BRAF mutations are detected in approximately 3% to 8% of patients with NSCLC. In contrast to melanoma, in which most BRAF mutations occur at the V600 codon, only approximately 35% of BRAF-mutant NSCLC tumors harbor V600 mutations. Among the remaining cases, 60% to 70% present non-V600 mutations, primarily in exons 11 and 15. BRAF mutations are classified into three classes according to their kinase activity and their dependence on RAS activation. Compared with class I (V600), patients with class II and class III mutations are associated with poorer clinical outcomes partly due to the lack of effective targeted therapeutic strategies. Indeed, although dual BRAF and MEK inhibition has demonstrated clinical benefit in BRAF V600-mutant NSCLC, there is currently no consensus on treatment strategies for patients with class II and class III mutations. Beyond point mutations, other BRAF alterations (e.g., gene fusions, deletions, and amplifications) have been identified in treatment-naive tumors and in the context of acquired resistance to targeted therapies in other oncogene-driven NSCLC subtypes. However, the biology and clinical implications of these alterations remain poorly characterized. In this review, we provide a comprehensive overview on the biology, epidemiology, and therapeutic strategies of class II/III BRAF mutations, fusions, deletions, and amplifications in NSCLC. We highlight current challenges in the clinical management of BRAF-mutant NSCLC, emerging inhibitors, and combinatorial therapeutic strategies developed to treat non-V600E BRAF-driven cancers. Finally, we briefly discuss BRAF alterations in the context of resistance to targeted therapies in other oncogene-driven NSCLC.

BACKGROUND:This study describes clinical characteristics, treatment patterns as well as safety outcomes of NSCLC patients harboring PD-L1 ≥ 50% who received adjuvant atezolizumab within the Italian real-world scenario. METHODS:Patients with surgically resected NSCLC harboring EGFR/ALK wild type disease and PD-L1 TPS ≥ 50%, who received at least one cycle of adjuvant atezolizumab were included. Clinical-pathological and molecular data, safety and efficacy outcomes were collected from the Italian ATLAS real-world registry. RESULTS:A total of 132 patients were included across 45 Italian centers between July 2022 and August 2024. Lobectomy was performed in 81.1% of cases, with 8.3% pathological stage IIA, 40.2% stage IIB, 43.9% stage IIIA, and 7.6% stage IIIB, according to the eighth TNM staging edition. The median number of atezolizumab cycles was 12.5 (range: 1-20). Treatment related adverse events (TRAEs) during atezolizumab were reported in 44 patients (33.3%), including 11 (8.3%) who experienced multiple TRAEs. Grade ≥ 3 TRAEs were reported in 21 cases (15.9%), leading to treatment discontinuation in 18 (13.6%). The median time to the first onset of TRAEs was 89 days (range: 3-390 days). 15 patients experienced a disease recurrence, including 6 locoregional-only and 9 distant relapses, with a median time since surgery of 13.3 months. CONCLUSION:This study showed that the safety profile of adjuvant atezolizumab outside of a clinical trial context was comparable to the IMPower-010 study, highlighting the value of the Italian ATLAS registry as source of real-word evidence to optimize the clinical management of NSCLC patients.

PURPOSE:Tumor comprehensive genomic profiling (CGP) may detect potential germline pathogenic/likely pathogenic (P/LP) alterations as secondary findings. We analyzed the frequency of potentially germline variants and large rearrangements (LRs) in the RATIONAL study, an Italian multicenter, observational clinical trial that collects next-generation sequencing-based tumor profiling data, and evaluated how these findings were managed by the enrolling centers. PATIENTS AND METHODS:Patients prospectively enrolled in the pathway-B of the RATIONAL study and undergoing CGP with the FoundationOne CDx assays were included in the analysis. Potentially germline variants detected in 40 cancer susceptibility genes (CSGs) were classified in three classes with different actionability, most (MA), high (HA), and standard (SA), on the basis of penetrance, mutational spectrum, and intervention for prevention/early detection. RESULTS:On the basis of the European Society of Medical Oncology recommendations, we identified 225 potentially germline P/LP variants in 193/1,339 (14.4%) enrolled patients. In particular, 62/225 (27.5%) variants were detected in genes classified as MA-CSG class, 53/225 (23.6%) in genes belonging to the HA-CSG class, and 110/225 (48.9%) in the SA-CSG class. In addition, we detected 58 LRs in the 16/40 CSGs in 53/1,339 (3.95%) patients. Information about germline-focused analysis and follow-up was available for 99 patients with potentially germline variants. Surprisingly, 95/99 (96%) patients were not referred to oncogenetic consultation and follow-up, including 30/32 (93.75%) patients with variants in the MA-CSG class. CONCLUSION:Our data confirm the utility of CGP for the identification of potentially germline variants in CSGs, highlighting the importance of reporting LRs in addition to single-nucleotide variants and insertions/deletions. However, our findings also demonstrate a relative lack of knowledge of the implications of germline findings detected on tumor-only sequencing among oncologists and underline the need for specific training in this area.

Double positive OCT3/4-PD1 subsets in the bulk NSCLC cells after cisplatin treatment. OCT3/4 expression preferentially co-localized on PD-1+ NSCLC cells as compared with the PD-1- counterpart (14% vs 0.1%). Representative flow cytometry histograms of OCT3/4 expression are reported in figure.

Activity of ERβ on CD274/PD-L1 promoter. Chromatin immunoprecipitation of ERE1 and ERE2 regions from CD274/PD-L1 promoter, using an anti-ERα or an anti-ERβ antibody, followed by RT-PCR (technical triplicates) of the precipitated products, in ERα highNCI-H1975 cells, grown 24 h in fresh medium (Ctrl) or in medium containing 10 nM fluvestrant (Fulv), an ERα inhibitor, or 10 nM letrozole (Letr), an aromatase inhibitor. Data are means+SD (n=3). **p<0.01, ***p<0.001: treated cells versus Ctrl cells; °°°p<0.001: anti-ERβ versus anti-ERα.

Dose-dependent inhibition of Akt, ERK1/2 and EGFR. ERα highNCI-H1975 cells were grown 24 h in fresh medium (Ctrl) or in medium containing 10 nM, 100 nM, 1 μM of the Akt inhibitor MK-2206 (Akti, panel a), 0.1 μM, 1 μM, 10 μM of the ERK1/2 inhibitor U-0126 (ERKi, panel b), 10 nM, 100 nM, 1 μM of the EGFR inhibitor AZD9291/Osimertinib (EGFRi, panel c). As read-out assays, Akt and ERK1/2 activity were measured by ELISA (technical duplicates), EGFR kinase activity was measured by a chemiluminescence-based assay (technical duplicates). Data are means + SD (n=3, biological replicates). *p<0.05, ***p<0.001: treated cells versus Ctrl cells (ANOVA).

Levels of FSHR and LHR in non-small cell lung cancer cells FSHR (panel a) and LHR (panel b) mRNA, measured by RT-PCR (technical triplicates), in female (F)-derived NCI-H1385 and NCI-H1975 cells, and in male (M)-derived A549 and NCI-H1650 cells. The relative expression of each gene in NCI-H1385 cells was considered 1. Data are means+SD (n=3, biological replicates).

Background:Non-small cell lung cancer (NSCLC) is the predominant histological subtype of lung cancer, whose diverse genomic landscape complicates prognosis and outcome prediction. In this context, immunogenic cell death (ICD), a distinct mechanism of cell death, may exert important antitumor effects. However, the specific role of ICD in NSCLC has not been clarified, and there is no suitable method for using ICD to achieve the treatment and prognosis assessment of NSCLC. The purpose of the current research is to develop a new approach to predict the survival prognosis and response to chemotherapy and targeted therapy in patients with NSCLC. Methods:We used 101 combinations of 10 machine learning algorithms to construct an ICD-related signature (ICDRS). The predictive potential of this specific ICDRS for immune cell infiltration and therapeutic response was evaluated. We also examined the molecular mechanisms underlying the various responses of different ICDRS subpopulations, characterized the mutational landscape and tumor mutational burden (TMB), and assessed the applicability of the ICDRS in single-cell transcriptomic datasets. Gene expression patterns were subsequently validated with quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Results:We screened out five key ICDRS genes (MMP14, ALDH2, FBP1, HLA-DRA, KCTD12). Patients were classified into high-risk and low-risk groups according to the ICDRS score determined by the expression levels of these five genes. The high-risk group exhibited less favorable prognoses compared with the low-risk group, which demonstrated more positive outcomes. The low-risk group had more anticancer immune-cell infiltration and showed better response to chemotherapy and targeted therapy than the high-risk group (P<0.05). Conclusions:The ICDRS exhibited excellent predictive performance and broad applicability, suggesting it as a powerful tool for prognosis and therapy response. The current study may contribute to more adequate patient selection in the context of tailored therapies.

Molecular circuitries linking ERα and PD-L1. ERα, activated by the binding of 17-β-estradiol and by the phosphorylation on Ser118 via the EGFR/Akt and EGFR/ERK1/2 axes, upregulates PD-L1 and predicts a better response to pembrolizumab. However, lowering the synthesis of 17-β-estradiol with letrozole or the activity of ERα with fulvestrant sensitizes non-small cell lung cancer to pembrolizumab, relieving the PD-L1-dependent immune-suppression, allowing the expansion and activity of anti-tumor infiltrating lymphocytes, such as CD8+T-cells, NK cells and Vγ9Vδ2+T cells. We suggest the existence of four possible scenarios, depending on sex, EGFR activity, 17-β-estradiol/ERα and PD-L1 status, with a differential sensitivity to pembrolizumab and aromatase inhibitors:1) male patients with low 17-β-estradiol synthesis, low EGFR signaling, low ERα amount/activity and low PD-L1 amount; 2) male patients with high EGFR signaling, high ERα amount/activity but low 17-β-estradiol synthesis, with intermediate PD-L1 amount; 3) female patients with low EGFR signaling, low ERα amount/activity but high 17-β-estradiol synthesis, with intermediate PD-L1 amount; 4) female patients with high EGFR signaling, high ERα amount/activity, high 17-β-estradiol synthesis and high PD-L1 amount. The efficacy of pembrolizumab and the additional benefit of aromatase inhibitors increased progressively from scenario 1 to 4.

Outcome of non-small cell lung cancer patients, disaggregated according to gender, PD-L1 and ERα levels. Best response (PD: progressive disease; PR: partial response; SD: stable disease) after 6 months from the beginning of pembrolizumab treatment as first-line monotherapy, PFS and OS (Kapan-Meier and log-rank test) were analyzed in 35 NSCLC patients (15 females; 20 males). a-c. Female (F) and male (M) patients were stratified in CD274/PD-L1low and CD274/PD-L1high, according to the median value of the CD274/PD-L1mRNA, measured by RT-PCR in the tumor samples (technical triplicates). d-f. F and M patients were stratified in ESR1/ERαlow and ESR1/ERαhigh, according to the median value of the ESR1/ERα mRNA, measured by RT-PCR in the tumor samples (technical triplicates).

Effects of letrozole on the efficacy of chemo-immuno-therapy in non-small cell lung cancer xenografts. 1×106 female-derived ERα lowNCI-H1385 cells and ERα highNCI-H1975 cells, male-derived ERα lowA549 cells and ERα highNCI-H1650 cells were implanted subcutaneously (s.c.) in Hu-CD34+NSG mice. When tumors reached the volume of 100 mm3, mice (n= 5/group) were randomized in the following groups: 1) Vehicle group, treated intraperitoneally (i.p.) with 100 µl saline solution (days 1, 7, 14, 21, 28, 35 after randomization); 2) cisplatin (PT) group, treated with 2 mg/kg intravenously (i.v.) (days 7, 14, 21, 28, 35); 3) cisplatin + pembrolizumab (PT+Pemb) group, treated with 2 mg/kg cisplatin i.v. (days 7, 14, 21, 28, 35) and 10 mg/kg i.p. of 100 µl saline solution of pembrolizumab (day 1) followed by 5 mg/kg i.p. (days 7, 14, 21, 28, 35); 4) cisplatin + pembrolizumab + letrozole (PT+Pemb+Letr) group, treated with 2 mg/kg cisplatin i.v. (days 7, 14, 21, 28, 35), 10 mg/kg i.p. of 100 µl saline solution of pembrolizumab (day 1) followed by 5 mg/kg i.p. (days 7, 14, 21, 28, 35) and 1 mg/kg per os of 100 µl saline solution of letrozole daily (days 1-35). Tumor growth was monitored daily with a caliper. Mice were euthanized on day 40. Data are mean volumes+SD. **p<0.01, ***p<0.001: treatments group versus Vehicle group (day 40); °°°p<0.001: PT+Pemb+Letr group versus Pemb+Letr group (day 40; ANOVA).

Thymic epithelial tumors (TETs) are a heterogeneous group of rare tumors that arise from thymic epithelial cells in the anterior mediastinum. They can be divided into three different histological subtypes: thymomas, thymic carcinomas (TC), and neuroendocrine carcinomas (TNET). TCs and TNETs are rarer but more aggressive entities with frequent distant metastasis. Thymomas occur in 90 % of cases in a localized/locally advanced stage, on the other hand about 70 % of TCs are locally advanced at the time of diagnosis. Surgery plays a primary role in the management of patients in whom complete resection is feasible. The benefit of post-operative radiotherapy (PORT) is still controversial, since it could be related to stage, histotype, and preoperative chemotherapy. If the tumor is unresectable at diagnosis, radiotherapy or concurrent chemoradiotherapy is the most commonly used approach. Cisplatin and anthracycline-based regimens are standard of care in patients with unresectable or metastatic thymomas, but, at the same time, regimens with carboplatin and paclitaxel are the most widely used especially in patients with contraindications to cisplatin/anthracyclines, due to better tolerance. Recently, the anti-VEGFR antibody Ramucirumab has shown promising activity in combination with carboplatin plus paclitaxel in previously untreated advanced TCs. Several clinical trials with chemotherapy combination, target therapy and immunotherapy are still ongoing to define the best therapeutic strategy in this disease, also for the second line treatment, for which in daily practice there is currently no standard of care for patients who went into progression to the first line.

Representativeness of Study Participants

EML4-ALK fusion has emerged as a significant oncogenic driver in Non-Small Cell Lung Cancer (NSCLC), promoting abnormal cell proliferation and survival. Despite advances with specific tyrosine-kinase inhibitors (TKIs), resistance and relapse still pose significant challenges. Notably, patients with ALK+ NSCLC and co-occurring TP53 mutations experience poorer outcomes after treatment, underscoring an unmet medical need. To anticipate evolving resistance, it is essential to identify and target new vulnerabilities, both on-target and off-target. Exportin 1 (XPO1), a nuclear export protein involved in tumor growth and survival, has emerged as a promising target, and selective XPO1 inhibitors (XPO1i) have been developed. This study evaluates the preclinical efficacy of combining XPO1i with first, second, and third-generation ALK-TKIs in both TKI-sensitive and TKI-resistant NSCLC models. To explore the clinical relevance of XPO1 in ALK+ NSCLCs, we examined its expression in relation to patient survival using data from the Cancer Genome Atlas and Genotype-Tissue Expression datasets. We observed overexpression of XPO1 in tumor tissues, which was positively correlated with a higher risk of disease progression in lung adenocarcinoma. Consistently, patients with low XPO1 expression had significantly better overall survival than those with high XPO1 expression, indicating that elevated XPO1 levels are associated with a poorer prognosis. To investigate the potential vulnerability of ALK+NSCLC to XPO1 inhibition, we tested the XPO1i KPT-330 alone and in combination with ALK-TKIs in various ALK+NSCLC models, including in vitro-generated and patient-derived TKI-resistant cell lines and tumor organoids (PDTOs). Cell viability and proliferation were evaluated using the CellTiter-Glo® assay and the Incucyte Live Imaging system. In vitro, KPT-330 significantly affected cell viability as a single agent and potentiated the antiproliferative effect of ALK-TKIs when combined. Synergy analysis confirmed a synergistic interaction between the two drugs. Cell cycle distribution analysis revealed that XPO1i induced G1 phase arrest, promoting the accumulation of p53, along with a proportional decrease in anti-apoptotic proteins. Notably, KPT-330 demonstrated efficacy regardless of p53 status, inducing apoptosis in both wild-type and mutant cells. ALK+NSCLC cell-derived xenograft (CDX) and PDTO-derived xenograft (PDOXs) mouse models were used to evaluate the efficacy of the combination in vivo. Consistent with in vitro results, the combination of TKIs with KPT-330 displayed enhanced tumor growth inhibition compared to single-agent treatments in ALK+ NSCLC mouse models, indicating its potential to overcome resistance mechanisms and prevent relapse occurrence. Overall, these data support the therapeutic efficacy of XPO1 inhibition, both alone and in combination with ALK-TKIs, in treating ALK+ NSCLCs, despite the development of resistance to targeted therapy and regardless of the patients' p53 mutation status. Maria Vittoria Di Marco, Francesca Picca, Mariantonia Costanza, Enrico Patrucco, Lisa Bonello, Francesca Bersani, Riccardo Taulli, Chiara Ambrogio, Fabrizio Tabbò, Luisella Righi, Aaron Hata, Silvia Novello, Giorgio Vittorio. Scagliotti, Stephan Mathas, Roberto Chiarle, Claudia Voena. XPO1-dependent nuclear export as a therapeutic target for ALK-positive non-small cell lung cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B055.

Switch-off/on EGFR activity in NCI-H1385 cells. a. Experimental scheme: Low EGFR activitywild-type NCI-H1385 cells were knocked-out (KO) for endogenous EGFR by a CRISPR/Cas vector, then stably transfected with an expression vector encoding L858R EGFR. b. Immunoblot of EGFR in wild-type (wt) NCI-H1385 cells and in clones subjected to two (#1, #2) EGFR-CRISPR/Cas vectors. Tubulin is included as control of equal protein loading. The image is representative of one of three experiments. c. L858R EGFR mRNA levels in wt NCI-H1385 cells and in cells stably transfected with the L858R EGFR-expression vector, measured by RT-PCR (technical triplicates). Data are means + SD (n=3, biological replicates). ***p<0.001: L858R cells versus wt cells (ANOVA). d. Immunoblot of EGFR in wt and L858R NCI-H1385 cells. Tubulin is included as control of equal protein loading. The image is representative of one of three experiments. e. EGFR kinase activity, measured in wt and L858R NCI-H1385 cells by a chemiluminescence-based assay in technical duplicates. Data are means+SD (n=3, biological replicates). ***p<0.001: L858R cells versus wt cells (ANOVA).

PD-L1 inhibition does not affect amount and phosphorylation of estrogen receptor α. Female-derived ERα lowNCI-H1385 and ERα highNCI-H1975 cells, male-derived ERα lowA549 and ERα highNCI-H1650 cells were treated 24 h in the absence (-) or presence (+) of 1 μg/mL atezolizumab (Atezo), a PD-L1 inhibitor, then lysed and analyzed for the indicated proteins in nuclear extracts. TBP is included as control of equal protein loading. The image is representative of one of three experiments.

Disaggregated data of ERα, 17-β-estradiol and PD-L1 levels in non-small cell lung cancer cells. a-c. Expression of ERα mRNA, measured by RT-PCR (technical triplicates) (panel a), levels of 17-β-estradiol, measured by ELISA (technical triplicates) (panel b), expression of surface PD-L1 (panel c), measured by flow cytometry (technical triplicates) in 29 human NSCLC cell lines, divided in female (F)- or male (M)-derived cell lines, primary tumor (P) or metastatic (MTS) localization-derived cell lines, non-smoker (NSm) or smoker (Sm) patients. *p<0.05, **p<0.01: F versus M, P versus MTS, NSm versus Sm (ANOVA).