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BOB.1, encoded by POU2AF1 , is one of many factors regulating physiological B-cell maturation in the germinal center. Recently, several studies have described recurrent mutations in a three-nucleotide region in the POU2AF1 splice site in the two most common B-cell non-Hodgkin lymphomas: diffuse large B-cell lymphoma and, more frequently, follicular lymphoma. In this study, we introduced a C→G mutation at the + 1 position of the POU2AF1 splice site in two B-cell lymphoma cell lines (WSU-NHL and SUDHL4) using CRISPR/Cas9 gene editing. Our results demonstrate how point mutations in the POU2AF1 splice site decreased BOB.1 expression levels. The mutation did not produce significant changes in cell proliferation, migration, or invasiveness, but did affect cell morphology, aggregation, and cell survival in a cell-line-dependent manner. Lastly, we found that the POU2AF1 mutation c.16 + 1G > C increased BCR activation, especially in SUDHL4 cells, downregulated oxidative phosphorylation (OxPhos) metabolism, and modified therapy sensitivities in both cell lines. Mutated B-cells were more sensitive to the BTK inhibitor ibrutinib. In conclusion, mutations in the POU2AF1 splice site impact B-cell lymphomagenesis at multiple levels and represent a potential therapeutic target for patients with tumors harboring this mutation.

MT1‐matrix metalloproteinase (MT1‐MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1‐MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1‐MMP occurs during MDA‐MB‐231 adenocarcinoma cell migration into collagen type I three‐dimensional matrices. Polarized trafficking of MT1‐MMP is triggered by β1 integrin‐mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1‐MMP within VSV‐G/Rab8‐positive vesicles, but not in Rab11/Tf/TfRc‐positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1‐MMP exocytic traffic, collagen degradation and invasion, whereas Rab8‐ but not Rab11‐knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1‐MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1‐MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.

Dimethyl fumarate (DMF) is an immunomodulatory drug approved for the therapy of multiple sclerosis (MS). The identification of response biomarkers to DMF is a necessity in the clinical practice. With this aim, we studied the immunophenotypic and transcriptomic changes produced by DMF in peripheral blood mononuclear cells (PBMCs) and its association with clinical response. PBMCs were obtained from 22 RRMS patients at baseline and 12 months of DMF treatment. Lymphocyte and monocyte subsets, and gene expression were assessed by flow cytometry and next-generation RNA sequencing, respectively. Clinical response was evaluated using the composite measure \"no evidence of disease activity\" NEDA-3 or \"evidence of disease activity\" EDA-3 at 2 years, classifying patients into responders (n=15) or non-responders (n=7), respectively. In the whole cohort, DMF produced a decrease in effector (TEM) and central (TCM) memory T cells in both the CD4+ and CD8+ compartments, followed by an increase in CD4+ naïve T cells. Responder patients presented a greater decrease in TEM lymphocytes. In addition, responder patients showed an increase in NK cells and were resistant to the decrease in the intermediate monocytes shown by non-responders. Responder patients also presented differences in 3 subpopulations (NK bright, NK dim and CD8 TCM) at baseline and 4 subpopulations (intermediate monocytes, regulatory T cells, CD4 TCM and CD4 TEMRA) at 12 months. DMF induced a mild transcriptional effect, with only 328 differentially expressed genes (DEGs) after 12 months of treatment. The overall effect was a downregulation of pro-inflammatory genes, chemokines, and activators of the NF-kB pathway. At baseline, no DEGs were found between responders and non-responders. During DMF treatment a differential transcriptomic response was observed, with responders presenting a higher number of DEGs (902 genes) compared to non-responders (189 genes). Responder patients to DMF exhibit differences in monocyte and lymphocyte subpopulations and a distinguishable transcriptomic response compared to non-responders that should be further studied for the validation of biomarkers of treatment response to DMF.

Fingolimod is a functional sphingosine-1-phosphate antagonist approved for the treatment of multiple sclerosis (MS). Fingolimod affects lymphocyte subpopulations and regulates gene expression in the lymphocyte transcriptome. Translational studies are necessary to identify cellular and molecular biomarkers that might be used to predict the clinical response to the drug. In MS patients, we aimed to clarify the differential effects of fingolimod on T, B, and natural killer (NK) cell subsets and to identify differentially expressed genes in responders and non-responders (NRs) to treatment. Samples were obtained from relapsing-remitting multiple sclerosis patients before and 6 months after starting fingolimod. Forty-eight lymphocyte subpopulations were measured by flow cytometry based on surface and intracellular marker analysis. Transcriptome sequencing by next-generation technologies was used to define the gene expression profiling in lymphocytes at the same time points. NEDA-3 (no evidence of disease activity) and NEDA-4 scores were measured for all patients at 1 and 2 years after beginning fingolimod treatment to investigate an association with cellular and molecular characteristics. Fingolimod affects practically all lymphocyte subpopulations and exerts a strong effect on genetic transcription switching toward an anti-inflammatory and antioxidant response. Fingolimod induces a differential effect in lymphocyte subpopulations after 6 months of treatment in responder and NR patients. Patients who achieved a good response to the drug compared to NR patients exhibited higher percentages of NK bright cells and plasmablasts, higher levels of FOXP3, glucose phosphate isomerase, lower levels of FCRL1, and lower Expanded Disability Status Scale at baseline. The combination of these possible markers enabled us to build a probabilistic linear model to predict the clinical response to fingolimod. MS patients responsive to fingolimod exhibit a recognizable distribution of lymphocyte subpopulations and a different pretreatment gene expression signature that might be useful as a biomarker.

Background Immunotherapy is being tested in early‐stage non‐small cell lung cancer (NSCLC), and achieving higher rates of complete pathological responses (CPR) as compared to standard of care. Early identification of CPR patients has vital clinical implications. In this study, we focused on basal peripheral immune cells and their treatment‐related changes to find biomarkers associated to CPR. Methods Blood from 29 stage IIIA NSCLC patients participating in the NADIM trial (NCT03081689) was collected at diagnosis and post neoadjuvant treatment. More than 400 parameters of peripheral blood mononuclear cells (PBMCs) phenotype and plasma soluble factors were analyzed. Results Neoadjuvant chemoimmunotherapy altered more than 150 immune parameters. At diagnosis, 11 biomarkers associated to CPR were described, with an area under the ROC curve >0.70 and p‐value <.05. CPR patients had significantly higher levels of CD4+PD‐1+ cells, NKG2D, and CD56 expression on T CD56 cells, intensity of CD25 expression on CD4+CD25hi+ cells and CD69 expression on intermediate monocytes; but lower levels of CD3+CD56–CTLA‐4+ cells, CD14++CD16+CTLA‐4+ cells, CTLA‐4 expression on T CD56 cells and lower levels of b‐NGF, NT‐3, and VEGF‐D in plasma compared to non‐CPR. Post treatment, CPR patients had significantly higher levels of CD19 expression on B cells, BCMA, 4‐1BB, MCSF, and PARC and lower levels of MPIF‐1 and Flt‐3L in plasma compared to non‐CPR. Conclusions Patients achieving CPR seem to have a distinctive peripheral blood immune status at diagnosis, even showing different immune response to treatment. These results reinforce the different biology behind CPR and non‐CPR responses. 1. Non‐small cell lung cancer (NSCLC) patients achieving complete pathologic response (CPR) after neoadjuvant chemoimmunotherapy seem to have a distinctive peripheral blood immune status at diagnosis and surgery. 2. At diagnosis, CPR patients are characterized by a stronger previously induced immune response with a higher cytotoxic profile and lower levels of inhibitory cytokines and cells. 3. This exploratory analysis of the NADIM study supports the use of blood as a valid source for response biomarkers and may serve as a first step to elucidate response mechanisms to chemoimmunotherapy in NSCLC.

BackgroundPneumonitis (Pn) is one of the main immune-related adverse effects, having a special importance in lung cancer, since they share affected tissue. Despite its clinical relevance, Pn development remains an unpredictable treatment adverse effect, whose mechanisms are mainly unknown, being even more obscure when it is associated to chemoimmunotherapy.MethodsIn order to identify parameters associated to treatment related Pn, we analyzed clinical variables and molecular parameters from 46 patients with potentially resectable stage IIIA non-small-cell lung cancer treated with neoadjuvant chemoimmunotherapy included in the NADIM clinical trial (NCT03081689). Pn was defined as clinical or radiographic evidence of lung inflammation without alternative diagnoses, from treatment initiation to 180 days.ResultsAmong 46 patients, 12 developed Pn (26.1%). Sex, age, smoking status, packs-year, histological subtype, clinical or pathological response, progression-free survival, overall survival and number of nivolumab cycles, were not associated to Pn development. Regarding molecular parameters at diagnosis, Pn development was not associated to programmed death ligand 1, TPS, T cell receptor repertoire parameters, or tumor mutational burden. However, patients who developed Pn had statistically significant lower blood median levels of platelet to monocyte ratio (p=0.012) and teratocarcinoma-derived growth factor 1 (p=0.013; area under the curve (AUC) 0.801), but higher median percentages of natural killers (NKs) (p=0.019; AUC 0.786), monocytes (p=0.017; AUC 0.791), MSP (p=0.006; AUC 0.838), PARN (p=0.017; AUC 0.790), and E-Cadherin (p=0.022; AUC 0.788). In addition, the immune scenario of Pn after neoadjuvant treatment involves: high levels of neutrophils and NK cells, but low levels of B and T cells in peripheral blood; increased clonality of intratumoral T cells; and elevated plasma levels of several growth factors (EGF, HGF, VEGF, ANG-1, PDGF, NGF, and NT4) and inflammatory cytokines (MIF, CCL16, neutrophil gelatinase-associated lipocalin, BMP-4, and u-PAR).ConclusionsAlthough statistically underpowered, our results shed light on the possible mechanisms behind Pn development, involving innate and adaptative immunity, and open the possibility to predict patients at high risk. If confirmed, this may allow the personalization of both, the surveillance strategy and the therapeutic approaches to manage Pn in patients receiving chemoimmunotherapy.

Lung cancer remains the leading cause of cancer-related death worldwide, with one-third diagnosed with locally advanced (stage III) disease. Preoperative induction chemo-radiotherapy is key for the treatment of these patients, however conventional cisplatin based approaches has apparently reached a plateau of effectiveness. In the search for new therapies, the targeting of tumor metabolism is revealed as an interesting option to improve the patient’s responses. Here we describe the importance of PGC-1alpha and GAPDH/MT-CO1 ratio levels as surrogates of the Warburg effect from a series of 28 stage III NSCLC patients, on PFS, OS and PET uptake. Moreover, our results show a great variability between tumors of different individuals, ranging from very glycolytic to more OXPHOS-dependent tumors, which compromises the success of therapies directed to metabolism. In this sense, using 3 different cell lines, we describe the relevance of Warburg effect on the response to metabolism-targeted therapies. Specifically, we show that the inhibitory effect of metformin on cell viability depends on cell’s dependence on the OXPHOS system. The results on cell lines, together with the results of PGC-1alpha and GAPDH/MT-CO1 as biomarkers on patient’s biopsies, would point out what type of patients would benefit more from the use of these drugs.

Background Immunotherapy (IO) has improved prognosis of non-small cell lung cancer (NSCLC); however, some patients experience primary IO resistance (PIR). CXCL13 could be a promising PIR biomarker due to its role in antitumor immune responses. Methods 177 patients with stage IV NSCLC receiving IO from the observational, multicenter, real-world study BLI-O were included from 15 centers in Spain. CXCL13 and 39 other cytokines were measured in 158 baseline (BS1) and 98 post-first-cycle of IO (BS2) plasma samples. Additionally, peripheral T and B cell BS2 immunophenotypes were determined. CXCL13 levels were also measured in plasma samples from 38 stage IIIA NSCLC patients treated with neoadjuvant chemoimmunotherapy from the NADIM II trial (NCT03838159). PIR was defined as disease progression within 3 months in non-surgical cases or incomplete pathological response in surgical cases. Results Metastatic PIR patients had higher CXCL13 plasma levels compared to non-PIR patients, especially in BS2 samples, and exhibited a greater increase of CXCL13 levels after the first cycle of IO. Patients with high BS2 CXCL13 levels showed shorter PFS ( p  = 0.0002) and OS ( p  = 0.0007). Females showed a lower percentage of high CXCL13 cases compared to male cases. CXCL13 did not influence the growth of NSCLC cell lines in vitro. Moreover, the expression of the CXCL13/CXCR5 axis was restricted to the immune compartment of tumors. Plasmatic CXCL13 levels were not associated with PD-L1 TPS expression nor TLS density in tumor tissue. However, at systemic level, patients with high CXCL13 levels exhibited impaired B and T cell phenotypes, along with elevated levels of inflammatory cytokines, after first IO cycle. Finally, high BS2 CXCL13 levels in resectable cases were also associated with PIR. Conclusions Elevated CXCL13 levels after the first cycle of IO are associated with PIR and an altered peripheral immune profile in NSCLC, supporting its potential as a prognostic biomarker in these patients.

BackgroundNon-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths, often diagnosed at advanced stages. While immunotherapy (IO) targeting the PD-1/PD-L1 axis has improved survival, some patients experience primary IO resistance (PIR).1 2 In this sense, CXCL13 could be a promising PIR biomarker due to its role in antitumor immune responses.3 MethodsThis study included 177 patients with advanced NSCLC receiving IO from the BLI-O study. CXCL13 and 39 other cytokines were measured in 158 baseline (BS1) and 98 post-first-cycle (BS2) plasma samples. Peripheral T- and B-cell immunophenotypes were assessed by flow cytometry in 83 and 30 blood samples, respectively. Additionally, CXCL13 levels were measured in 35 BS2 plasma samples from resectable locally advanced NSCLC patients treated with neoadjuvant chemoimmunotherapy from the NADIM II trial (NCT03838159). PIR was defined as disease progression within 3 months in non-surgical cases or incomplete pathological response in surgical cases.ResultsMetastatic PIR patients (41 cases, 23.2%) had higher BS1 CXCL13 levels compared to non-PIR patients (136 cases, 76.8%) (p=0.042), being this more evident in BS2 samples (p=0.0002). Furthermore, PIR patients exhibited a greater increase of CXCL13 levels after the first cycle of IO (p=0.037). Patients with High BS2 CXCL13 levels (65 cases, 66.3%) were associated with significantly shorter PFS (HR=2.25, p=0.0002) and OS (HR=2.32, p=0.0007), compared to low CXLC13 cases (33, 33.7%). The prognostic value of BS2 CXCL13 levels was independent of IO combination, line, ECOG and sex. However, stratifying by sex, females showed a higher percentage of low CXCL13 cases (p=0.039) compared to male cases (figure 1).Regarding peripheral immune status at BS2 (figure 2), CXCL13 high patients showed impaired B-cell profiles, including a decreased percentage of transitional B-cells, a reduced expression of CD24 and CD38 in naïve B-cells cells, and lower percentage of memory B-cells with lower CD27 expression compared to CXCL13 low patients. Additionally, they exhibited reduced percentages of CD8+CD28+ T cells and CD8+CD62L+, and showed elevated levels of inflammatory cytokines (e.g., IFN-γ, IL-1a, G-CSF).Finally, higher BS2 CXCL13 levels in potentially resectable neoadjuvant IO cases were associated with PIR (p=0.005). Patients with ≥311pg/mL in BS2 samples showed 72.7% of PIR compared to 22.2% in patients with <311pg/mL (p=0.007).ConclusionsElevated CXCL13 levels after the first cycle of immunotherapy are associated with Primary IO Resistance (PIR) in metastatic and locally advanced NSCLC, as well as with a weakened peripheral immune profile, supporting its potential as a prognostic biomarker in these patients.Trial RegistrationNADIM II ClinicalTrials.gov number, NCT03838159ReferencesReck M, Rodríguez-Abreu D, Robinson AG, et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. New England Journal of Medicine 2016;375:1823–33.Provencio M, Nadal E, González-Larriba JL, et al. Perioperative nivolumab and chemotherapy in stage III non-small-cell lung cancer. New England Journal of Medicine 2023;389:504–13.Gu X, Li D, Wu P, et al. Revisiting the CXCL13/CXCR5 axis in the tumor microenvironment in the era of single-cell omics: Implications for immunotherapy. Cancer Lett. Elsevier Ireland Ltd; 2024.Ethics ApprovalThe study was conducted in accordance with the precepts of the Code of Ethics of the World Medical Association (Declaration of Helsinki).ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images.Abstract 755 Figure 1[Image Omitted. See PDF.]Abstract 755 Figure 2[Image Omitted. See PDF.]