vs 39,2 mins). Discussion: The combination of the ultrathin instrument and the 1.1 mm cryoprobe is a safe and feasable alternative to what is considered standard practice. It could also prove to be superior given the bronchial selectivity of the instrument and the size and quality of the sample provided using the 1.1mm cryoprobe but further research is needed.

The incidental discovery of pre-clinical interstitial lung disease (ILD) has led to the designation of interstitial lung abnormalities (ILA), a radiological entity defined as the incidental finding of computed tomography (CT) abnormalities affecting more than 5% of any lung zone. Two recent documents have redefined the borders of this entity and made the recommendation to monitor patients with ILA at risk of progression. In this narrative review, we will focus on some of the limits of the current approach, underlying the potential for progression to full-blown ILD of some patients with ILA and the numerous links between subpleural fibrotic ILA and idiopathic pulmonary fibrosis (IPF). Considering the large prevalence of ILA in the general population (7%), restricting monitoring only to cases considered at risk of progression appears a reasonable approach. However, this suggestion should not prevent pulmonary physicians from pursuing an early diagnosis of ILD and timely treatment where appropriate. In cases of suspected ILD, whether found incidentally or not, the pulmonary physician is still required to make a correct ILD diagnosis according to current guidelines, and eventually treat the patient accordingly.

Pulmonary sequelae after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection range from limited abnormalities to major interstitial lung diseases (ILD). Bronchoalveolar lavage (BAL) and cryobiopsy findings, integrated with the clinical and radiological scenario, may help clinicians to correctly manage patients and aid researchers in better understanding the features and pathogenic mechanisms of post-COVID-19 fibrosis.

Background:KRAS is commonly mutated in non-small cell lung cancer (NSCLC); however, the prognostic and predictive impact of each G12 substitution has not been fully elucidated. The approval of specific G12C inhibitors has modified the idea of KRAS "undruggability", and although the first-line standard consists of immune checkpoint inhibitors (ICIs) with or without chemotherapy, as suggested at ASCO 2022, the outcome in KRAS-mutated population is still controversial.Methods:We retrospectively described the clinical and pathological characteristics of a homogeneous G12 mutated cohort of 219 patients treated in four Italian oncologic units. We evaluated the outcome (PFS at 18 months and OS at 30 months) of those who underwent standard first-line treatment according to PD-L1 status, focusing on differences across single mutations.Results:In the study population, 47.9% of patients harbor the KRAS G12C mutation; 20.5%, G12V; 17.4%, G12D; and 8.2%, G12A. Smoking was a common behavior of patients harboring transversions and transition mutations. PD-L1 expression does not show particular distribution in the case series, although we recorded a prevalence of PD-L1 <1% in G12V (51.4%) compared to G12A (26.7%). ICIs alone was the clinician's choice in 32.7% of patients, and the chemo-immune combination in 17.3% of patients. We described the independent prognostic role of young age (p = 0.007), female gender (p = 0.016), and an ICI-based regimen (p = 0.034) regardless of mutations. Overall, our data confirm the worst prognostic value of G12V mutation apart from treatment choice unlike the other major mutations (C, D, and A) that showed a favorable trend in PFS.Conclusions:KRAS G12 mutations are confirmed to have different characteristics, and the outcome is influenced by ICI first-line regimen. This study provides valuable information for further analysis in the future.

Rationale: Lung cancer is the most common cause of cancer-related deaths worldwide. Approximately 50% of patients is metastatic at diagnosis and the most common metastatic sites are bone, lungs, brain, adrenal glands, liver, and extra thoracic lymph nodes. The occurrence of gastrointestinal metastasis from lung carcinoma is rare and seems more commonly related to small cell lung cancer compared to non-small cell lung cancer (NSCLC). Patient information and diagnosis: A 78-year-old man with completely surgically resected NSCLC and no initial evidence of distant metastases developed colon and gastric metastases 7 months after diagnosis, confirmed by serial radiological examinations and endoscopic biopsies. Interventions: The patient was subjected to total gastrectomy with D2 lymph node dissection plus partial colectomy for intraoperative detection of a transverse colon neoformation. Subsequent instrumental imaging showed bilateral lung tumor recurrence, treated with gemcitabine monotherapy for 8 months as first line chemotherapy for lung adenocarcinoma. Results: The patient presented complete response to therapy and was disease-free for 4 years. Lessons: Colonic and gastric metastasis are very infrequent in NSCLC. The resection of gastrointestinal metastasis may provide benefits in terms of both symptom control and survival in patients properly selected.