Targeting glucosylceramide synthase induces antiproliferative and proapoptotic effects in osimertinib-resistant NSCLC cell models.

The EGFR tyrosine kinase inhibitor osimertinib has been approved for the first-line treatment of EGFR-mutated Non-Small Cell Lung Cancer (NSCLC) patients. Despite its efficacy, patients develop resistance. Mechanisms of resistance are heterogeneous and not fully understood, and their characterization is essential to find new strategies to overcome resistance. Ceramides are well-known regulators of apoptosis and are converted into glucosylceramides (GlcCer) by glucosylceramide synthase (GCS). A higher content of GlcCers was observed in lung pleural effusions from NSCLC patients and their role in osimertinib-resistance has not been documented. The aim of this study was to determine the therapeutic potential of inhibiting GCS in NSCLC EGFR-mutant models resistant to osimertinib in vitro and in vivo. Lipidomic analysis showed a significant increase in the intracellular levels of glycosylceramides, including GlcCers in osimertinib resistant clones compared to sensitive cells. In resistant cells, the GCS inhibitor PDMP caused cell cycle arrest, inhibition of 2D and 3D cell proliferation, colony formation and migration capability, and apoptosis induction. The intratumoral injection of PDMP completely suppressed the growth of OR xenograft models. This study demonstrated that dysregulation of ceramide metabolism is involved in osimertinib-resistance and targeting GCS may be a promising therapeutic strategy for patients progressed to osimertinib.

Targeting metabolic adaptive responses induced by glucose starvation inhibits cell proliferation and enhances cell death in osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines.

Osimertinib, a tyrosine kinase inhibitor targeting mutant EGFR, has received approval for initial treatment in patients with Non-Small Cell Lung Cancer (NSCLC). While effective in both first- and second-line treatments, patients eventually develop acquired resistance. Metabolic reprogramming represents a strategy through which cancer cells may resist and adapt to the selective pressure exerted by the drug. In the current study, we investigated the metabolic adaptations associated with osimertinib-resistance in NSCLC cells under low glucose culture conditions. We demonstrated that, unlike osimertinib-sensitive cells, osimertinib-resistant cells were able to survive under low glucose conditions by increasing the rate of glucose and glutamine uptake and by shifting towards mitochondrial metabolism. Inhibiting glucose/pyruvate contribution to mitochondrial respiration, glutamine deamination to glutamate, and oxidative phosphorylation decreased the proliferation and survival abilities of osimertinib-resistant cells to glucose starvation. Our findings underscore the remarkable adaptability of osimertinib-resistant NSCLC cells in a low glucose environment and highlight the pivotal role of mitochondrial metabolism in mediating this adaptation. Targeting the metabolic adaptive responses triggered by glucose shortage emerges as a promising strategy, effectively inhibiting cell proliferation and promoting cell death in osimertinib-resistant cells.

Genetic Basis of Breast and Ovarian Cancer: Approaches and Lessons Learnt from Three Decades of Inherited Predisposition Testing.

Germline variants occurring in BRCA1 and BRCA2 give rise to hereditary breast and ovarian cancer (HBOC) syndrome, predisposing to breast, ovarian, fallopian tube, and peritoneal cancers marked by elevated incidences of genomic aberrations that correspond to poor prognoses. These genes are in fact involved in genetic integrity, particularly in the process of homologous recombination (HR) DNA repair, a high-fidelity repair system for mending DNA double-strand breaks. In addition to its implication in HBOC pathogenesis, the impairment of HR has become a prime target for therapeutic intervention utilizing poly (ADP-ribose) polymerase (PARP) inhibitors. In the present review, we introduce the molecular roles of HR orchestrated by BRCA1 and BRCA2 within the framework of sensitivity to PARP inhibitors. We examine the genetic architecture underneath breast and ovarian cancer ranging from high- and mid- to low-penetrant predisposing genes and taking into account both germline and somatic variations. Finally, we consider higher levels of complexity of the genomic landscape such as polygenic risk scores and other approaches aiming to optimize therapeutic and preventive strategies for breast and ovarian cancer.

Prognostic Impact of Blood Lipid Profile in Patients With Advanced Solid Tumors Treated With Immune Checkpoint Inhibitors: A Multicenter Cohort Study.

BACKGROUND: Specific components of lipid profile seem to differently impact on immune activity against cancer and unraveling their prognostic role in patients with solid cancer treated with immune checkpoint inhibitors (ICIs) is needed. MATERIALS AND METHODS: We retrospectively collected baseline clinicopathological characteristics including circulating lipid profile (total cholesterol [TC], triglycerides [TG], low-density lipoproteins [LDL], high-density lipoproteins [HDL]) of patients with consecutive solid cancer treated with ICIs, and we investigated their role in predicting clinical outcomes. RESULTS: At a median follow-up of 32.9 months, among 430 enrolled patients, those with TC ≥ 200 mg/dl showed longer median progression-free survival (mPFS; 6.6 vs. 4.7 months, P = .4), although not reaching statistical significance, and significantly longer median overall survival (mOS; 19.4 vs. 10.8 months, P = .02) compared to those with TC < 200 mg/dl. Conversely, patients with TG ≥150 mg/dl displayed shorter PFS (3.4 vs. 5.1 months, P = .02) and OS (7.1 vs. 12.9 months, P = .009) compared to those with TG <150 mg/dl. TC and TG were then combined in a "LIPID score" identifying three subgroups: good-risk (GR) (TC ≥200 mg/dl and TG <150 mg/dl), intermediate-risk (IR) (TC <200 mg/dl and TG <150 mg/dl or TC ≥200 mg/dl and TG ≥150 mg/dl) and poor-risk (PR) (TC <200 mg/dl and TG ≥150 mg/dl). The mPFS of GR, IR, and PR groups was 7.8, 4.3, and 2.5 months, respectively (P = .005); mOS of GR, IR, and PR was 20.4, 12.4, and 5.3 months, respectively (P < .001). At multivariable analysis, the PR profile represented an independent poor prognostic factor for both PFS and OS. CONCLUSIONS: We developed a lipid score that defined subgroups of patients with cancer who differently benefit from ICIs. Further mechanistic insights are warranted to clarify the prognostic and predictive role of lipid profile components in patients treated with ICIs.

Intrinsic Resistance to Osimertinib in EGFR Mutated NSCLC Cell Lines Induced by Alteration in Cell-Cycle Regulators.

BACKGROUND: Cell-cycle regulators are mutated in approximately 40% of all cancer types and have already been linked to worse outcomes in non-small cell lung cancer adenocarcinomas treated with osimertinib. However, their exact role in osimertinib resistance has not been elucidated. OBJECTIVE: In this study, we aimed to evaluate how the CDK4/6-Rb axis may affect the sensitivity to osimertinib. METHODS: We genetically increased the level of CCND1 (Cyclin D1) and reduced the levels of CDKN2A (p16) in two different adenocarcinoma cell lines, PC9 and HCC827. We also retrospectively evaluated the outcome of patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer depending on their level of Cyclin D1 and p16. RESULTS: The modified clones showed higher proliferative capacity, modifications in cell-cycle phases, and higher migratory capacity than the parental cells. Cyclin D1-overexpressing clones were highly resistant to acute osimertinib treatment. CDKN2A knockdown conferred intrinsic resistance as well, although a longer time was required for adaption to the drug. In both cases, the resistant phenotype was epidermal growth factor receptor independent and associated with a higher level of Rb phosphorylation, which was unaffected by osimertinib treatment. Blocking the phosphorylation of Rb using abemaciclib, a CDK4/6 inhibitor, exerted an additive effect with osimertinib, increasing sensitivity to this drug and reverting the intrinsic resistant phenotype. In a group of 32 patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer, assessed for Cyclin D1 and p16 expression, we found that the p16-deleted group presented a lower overall response rate compared with the control group. CONCLUSIONS: We conclude that perturbation in cell-cycle regulators leads to intrinsic osimertinib resistance and worse patient outcomes.

Liquid biopsy comprehensive genomic profiling of lung cancer in the Italian population: A real-world experience.

OBJECTIVES: Liquid biopsy with next-generation sequencing (NGS) has emerged as a promising tool for tumor mutation profiling. In this study, we describe the genomic profile of Italian lung cancer patients tested with blood-based comprehensive genomic profiling (CGP) to assess the genomic landscape complexity and its impact on enhancing treatment options for patients. MATERIALS AND METHODS: Between January 2021 and December 2021, a total of 229 lung cancer patients were profiled by FoundationOne®Liquid CDx (F1LCDx®) assay on circulating tumor DNA (ctDNA). F1LCDx® reports alterations across 324 cancer-related genes and genomic signatures, including tumor fraction (TF) and blood-based tumor mutational burden (bTMB). Detected variants were classified according to the ESMO Scale of Clinical Actionability for molecular Targets (ESCAT). RESULTS: 90.4% of patients had at least one detectable alteration in plasma. The most frequently mutated genes were TP53 (47.6%), DNMT3A (33.2%), EGFR (20.1%), and KRAS (15.7%). Elevated TF was detected in 18.3% of patients, suggesting high reliability of test results. According to the ESCAT classification, potentially actionable alterations (Tier I-II) were identified in 27.1% of samples. An additional 5.2% harbored an alteration for which an approved drug is available in other cancer types (Tier III). Furthermore, 13.1% of tumors exhibited high bTMB, which may predict response to immunotherapy. Overall, 156 (68.1%) patients were eligible for enrolment in clinical trials. CONCLUSION: Liquid biopsy NGS is a viable and valuable approach to guide personalized therapy. The use of blood-based CGP may help identify a larger number of actionable mutations and increase chances of enrolment in clinical trials.

Relationship Between the Diffusing Capacity of the Lung for Carbon Monoxide (DLCO) and Lung Adenocarcinoma Patterns: New Possible Insights

Introduction: This study aimed to evaluate a potential relationship between the diffusing capacity of the lung for carbon monoxide (DLCO) and the aggressiveness of lung adenocarcinoma (ADC). Methods: Patients who underwent radical surgery for lung ADC between 2001 and 2018 were retrospectively reviewed. DLCO values were dichotomized into DLCOlow (<80% of predicted) and DLCOnormal (≥80%). Relationships between DLCO and ADC histopathological features, clinical features, as well as with overall survival (OS), were evaluated. Results: Four-hundred and sixty patients were enrolled, of which 193 (42%) were included in the DLCOlow group. DLCOlow was associated with smoking status, low FEV1, micropapillary and solid ADC, tumour grade 3, high tumour lymphoid infiltrate and presence of tumour desmoplasia. In addition, DLCO values were higher in low-grade ADC and progressively decreased in intermediate and high-grade ADC (p=0.024). After adjusting for clinical variables, at multivariable logistic regression analysis, DLCOlow still showed a significant correlation with high lymphoid infiltrate (p=0.017), presence of desmoplasia (p=0.065), tumour grade 3 (p=0.062), micropapillary and solid ADC subtypes (p=0.008). To exclude the association between non-smokers and well-differentiated ADC, the relationship between DLCO and histopathological ADC patterns was confirmed in the subset of 377 former and current smokers (p=0.021). At univariate analysis, gender, DLCO, FEV1, ADC histotype, tumour grade, stage, pleural invasion, tumour necrosis, tumour desmoplasia, lymphatic and blood invasion were significantly related with OS. At multivariate analysis, only gender (p<0.001), tumour stage (p<0.001) and DLCO (p=0.050) were significantly related with the OS. Conclusions: We found a relationship between DLCO and ADC patterns as well as with tumour grade, tumour lymphoid infiltrate and desmoplasia, suggesting that lung damage may be associated with tumour aggressiveness. (AU)

Relationship Between the Diffusing Capacity of the Lung for Carbon Monoxide (DLCO) and Lung Adenocarcinoma Patterns: New Possible Insights.

INTRODUCTION: This study aimed to evaluate a potential relationship between the diffusing capacity of the lung for carbon monoxide (DLCO) and the aggressiveness of lung adenocarcinoma (ADC). METHODS: Patients who underwent radical surgery for lung ADC between 2001 and 2018 were retrospectively reviewed. DLCO values were dichotomized into DLCOlow (<80% of predicted) and DLCOnormal (≥80%). Relationships between DLCO and ADC histopathological features, clinical features, as well as with overall survival (OS), were evaluated. RESULTS: Four-hundred and sixty patients were enrolled, of which 193 (42%) were included in the DLCOlow group. DLCOlow was associated with smoking status, low FEV1, micropapillary and solid ADC, tumour grade 3, high tumour lymphoid infiltrate and presence of tumour desmoplasia. In addition, DLCO values were higher in low-grade ADC and progressively decreased in intermediate and high-grade ADC (p=0.024). After adjusting for clinical variables, at multivariable logistic regression analysis, DLCOlow still showed a significant correlation with high lymphoid infiltrate (p=0.017), presence of desmoplasia (p=0.065), tumour grade 3 (p=0.062), micropapillary and solid ADC subtypes (p=0.008). To exclude the association between non-smokers and well-differentiated ADC, the relationship between DLCO and histopathological ADC patterns was confirmed in the subset of 377 former and current smokers (p=0.021). At univariate analysis, gender, DLCO, FEV1, ADC histotype, tumour grade, stage, pleural invasion, tumour necrosis, tumour desmoplasia, lymphatic and blood invasion were significantly related with OS. At multivariate analysis, only gender (p<0.001), tumour stage (p<0.001) and DLCO (p=0.050) were significantly related with the OS. CONCLUSIONS: We found a relationship between DLCO and ADC patterns as well as with tumour grade, tumour lymphoid infiltrate and desmoplasia, suggesting that lung damage may be associated with tumour aggressiveness.

The role of blood cholesterol quality in patients with advanced cancer receiving immune checkpoint inhibitors.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) became the standard of care for several solid tumors. A limited fraction of patients (pts) achieves a long-term benefit. Plasmatic and intracellular cholesterol levels have emerged as promising biomarkers. The aim of the present study was to determine whether cholesterol efflux capacity (CEC), mediated by serum transporters (ABCA1 and ABCG1) and passive diffusion (PD), impacts on clinical outcome of advanced non-small cell lung cancer (NSCLC) and metastatic renal cell carcinoma (mRCC) pts treated with ICIs. MATERIAL AND METHODS: We retrospectively enrolled advanced NSCLC and mRCC pts consecutively treated with ICIs between October 2013 and October 2018. CEC and cholesterol loading capacity (CLC) were assessed by well-established specific cell models. As primary endpoint, CEC, PD and CLC were correlated with overall survival (OS) while the effects of these parameters on progression-free survival (PFS) and clinical benefit (CB), defined as complete/partial response or stable disease, represented secondary endpoints. RESULTS: NSCLC accounted for 94.2% of 70 enrolled cases, and serum sample suitable for CEC and PD determination was available in 68. Blood cholesterol and serum ABCA1, ABCG1, PD and CLC were associated with outcomes (OS, PFS and CB) at univariate analysis. At the multivariate analysis, only PD confirmed its positive prognostic value in terms of OS, PFS and CB. CONCLUSION: The favorable impact of cholesterol PD on clinical outcome might reflect its main conformation in mature HDL particles which potentially shape an inflamed context, ultimately promoting ICI efficacy. Further prospective studies are needed to support our findings and uncover targetable pathways.

Therapeutic Outcomes and Clinical Features of Advanced Non-Small Cell Lung Cancer Carrying KRAS Mutations: A Multicenter Real-life Retrospective Study.

INTRODUCTION: Targeting Kirsten Rat Sarcoma (KRAS) has been deemed impossible for long time, but new drugs have recently demonstrated promising results. Evidence on the outcome of KRAS-mutant advanced-NSCLC treated with new standard regimens are still scarce. Thus, we aimed at assessing the incidence and clinical impact of KRAS mutations in a real-life population of advanced-NSCLC, exploring the prognostic significance of distinct alterations. MATERIALS AND METHODS: The present multicenter retrospective study, conducted by 5 Italian Centers from January 2018 to February 2020, involved 297 advanced KRAS mutant NSCLC. Complete clinico-pathological data were evaluated. RESULTS: Out of 297 patients, 130 carried KRAS_G12C mutation, while 167 presented with mutations other than G12C. Within KRAS_non-G12C group, 73%, 16.8% and 8.9% harboured G12X, codon 13 and Q61H alterations, respectively. No significant differences in survival outcome and treatment response were documented according to KRAS_G12C versus non-G12C, nor KRAS_G12C versus G12X versus other mutations. On univariate analysis ECOG PS, number and sites of metastatic lesions and PD-L1 status significantly impacted on survival. A clear trend towards worse prognosis was apparent in chemotherapy-treated patients, while immunotherapy-based regimens were associated to prolonged survival. Investigating the outcome of PD-L1 ≥ 50% population, we did not detect any significant difference between KRAS_G12C and non-G12C subsets. CONCLUSION: Here, we report on real-life data from a large retrospective cohort of advanced NSCLC harbouring KRAS alterations, with particular attention to G12C mutation. Our study offers useful clues on survival outcome, therapeutic response and clinico-pathological correlations in KRAS-mutant setting, especially in the upcoming era of KRAS G12C targeting therapy.

It Takes Two to Tango: Potential Prognostic Impact of Circulating TGF-Beta and PD-L1 in Pancreatic Cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with rising incidence and poor prognosis. The lack of reliable prognostic biomarkers hampers the individual evaluation of the survival and recurrence potential. Methods: Here, we investigate the value of plasma levels of two potential key players in molecular mechanisms underlying PDAC aggressiveness and immune evasion, soluble TGF-beta (sTGF-beta) and sPD-L1, in both metastatic and radically-resected PDAC. To this aim we prospectively enrolled 38 PDAC patients and performed appropriate statistical analyses in order to evaluate their correlation, and role in the prediction of disease relapse/progression, and patients' outcome. Results: Metastatic patients showed lower levels of circulating sTGF-beta and higher levels of sPD-L1 compared to radically-resected patients. Moreover, a decrease in sTGF-beta levels (but not sPD-L1) was significantly associated with disease relapse in radically-resected patients. We also observed lower sTGF-beta at disease progression after first-line chemotherapy in metastatic patients, though this change was not statistically significant. We found a significant correlation between the levels of sTGF-beta and sPD-L1 before first-line chemotherapy. Conclusions: These findings support the possible interaction of TGF-beta and PD-L1 pathways and suggest that sTGF-beta and sPD-L1 might synergize and be new potential blood-based biomarkers.

Multicenter Observational Study on Metastatic Non-Small Cell Lung Cancer Harboring BRAF Mutations: Focus on Clinical Characteristics and Treatment Outcome of V600E and Non-V600E Subgroups.

INTRODUCTION: BRAF mutation involved 2-4% of lung adenocarcinoma. Differences in clinicopathologic features and patient outcome exist between V600E and non-V600E BRAF mutated NSCLC. Thus, we sought to assess the frequency and clinical relevance of BRAF mutations in a real-life population of advanced-NSCLC, investigating the potential prognostic significance of distinct genetic alterations. MATERIALS AND METHODS: The present multicenter Italian retrospective study involved advanced BRAF mutant NSCLC. Complete clinicopathologic data were evaluated for BRAF V600E and non-V600E patients. RESULTS: A total of 44 BRAFmut NSCLC patients were included (V600E, n = 23; non-V600E, n = 21). No significant differences in survival outcome and treatment response were documented, according to V600E vs. non-V600E mutations, although a trend towards prolonged PFS was observed in the V600E subgroup (median PFS = 11.3 vs. 6.0 months in non-V600E). In the overall population, ECOG PS and age significantly impacted on OS, while bone lesions were associated with shorter PFS. Compared to immunotherapy, first-line chemotherapy was associated with longer OS in the overall population, and especially in the BRAF V600E subtype. CONCLUSIONS: Here, we report on real-life data from a retrospective cohort of advanced-NSCLC harboring BRAF alterations. Our study offers relevant clues on survival outcome, therapeutic response, and clinicopathologic correlations of BRAF-mutant NSCLC.

YES1 and MYC Amplifications as Synergistic Resistance Mechanisms to Different Generation ALK Tyrosine Kinase Inhibitors in Advanced NSCLC: Brief Report of Clinical and Preclinical Proofs.

INTRODUCTION: ALK tyrosine kinase inhibitors (TKIs) are the standard treatment for advanced ALK-positive NSCLC. Nevertheless, drug resistance inevitably occurs. Here, we report a case of a patient with metastatic ALK-positive lung adenocarcinoma with an impressive resistance to sequential treatment with ALK TKIs mediated by YES1 and MYC amplification in a contest of epithelial-to-mesenchymal transition and high progressive chromosomal instability. METHODS: The patient received, after chemotherapy and 7 months of crizotinib, brigatinib and lorlatinib with no clinical benefit to both treatments. A study of resistance mechanisms was performed with whole exome sequencing on different biological samples; primary cell lines were established from pleural effusion after lorlatinib progression. RESULTS: At whole exome sequencing analysis, YES1 and MYC amplifications were observed both in the pericardial biopsy and the pleural effusion samples collected at brigatinib and lorlatinib progression, respectively. Increasing chromosomal instability from diagnostic biopsy to pleural effusion was also observed. The addition of dasatinib to brigatinib or lorlatinib restored the sensitivity in primary cell lines; data were confirmed also in H3122_ALK-positive model overexpressing both YES1 and MYC. CONCLUSIONS: In conclusion, YES1 and MYC amplifications are candidates to justify a rapid acquired resistance to crizotinib entailing primary brigatinib and lorlatinib resistance. In this context, a combination strategy of ALK TKI with dasatinib could be effective to overcome a rapid resistance.

Role of ctDNA for the detection of minimal residual disease in resected non-small cell lung cancer: a systematic review.

Background: Operable stage I-III non-small cell lung cancer (NSCLC) has a high risk of recurrence, mainly due to remnant clones of the disease defined as minimal residual disease (MRD). Adjuvant chemotherapy has a limited efficacy in reducing the risk of relapse, and prognostic as well as predictive biomarkers in this context are currently missing. Methods: We performed a systematic review to evaluate the state of the art about the role of circulating tumor DNA detection through liquid biopsy for the assessment of MRD in resected early-stage NSCLC patients. Results: Among the 650 studies identified, 13 were eligible and included. Although highly heterogeneous, all the studies demonstrated a poor prognosis in patients with post-operative MRD, with a detection rate ranging from 6% to 45%. MRD detection preceded radiographic/clinical recurrence by a mean of 5.5 months. MRD positive patients were most likely to benefit from adjuvant treatment in terms of recurrence-free survival (RFS). Consistently, adjuvant therapy did not minimize the risk of relapse in the MRD negative group. Conclusions: Liquid biopsy has a relevant role in assessing post-surgical MRD in resected NSCLC. Since currently there are no criteria other than stage and risk factors for the choice of adjuvant treatment in this setting, post-operative assessment of MRD through liquid biopsy might be a promising approach to guide the decision.

PD-L1 SNPs as biomarkers to define benefit in patients with advanced NSCLC treated with immune checkpoint inhibitors.

OBJECTIVE: To investigate the role of CTLA-4, PD-1 (programmed death-1), and PD-L1 (programmed death-ligand 1) single nucleotide polymorphisms (SNPs) in predicting clinical outcome of patients with advanced non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). METHODS: A total of 166 consecutive patients were included. We correlated SNPs with clinical benefit, progression-free survival, time to treatment failure, and overall survival and evaluated the incidence of SNPs in nonresponder and long clinical benefit groups. RESULTS: Considering the entire cohort, no correlation was found between SNPs and clinical outcome; however, PD-L1 rs4143815 SNP and the long clinical benefit group showed a statistically significant association (p = 0.02). The nonresponder cohort displayed distinctive PD-L1 haplotype (p = 0.05). CONCLUSION: PD-L1 SNPs seem to be marginally involved in predicting clinical outcome of NSCLC treated with ICI, but further investigations are required.

Molecular profiling of advanced non-small cell lung cancer in the era of immunotherapy approach: a multicenter Italian observational prospective study of biomarker screening in daily clinical practice.

AIMS: Heterogeneous implementation of molecular tests in current diagnostic algorithm at a European and international level is emerging as a major issue for efficient lung cancer molecular profiling. METHODS: From May 2017 until October 2017, N=1612 patients referring to 13 Italian institutions were selected, at advanced stage non-small cell lung cancer (NSCLC), and prospectively evaluated. Principal endpoints were: the percentage of diagnoses performed on cytological and histological material, the proportion of requests for epidermal growth factor receptor (EGFR) mutational status, and resistance mutations detected on tissue and/or liquid biopsy samples after first-generation or second-generation tyrosine kinase inhibitors, the proportion of requests for anaplastic lymphoma kinase (ALK) gene rearrangements, ROS proto-oncogene 1 (ROS1) and Kirsten Rat Sarcoma (KRAS) determinations, the proportion of requests for programmed death-ligand1 (PD-L1) evaluation and, finally, the different assays used for the detection of EGFR mutations, ALK and ROS1 gene rearrangements and PD-L1 expression. RESULTS: Of 1325 patients finally included, only 50.8% requests were related to driver mutations with target agents already available in first-line at that preplanned time, while 49.2% were associated with PD-L1, ROS1, KRAS and others. Multiplex genomic assays (such as next-generation sequencing) were considered by all participating centres. CONCLUSIONS: To the best of our knowledge, this is the first study in a 'real-life daily practice' involving both pathologists and oncologists evaluating routinely workflow and trends towards improvements in molecular requests. Collected data aim to describe the applied algorithms and evolution of molecular screening for stage IV NSCLC in clinical practice.

Validation of a radiomic approach to decipher NSCLC immune microenvironment in surgically resected patients.

Radiomics has emerged as a noninvasive tool endowed with the potential to intercept tumor characteristics thereby predicting clinical outcome. In a recent study on resected non-small cell lung cancer (NSCLC), we identified highly prognostic computed tomography (CT) -derived radiomic features (RFs), which in turn were able to discriminate hot from cold tumor immune microenvironment (TIME). We aimed at validating a radiomic model capable of dissecting specific TIME profiles bearing prognostic power in resected NSCLC. The validation cohort included 31 radically resected NSCLCs clinicopathologically matched with the training set (n = 69). TIME was classified in hot and cold according to a multiparametric immunohistochemical analysis involving PD-L1 score and incidence of immune effector phenotypes among tumor infiltrating lymphocytes (TILs). High- throughput radiomic features (n = 841) extracted from CT images were correlated to TIME parameters to ultimately define prognostic classes. We confirmed PD-1 to CD8 ratio as best predictor of clinical outcome among TIME characteristics. Significantly prolonged overall survival (OS) was observed in patients carrying hot (median OS not reached) vs cold (median OS 22 months; hazard ratio 0.28, 95% confidence interval 0.09 -0.82; p = 0.015) immune background, thus validating the prognostic impact of these two TIME categories in resected NSCLC. Importantly, in the validation setting, three out of eight previously identified RFs sharply distinguishing hot from cold TIME were endorsed. Among signature-related RFs, Wavelet-HHH_gldm_HighGrayLevelEmphasis highly performed as descriptor of hot immune contexture (area under the receiver operating characteristic curve 0.94, 95% confidence interval 0.81 -1.00; p = 0.01). Based on our findings, Radiomics may decipher specific TIME profiles providing a noninvasive prognostic approach in resected NSCLC and an exploitable predictive strategy in advanced cases.

Monitoring cfDNA in Plasma and in Other Liquid Biopsies of Advanced EGFR Mutated NSCLC Patients: A Pilot Study and a Review of the Literature.

In order to study alternatives at the tissue biopsy to study EGFR status in NSCLC patients, we evaluated three different liquid biopsy platforms (plasma, urine and exhaled breath condensate, EBC). We also reviewed the literature of the cfDNA biological sources other than plasma and compared our results with it about the sensitivity to EGFR mutation determination. Twenty-two EGFR T790M-mutated NSCLC patients in progression to first-line treatment were enrolled and candidate to osimertinib. Plasma, urine and EBC samples were collected at baseline and every two months until progression. Molecular analysis of cfDNA was performed by ddPCR and compared to tissue results. At progression NGS analysis was performed. The EGFR activating mutation detection reached a sensitivity of 58 and 11% and for the T790M mutation of 45 and 10%, in plasma and urine samples, respectively. Any DNA content was recovered from EBC samples. Considering the plasma monitoring study, the worst survival was associated with positive shedding status; both plasma and urine molecular progression anticipated the radiological worsening. Our results confirmed the role of plasma liquid biopsy in testing EGFR mutational status, but unfortunately, did not evidence any improvement from the combination with alternative sources, as urine and EBC.

Modulating Tumor Microenvironment: A Review on STK11 Immune Properties and Predictive vs Prognostic Role for Non-small-cell Lung Cancer Immunotherapy.

OPINION STATEMENT: The quest for immunotherapy (IT) biomarkers is an element of highest clinical interest in both solid and hematologic tumors. In non-small-cell lung cancer (NSCLC) patients, besides PD-L1 expression evaluation with its intrinsic limitations, tissue and circulating parameters, likely portraying the tumor and its stromal/immune counterparts, have been proposed as potential predictors of IT responsiveness. STK11 mutations have been globally labeled as markers of IT resistance. After a thorough literature review, STK11 mutations condition the prognosis of NSCLC patients receiving ICI-containing regimens, implying a relevant biological and clinical significance. On the other hand, waiting for prospective and solid data, the putative negative predictive value of STK11 inactivation towards IT is sustained by less evidence. The physiologic regulation of multiple cellular pathways performed by STK11 likely explains the multifaceted modifications in tumor cells, stroma, and tumor immune microenvironment (TIME) observed in STK11 mutant lung cancer, particularly explored in the molecular subgroup of KRAS co-mutation. IT approaches available thus far in NSCLC, mainly represented by anti-PD-1/PD-L1 inhibitors, are not promising in the case of STK11 inactivation. Perceptive strategies aimed at modulating the TIME, regardless of STK11 status or specifically addressed to STK11-mutated cases, will hopefully provide valid therapeutic options to be adopted in the clinical practice.

A sulfonyl fluoride derivative inhibits EGFRL858R/T790M/C797S by covalent modification of the catalytic lysine.

The emergence of the C797S mutation in EGFR is a frequent mechanism of resistance to osimertinib in the treatment of non-small cell lung cancer (NSCLC). In the present work, we report the design, synthesis and biochemical characterization of UPR1444 (compound 11), a new sulfonyl fluoride derivative which potently and irreversibly inhibits EGFRL858R/T790M/C797S through the formation of a sulfonamide bond with the catalytic residue Lys745. Enzymatic assays show that compound 11 displayed an inhibitory activity on EGFRWT comparable to that of osimertinib, and it resulted more selective than the sulfonyl fluoride probe XO44, recently reported to inhibit a significant part of the kinome. Neither compound 11 nor XO44 inhibited EGFRdel19/T790M/C797S triple mutant. When tested in Ba/F3 cells expressing EGFRL858R/T790M/C797S, compound 11 resulted significantly more potent than osimertinib at inhibiting both EGFR autophosphorylation and proliferation, even if the inhibition of EGFR autophosphorylation by compound 11 in Ba/F3 cells was not long lasting.