Glyoxalase 2 (Glo2) is a key enzyme of the glyoxalase system that catalyzes the conversion of S-lactoylglutathione (LSG) into glutathione (GSH) and D-lactate. In prostate cancer (PCa), we previously demonstrated that the oncogenic PTEN-PI3K-AKT-mTOR-ERα signaling pathway upregulates Glo2, leading to intracellular D-lactate accumulation and enhanced cell migration, invasiveness, and expression of epithelial-to-mesenchymal transition (EMT)-associated markers. However, whether D-lactate acts as a bioactive metabolic signal contributing to tumor aggressiveness remains unclear. Here, after confirming our previous findings, we demonstrate-using Glo2 silencing, ectopic expression, pharmacological inhibitors, and exogenous D-lactate supplementation-that Glo2-dependent D-lactate accumulation promotes EMT-like plasticity, migration, and invasion in PTEN-deficient PCa cells via a functional link with FAK/Src signaling. Collectively, these results suggest that the Glo2-D-lactate axis may contribute to metabolic rewiring associated with aggressive behavior in PTEN-deficient PCa, warranting further in vivo studies to evaluate its potential as a therapeutic target to limit tumor progression.

In this retrospective study, the diagnostic performance of an individualized apparent diffusion coefficient (ADC) signal intensity (SI) scale for differentiating prostate cancer (PCa) from benign simplified Prostate Imaging Reporting and Data System (S-PI-RADS) 4 lesions detected on biparametric magnetic resonance imaging (bpMRI) was evaluated. A total of 102 patients with biopsy-confirmed PCa and 79 patients with benign lesions (inflammation, HGPIN, ASAP) presenting as S-PI-RADS 4 were included. An individualized ADC SI scale was generated for each patient using maximum and minimum ADC values from the bladder and femoral head, respectively. The midpoint (ADC_mean) and the percentage difference (PD) between ADC_mean and lesion intensity (ADC_lesion) were calculated. Differences and diagnostic performance were assessed using Mann-Whitney U tests and receiver operating characteristic (ROC) analysis. PCa lesions demonstrated significantly lower ADC values and higher PDs compared with benign lesions (P<0.001). ROC analysis of PD yielded an area under the curve (AUC) of 0.798. Using a PD cut-off of 14.67%, sensitivity and specificity for detecting PCa were 73.5% and 94.9%, respectively. Intra-reader repeatability was high [intraclass correlation coefficient (ICC) 0.92-0.94], and PD values remained robust across variations in ADC fitting. The individualized ADC SI scale provides a patient-specific quantitative measure that effectively distinguishes malignant from benign S-PI-RADS 4 lesions. This approach complements visual bpMRI assessment, demonstrates high specificity, and has the potential to reduce unnecessary biopsies. Further prospective studies are warranted to validate its clinical utility.

Appendiceal abscesses are a heterogeneous manifestation of acute appendicitis, with diverse pathological substrates and imaging phenotypes. The lack of standardized definitions hampers cross-study comparability and consistent diagnostic pathways. The Italian Society of Surgical Research (SIRC) and the Italian Society of Emergency Surgery and Trauma (SICUT) conducted a four-round modified Delphi, culminating in an in-person consensus conference (Rome, November 6, 2025). A multidisciplinary panel refined the statements on pathological definitions, classifications, and imaging correlates. Consensus was reached on 22 statements. Part 1 reports seven statements that delineate the spectrum from uncomplicated to complicated appendicitis (phlegmonous, gangrenous, and perforated) and distinguishes appendiceal abscesses from appendiceal masses. An imaging framework centered on CT descriptors was used to support consistent radiology–pathology correlations. These statements provide a unified taxonomy and diagnostic framework for appendiceal abscesses and related entities, improving interdisciplinary communication, and enabling cross-study comparability.

Metabolic renal cell carcinomas (RCC) deficient in succinate dehydrogenase (SDH) or fumarate hydratase (FH) are rare but clinically significant entities formalized in the last WHO classification. Their recognition typically starts from morphology and is corroborated by targeted immunohistochemistry (IHC) and, where appropriate, molecular and germline testing. In routine practice, however, implementation may be uneven. Hence, we conducted a nationwide, web-based survey (made by 25 items) among members of the Italian Study Group of Uropathology (GIUP) to map real-world awareness, diagnostic pathways, and test availability across Italian centers. Twenty-one pathologists responded; 18/21 (85.7

BACKGROUND:The prognostic impact of tumour microenvironment (TME) heterogeneity in primary cutaneous melanomas (CM) and paired distant metastases is unclear. Few studies have explored the impact of genetic features on TME immune cell distribution. This study assessed the prognostic impact of TME heterogeneity and investigated the correlation between genetic features and TME cell composition. METHODS:Demographics, treatments and outcome of melanoma patients - with paired samples - from five Italian Melanoma Intergroup (IMI) centers were collected. TME immune phenotypes and cell distribution in FFPE whole tumour sections were analyzed using 9 biomarkers (CD3, CD4, CD8, CD20, CD68, CD163, PD-1, PD-L1, FOXP3), classifying samples as desert (D), excluded (E), or inflamed (I). Additionally, genomic pathways were assessed by next-generation sequencing in both primary and paired samples. RESULTS:TME was evaluated in 221 samples from 91 patients.The distribution of immune phenotype (I vs E/D) of CD8 + cells (p < 0.0001), CD163 + cells (p < 0.0001), CD20 + cells (p = 0.0008), CD3 + cells (p < 0.0001), CD4 + cells (p < 0.0001), CD68 + cells (p < 0.0001), PD1 + cells (p = 0.00015) significantly differed between metastatic and primary melanomas. In primary tumors, BRAFV600 status did not correlate with immune phenotypes. However, in paired metastases, it was inversely associated with infiltration of CD8 + , CD3 + , CD68 + , and CD20 + cells. Patients in the CD8 + D/E TME group had significantly shorter median survival (21 months) compared to those with at least one inflamed (I) TME sample [(58 months); HR 2.35, 95 % CI 1.28-4.32]. CONCLUSIONS:Metastatic and primary melanomas show distinct immune phenotypes. Longitudinal TME assessment predicts overall survival.

PURPOSE:Lung cancer is the leading cause of cancer death worldwide which includes two main types of carcinoma distinguished in non-small cell lung cancer (NSCLC) involving epithelial cells, and small cell lung cancer (SCLC) affecting neuronal cells and hormone secreting cells. Studies have shown a causal link between inflammation/innate immunity and onset of NSCLC. The present study aimed to evaluate the expression of Toll-like receptors (TLRs) 4 and TLR8 in peripheral blood mononuclear cells (PBMC) and in lung tissues of patients with NSCLC, useful for future prognostic tools for NSCLC. METHODS:Patients surgically treated for NSCLC with anatomical resections and patients with benign disease were enrolled. The expression levels of TLR4 and TLR8 were determined by real time PCR and by immunohistochemical analysis in PBMC and in lung tissues, respectively. A preliminary in silico analysis including 1194 arrays from healthy and cancer tissues were extracted by Genevestigator database. The association between TLRs gene expression and survival outcome was also investigated. RESULTS:Bioinformatics analysis revealed that downregulation of TLR4 and TLR8 positively impacts the survival in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). However, no significant differences in TLR4 and TLR8 gene expression between case and control groups were observed in PBMC. A positive correlation was found in their expression levels. Interestingly, immunohistochemical analysis showed that the levels of TLR4 and TLR8 were higher in the lung tissues of patients with NSCLC than in the control group in terms of staining intensity and positive cells. CONCLUSION:Albeit the precise role of TLRs is not fully defined, this study identified the potential involvement of TLR4 and TLR8 in the pathogenesis of NSCLC. Our data led us to hypothesize their potential role in overall survival which deserves to be explored further to establish whether TLR4 and TLR8 can represent positive prognostic indicators of disease in NSCLC.

Glyoxalase 1 (Glo1) functions as a catalyst that neutralizes methylglyoxal (MG), a highly reactive glycating agent predominantly produced during glycolysis—a metabolic pathway upregulated in cancer cells. MG primarily reacts with the amino groups of proteins (especially at arginine residues), leading to the formation of a major advanced glycation end product known as MG-derived hydroimidazolone 1 (MG-H1). We previously demonstrated in PC3 human prostate cancer (PCa) cells that the PTEN/PKM2/ERα axis promotes their aggressive phenotype by regulating the Glo1/MG-H1 pathway. In this study, after confirming our earlier findings, we investigated the downstream mechanisms of the PTEN/PKM2/ERα/Glo1/MG-H1 axis in controlling PC3 cell growth, focusing on the role of RAGE, a high-affinity receptor for MG-H1; hydrogen peroxide (H2O2); and Krev interaction trapped 1 (KRIT1), an emerging tumor suppressor. Using genetic approaches and specific inhibitors/scavengers, we demonstrated that the PTEN/PKM2/ERα/Glo1/MG-H1 axis promotes PC3 cell growth—measured by proliferation and etoposide-induced apoptosis resistance—through a mechanism involving MG-H1/RAGE pathway desensitization that leads to H2O2-mediated KRIT1 downregulation. These findings support and expand the role of PTEN signaling in PCa progression and shed light on novel mechanistic pathways driven by MG-dependent glycative stress, involving KRIT1, in this still incurable stage of the disease.

In this work, we report on a clinically significant response of meningeal carcinomatosis to repotrectinib in a woman with a heavily pretreated ROS1-rearranged non-small cell lung cancer (NSCLC) that harbored the concomitant solvent front G2032R mutation. Meningeal carcinomatosis has a higher incidence in oncogene addicted NSCLC due to increased life expectancy, yet no report has ever documented the activity of repotrectinib in this context. In line with its activity, we documented the presence of the drug at potentially active concentrations in the cerebrospinal fluid. Nevertheless, the short-lived response reported by our patient highlights the importance for novel ROS1-tyrosine kinase inhibitors (TKIs) to be specifically developed to be able to penetrate the blood–brain barrier.

Lorlatinib is an active treatment for advanced anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) pretreated with ALK-tyrosine kinase inhibitors (-TKIs). However, there is paucity of data on the activity of platinum/pemetrexed chemotherapy administered at the time of progression on lorlatinib. In addition, it is uncertain whether continuation of lorlatinib beyond progression (LBP) would provide any additional clinical benefit. Here, we describe two cases experiencing an exceptional response to platinum/pemetrexed chemotherapy plus LBP and make an attempt to identify which patients' characteristics and biologic profiles of the tumor could predict benefit from such an approach. In this report, presence of controlled brain metastases, rapidly progressing extracranial disease, and presence of ALK-dependent mechanisms of resistance were associated with benefit from platinum/pemetrexed chemotherapy plus lorlatinib beyond progression.

Background:Urine cytology is useful to diagnose urinary neoplasms, whereas its role in the study of microhematuria is debatable. Usually, standard urinalysis (dipstick test and sediment examination with bright field microscope) detects the presence of microhematuria, but only urinalysis with phase-contrast microscopy (PCM) (dipstick test and sediment examination with PCM) allows the observation of red blood cell (RBC) morphology and identify their source. Usually glomerular diseases show RBCs with morphological alterations in high percentages, whereas on urologic bleeding, RBCs are rather homogeneous without morphological alterations. Aims:We compare, for the first time, RBC morphology observed in urine cytology and in urinalysis with PCM, to verify whether urinary cytology allows the recognition of the source of bleeding. Methods and Material:A total of 60 patients who had performed both urine cytology and urinalysis with PCM for microhematuria, detected with standard urinalysis, were investigated. Urine cytology showed RBCs and were negative for neoplastic cells or for inflammatory events. Urine samples were processed with the automated method ThinPrep®. RBCs with abnormal and variable shapes were defined as deformed. RBCs of the same spherical shape were defined as non-deformed. Results:Fifty-six urine cytology with RBCs deformed were confirmed in 55 urinalysis with PCM. One case showed RBCs non deformed in urine cytology and in urine sediment. Overall, agreement, between RBC morphology in urine cytology and urinalysis with PCM, was found in 56/60 cases (93%). Conclusions:Therefore, since sediment examination with PCM is available in only few laboratories, we propose that cytopathologist always reports, in urine cytology, any morphological abnormalities of RBCs in order to provide information of the hematuria origin and correctly refer the patient to a nephrologist rather than a urologist.

The oncogene and drug metabolism enzyme glutathione S-transferase P (GSTP) is also a GSH-dependent chaperone of signal transduction and transcriptional proteins with key role in liver carcinogenesis. In this study, we explored this role of GSTP in hepatocellular carcinoma (HCC) investigating the possible interaction of this protein with one of its transcription factor and metronome of the cancer cell redox, namely the nuclear factor erythroid 2-related factor 2 (Nrf2). Expression, cellular distribution, and function as glutathionylation factor of GSTP1-1 isoform were investigated in the mouse model of N-nitrosodiethylamine (DEN)-induced HCC and in vitro in human HCC cell lines. The physical and functional interaction of GSTP protein with Nrf2 and Keap1 were investigated by immunoprecipitation and gene manipulation experiments. GSTP protein increased its liver expression, enzymatic activity and nuclear levels during DEN-induced tumor development in mice; protein glutathionylation (PSSG) was increased in the tumor masses. Higher levels and a preferential nuclear localization of GSTP protein were also observed in HepG2 and Huh-7 hepatocarcinoma cells compared to HepaRG non-cancerous cells, along with increased basal and Ebselen-stimulated levels of free GSH and PSSG. GSTP activity inhibition with the GSH analogue EZT induced apoptotic cell death in HCC cells. Hepatic Nrf2 and c-Jun, two transcription factors involved in GSTP expression and GSH biosynthesis, were induced in DEN-HCC compared to control animals; the Nrf2 inhibitory proteins Keap1 and β-TrCP also increased and oligomeric forms of GSTP co-immunoprecipitated with both Nrf2 and Keap1. Nrf2 nuclear translocation and β-TrCP expression also increased in HCC cells, and GSTP transfection in HepaRG cells induced Nrf2 activation. In conclusion, GSTP expression and subcellular distribution are modified in HCC cells and apparently contribute to the GSH-dependent reprogramming of the cellular redox in this type of cancer directly influencing the transcriptional system Nrf2/Keap1.

Polyangiitis overlap syndrome is a rare clinical entity comprising patients with overlapping features of more than one vasculitis, usually eosinophilic granulomatosis with polyangiitis (EGPA) and granulomatosis with polyangiitis (GPA). Few cases of polyangiitis overlap syndrome have been described in the literature, mostly associated with c-ANCA, anti-proteinase (PR)-3 positivity, a protean clinical picture characterized by vasculitis, eosinophilia and eosinophilic infiltrates in tissues and a favorable response to steroids and immunosuppressant treatments. Herein, we present a case of a 66-year-old woman with nasal obstruction, external nose deformity, sensorineural hearing loss, peripheral blood eosinophilia, high titer anti-PR3 antibodies and lung involvement. Nasal septum biopsies showed inflammatory infiltrate with eosinophilic component; histopathology of the lung demonstrated necrotizing granulomas associated with inflammatory infiltrate composed of numerous neutrophils and some eosinophils. The patient was diagnosed with polyangiitis overlap syndrome and successfully treated with cyclophosphamide. Recognizing this entity is fundamental given the distinct clinical phenotype and outcomes to therapy in the complex scenario of ANCA-associated vasculitides.

Introduction: The onset of precision medicine has led to the integration of traditional morphologic tissues evaluation with biochemical and molecular data for a more appropriate pathological diagnosis. The preanalytic phase and, particularly, timing of cold ischemia are crucial to guarantee high-quality biorepositories of formalin-fixed paraffin-embedded (FFPE) tissues for patients' needs and scientific research. However, delayed fixation using the gold-standard and carcinogenic fixative neutral-buffered formalin (NBF) can be a significant limitation to diagnosis and biopathological characterization. HistoCold (patented; Bio-Optica Milano S.p.A., Milano, Italy) is a nontoxic, stable, and refrigerated preservative solution for tissue handling. This study examined HistoCold's potential role in improving the preanalytic phase of the pathological diagnostic process.Materials and Methods: Breast, lung, or colorectal cancers (20, 25, and 10 cases, respectively) that were to be surgically resected were recruited between 2019 and 2021. Once specimens were surgically removed, three residual samples for each patient were first promptly immersed into HistoCold for 24, 48, and 72 hours and then FFPE. These were compared with routine specimens regarding morphologic features (hematoxylin and eosin) and tissue antigenicity (immunohistochemical stains).Results: Good concordance regarding both the morphologic characteristics of the neoplasms and their proteins expression between the routine and HistoCold handled tissues were found. The tissue handling with the solution never affected the histopathological diagnosis.Conclusions: The use of HistoCold for samples transporting is easy, allows for improving the management of cold ischemia time, and monitoring the fixation times in NBF, resulting in good quality tissue blocks for biobanking. Moreover, it could be a candidate to eliminate formalin from operating theaters. HistoCold looks very promising for the preanalytic phase of human tissues handling in the era of precision medicine, to provide the best service to patients, and to scientific research.

Treatment with immune checkpoint inhibitors (ICIs) that target the programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) axis is usually ineffective in patients with epidermal growth factor receptor (EGFR)-mutated advanced non-small cell lung cancer (NSCLC), either as first-line treatment or in later lines. By contrast, especially for patients with common EGFR mutations (exon 19 deletion/L858R point mutation), an orally bioavailable EGFR tyrosine kinase inhibitor (EGFR-TKI) is the best upfront therapy, being able to provide response rates well above 50% and a median progression-free survival ranging from 11 to 19 months, depending on whether a second-generation (e.g., afatinib) or a third-generation (i.e., osimertinib) EGFR-TKI is used. Unfortunately, treatment options for these patients at the time of acquired resistance are limited. As for afatinib-pretreated patients, those who develop a T790M mutation may benefit from osimertinib, whereas platinum-based chemotherapy is the preferable therapeutic strategy for T790M-negative patients as well as for patients who progress on osimertinib administered as first-line therapy. Here, we describe the case of an exon-19-deleted patient who experienced a complete response to the anti-PD-1 agent pembrolizumab upon the development of T790M-negative acquired resistance to afatinib. Furthermore, we discuss this case in the context of the existing literature, especially focusing on the importance of evaluating multiple markers of immune response post-EGFR-TKI and prior to ICI treatment in order to select the best treatment strategy in this clinical scenario.

Clonal evolution and lineage plasticity are key contributors to tumor heterogeneity and response to treatment in cancer. However, capturing signal transduction events in coexisting clones remains challenging from a technical perspective. In this study, we developed and tested a signal-transduction-based workflow to isolate and profile coexisting clones within a complex cellular system like non-small cell lung cancers (NSCLCs). Cooccurring clones were isolated under immunohistochemical guidance using laser-capture microdissection, and cell signaling activation portraits were measured using the reverse-phase protein microarray. To increase the translational potential of this work and capture druggable vulnerabilities within different clones, we measured expression/activation of a panel of key drug targets and downstream substrates of FDA-approved or investigational agents. We isolated intermixed clones, including poorly represented ones (<5% of cells), within the tumor microecology and identified molecular characteristics uniquely attributable to cancer cells that undergo lineage plasticity and neuroendocrine transdifferentiation in NSCLCs.

Programmed death ligand 1 (PD-L1) expression is a predictive biomarker of the success of PD-1/PD-L1 inhibitor therapy for patients with advanced non-small cell lung cancer (NSCLC) but its role as a prognostic marker for early-stage resectable NSCLC remains unclear. We studied gene expression levels of immune-related genes PD-1, PD-L1, PD-L2, IDO-1, IDO-2 and INFγ in tumor tissue of surgically resected NSCLC and correlated the finding with clinicopathological features and patient outcomes. A total of 191 consecutive early-stage NSCLC patients who underwent curative pulmonary resection were studied. The mRNA expression levels of immune-related genes were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RT2 Profiler PCR Arrays (Qiagen). PD-1, PD-L2 and IDO-2 gene expression levels were significantly higher in patients with squamous histology (p = 0.001, p = 0.021 and p < 0.001; respectively). PD-1, PD-L1 and IDO-2 gene expression levels were significantly higher in patients with higher stage (p = 0.005, p = 0.048 and p = 0.002, respectively). The univariate analysis for recurrence-free survival (RFS) and overall survival (OS) showed that patients with higher levels of three-genes (PD-L1/PD-L2/INFγ) (hazard ratio (HR)) 1.90 (95% confidence interval (CI), 1.13–3.21), p = 0.015) were associated with a worse RFS, while patients with higher levels of both genes (PD-L1/IDO-2) or (PD-L2/IDO-1) were associated with a worse OS (HR 1.63 95% CI, 1.06–2.51, p = 0.024; HR 1.54 95% CI, 1.02–2.33, p = 0.04; respectively). The multivariate interaction model adjusted for histology and stage confirmed that higher levels of three genes (PD-L1/PD-L2/INFγ) were significantly associated with worse RFS (HR 1.98, p = 0.031) and higher levels of both genes (PD-L1/IDO-2) and (PD-L2/IDO-1) with worse OS (HR 1.98, p = 0.042, HR 1.92, p = 0.022). PD-L1/IDO-2 and PD-L2/IDO-1 co-expression high levels are independent negative prognostic factors for survival in early NSCLC. These features may have important implications for future immune-checkpoint therapeutic approaches.

Background Anti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples. Methods PD-L1 expression was measured using five antibody clones (22C3, 28–8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment. Results Median-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: −0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response. Conclusions Continuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.

Purpose Decades of quality control efforts have raised the standards of immunohistochemistry (IHC), the principle method used for biomarker testing in breast cancer; however, computational pathology and reverse transcription quantitative PCR (RT-qPCR) may also hold promise for additional substantial improvements. Methods Herein, we investigated discrepancies in the assessment of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and marker of proliferation Ki67 comparing routinely obtained IHC (and FISH) data (ORI) with the results of manual (REV) and semi-automated (DIA) re-evaluation of the original IHC slides and then with RNA expression data from the same tissue block using the MammaTyper® (MT) gene expression assay. Results Correlation for ER and PR was high between ORI IHC and the other three study methods (REV, DIA and RT-qPCR). For HER2, 10 out of 96 discrepant cases can be detected between ORI and REV that involved at least one call in the equivocal category (except for one case). For Ki67, 22 (29.1%) cases were categorized differently by either REV alone (n = 17), DIA alone (n = 15) or both (n = 10) and 28 cases (29.2%) for RT-qPCR. Most of the discrepant Ki67 cases changed from low to high between the original and following assessment and belonged to the intermediate Ki67 expression range (between 9 and 30%). Conclusions Determination of the breast cancer biomarkers ER, PR, HER2 and Ki67 at the mRNA level shows high degree of correlation with IHC and compares well with correlations between original with subsequent independent manual or semi-automated IHC assessments. The use of methods with wider dynamic range and higher reproducibility such as RT-qPCR may offer more precise assessment of endocrine responsiveness, improve Ki67 standardization and help resolve HER2 cases that remain equivocal or ambiguous by IHC/FISH. In summary, our findings seem to configure RT-qPCR as a complementary method to be used in cases of either equivocal results or presenting, at the traditional determination assays, biomarkers expressions close to the cut-off values.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of lung cancer. However, their clinical benefit is limited to a minority of patients. To unravel immune-related factors that are predictive of sensitivity or resistance to immunotherapy, we performed a gene expression analysis by RNA-Seq using the Oncomine Immuno Response Assay (OIRRA) on a total of 33 advanced NSCLC patients treated with ICI evaluating the expression levels of 365 immune-related genes. We found four genes (CD1C, HLA-DPA1, MMP2, and TLR7) downregulated (p < 0.05) and two genes (IFNB1 and MKI67) upregulated (p < 0.05) in ICI-Responders compared to ICI-Non-Responders. The Bayesian enrichment computational analysis showed a more complex interaction network that involved 10 other genes (IFNA1, TLR4, CD40, TLR2, IL12A, IL12B, TLR9, CD1E, IFNG, and HLA-DPB1) correlated with different functional groups. Five main pathways were identified (FDR < 0.0001). High TLR7 expression levels were significantly associated with a lack of response to immunotherapy (p < 0.0001) and worse outcome in terms of both PFS (p < 0.001) and OS (p = 0.03). The multivariate analysis confirmed TLR7 RNA expression as an independent predictor for both poor PFS (HR = 2.97, 95% CI, 1.16–7.6, p = 0.023) and OS (HR = 2.2, 95% CI, 1–5.08, p = 0.049). In conclusion, a high TLR7 gene expression level was identified as an independent predictor for poor clinical benefits from ICI. These data could have important implications for the development of novel single/combinatorial strategies TLR-mediated for an efficient selection of “individualized” treatments for NSCLC in the era of immunotherapy.