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Purpose:Follicular lymphoma (FL) is the most frequent indolent non-Hodgkin lymphoma. Around 20% of patients suffer early disease progression within 24 months (POD24) of diagnosis. This study examined the significance of circulating tumor DNA (ctDNA) in predicting response to therapy and POD24 in patients with FL.Experimental Design:We collected 100 plasma samples, before and during the treatment, from 36 patients with FL prospectively enrolled in 8 Spanish hospitals. They were treated with a chemotherapy-rituximab regimen and followed up for a median of 3.43 years. We performed targeted deep sequencing in cell-free DNA (cfDNA) and tumor genomic DNA from 31 diagnostic biopsy samples.Results:Of the alterations detected in the diagnostic tissue samples, 73% (300/411) were also identified in basal cfDNA. The mean numbers of alterations per basal cfDNA sample in patients who suffered progression of disease within 24 months (POD24-pos) or did not achieve complete response (non-CR) were significantly higher than in POD24-neg or CR patients (unpaired samples t test, P = 0.0001 and 0.001, respectively). Pretreatment ctDNA levels, as haploid genome equivalents per milliliter of plasma, were higher in patients without CR (P = 0.02) and in POD24-pos patients compared with POD24-neg patients (P < 0.001). Dynamic analysis showed that ctDNA levels decreased dramatically after treatment, although the reduction was more significant in patients with CR and POD24-neg patients.Conclusions:Basal ctDNA levels are associated with the risk of early progression and response to treatment in FL. cfDNA monitoring and genotyping during treatment and follow-up predict response to treatment and early progression.

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Purpose: Follicular lymphoma (FL) is the most frequent indolent non-Hodgkin lymphoma. Around 20% of patients suffer early disease progression within 24 months (POD24) of diagnosis. This study examined the significance of circulating tumor DNA (ctDNA) in predicting response to therapy and POD24 in patients with FL. Experimental Design: We collected 100 plasma samples, before and during the treatment, from 36 patients with FL prospectively enrolled in 8 Spanish hospitals. They were treated with a chemotherapy-rituximab regimen and followed up for a median of 3.43 years. We performed targeted deep sequencing in cell-free DNA (cfDNA) and tumor genomic DNA from 31 diagnostic biopsy samples. Results: Of the alterations detected in the diagnostic tissue samples, 73% (300/411) were also identified in basal cfDNA. The mean numbers of alterations per basal cfDNA sample in patients who suffered progression of disease within 24 months (POD24-pos) or did not achieve complete response (non-CR) were significantly higher than in POD24-neg or CR patients (unpaired samples t test, P = 0.0001 and 0.001, respectively). Pretreatment ctDNA levels, as haploid genome equivalents per milliliter of plasma, were higher in patients without CR (P = 0.02) and in POD24-pos patients compared with POD24-neg patients (P < 0.001). Dynamic analysis showed that ctDNA levels decreased dramatically after treatment, although the reduction was more significant in patients with CR and POD24-neg patients. Conclusions: Basal ctDNA levels are associated with the risk of early progression and response to treatment in FL. cfDNA monitoring and genotyping during treatment and follow-up predict response to treatment and early progression.

Author Correction: Identifcation of CRF89_BF, a new member of an HIV‑1 circulating BF intersubtype recombinant form family widely spread in South America. Sci Rep. 2021 Aug 3;11(1):16115. DOI: 10.1038/s41598-021-95679-z. Circulating recombinant forms (CRFs) contribute substantially to the HIV-1 pandemic. Among 105 CRFs described in the literature, 16 are BF intersubtype recombinants, most of South American origin, of which CRF12_BF is the most widely spread. A BF recombinant cluster identified in Bolivia was suggested to represent a new CRF_BF. Here we find that it belongs to a larger cluster incorporating 39 viruses collected in 7 countries from 3 continents, 22 of them in Spain, most from Bolivian or Peruvian individuals, and 12 in South America (Bolivia, Argentina, and Peru). This BF cluster comprises three major subclusters, two associated with Bolivian and one with Peruvian individuals. Near full-length genome sequence analyses of nine viruses, collected in Spain, Bolivia, and Peru, revealed coincident BF mosaic structures, with 13 breakpoints, 6 and 7 of which coincided with CRF12_BF and CRF17_BF, respectively. In a phylogenetic tree, they grouped in a clade closely related to these CRFs, and more distantly to CRF38_BF and CRF44_BF, all circulating in South America. These results allowed to identify a new HIV-1 CRF, designated CRF89_BF. Through phylodynamic analyses, CRF89_BF emergence was estimated in Bolivia around 1986. CRF89_BF is the fifth CRF member of the HIV-1 recombinant family related to CRF12_BF. This work was funded through Acción Estratégica en Salud Intramural (AESI), Instituto de Salud Carlos III, projects “Estudios sobre vigilancia epidemiológica molecular del VIH-1 en España”, PI16CIII/00033, and “Epidemiología molecular del VIH en España y su utilidad para investigaciones biológicas y en vacunas”, PI19CIII/00042; Red de Investigación en SIDA (RIS), Instituto de Salud Carlos III, Subdirección General de Evaluación y Fondo Europeo de Desarrollo Regional (FEDER), Plan Nacional I+D+I, project RD16ISCIII/0002/0004; and scientific agreement with Osakidetza-Servicio Vasco de Salud, Government of Basque Country, MVI 1001/16. Sí

Gene expression profiling; Lung neoplasms; Tumor biomarkers Perfil d'expressió gènica; Neoplàsies pulmonars; Biomarcadors tumorals Perfil de expresión génica; Neoplasias pulmonares; Biomarcadores tumorales Background Neoadjuvant chemoimmunotherapy for non-small cell lung cancer (NSCLC) has improved pathological responses and survival rates compared with chemotherapy alone, leading to Food and Drug Administration (FDA) approval of nivolumab plus chemotherapy for resectable stage IB-IIIA NSCLC (AJCC 7th edition) without ALK or EGFR alterations. Unfortunately, a considerable percentage of tumors do not completely respond to therapy, which has been associated with early disease progression. So far, it is impossible to predict these events due to lack of knowledge. In this study, we characterized the gene expression profile of tumor samples to identify new biomarkers and mechanisms behind tumor responses to neoadjuvant chemoimmunotherapy and disease recurrence after surgery. Methods Tumor bulk RNA sequencing was performed in 16 pretreatment and 36 post-treatment tissue samples from 41 patients with resectable stage IIIA NSCLC treated with neoadjuvant chemoimmunotherapy from NADIM trial. A panel targeting 395 genes related to immunological processes was used. Tumors were classified as complete pathological response (CPR) and non-CPR, based on the total absence of viable tumor cells in tumor bed and lymph nodes tested at surgery. Differential-expressed genes between groups and pathway enrichment analysis were assessed using DESeq2 and gene set enrichment analysis. CIBERSORTx was used to estimate the proportions of immune cell subtypes. Results CPR tumors had a stronger pre-established immune infiltrate at baseline than non-CPR, characterized by higher levels of IFNG, GZMB, NKG7, and M1 macrophages, all with a significant area under the receiver operating characteristic curve (ROC) >0.9 for CPR prediction. A greater effect of neoadjuvant therapy was also seen in CPR tumors with a reduction of tumor markers and IFNγ signaling after treatment. Additionally, the higher expression of several genes, including AKT1, BST2, OAS3, or CD8B; or higher dendritic cells and neutrophils proportions in post-treatment non-CPR samples, were associated with relapse after surgery. Also, high pretreatment PD-L1 and tumor mutational burden levels influenced the post-treatment immune landscape with the downregulation of proliferation markers and type I interferon signaling molecules in surgery samples. Conclusions Our results reinforce the differences between CPR and non-CPR responses, describing possible response and relapse immune mechanisms, opening the possibility of therapy personalization of immunotherapy-based regimens in the neoadjuvant setting of NSCLC. Work in the authors’ laboratories was supported by '‘Instituto de Salud Carlos III’' (ISCIII) PI19/01652 grant cofunded by European Regional Development Fund (ERDF), Bristol-Myers Squibb (BMS), Ministry of Science and Innovation RTC2017-6502-1 'INmunoSIGHT', RTC2019-007359-1 'BLI-O' and European Union’s Horizon 2020 research and innovation programme, CLARIFY 875160 grant, to MP. ThermoFisher provided reagents for RNA sequencing. AC-B received a Spanish Lung Cancer Group (SLCG) grant and is supported by a ISCIII-“Sara Borrell” contract CD19/00170. MCa is supported by PEJD-2019-PRE/BMD-17006 contract granted to AC-B.

The endocannabinoid system (ECS), a signalling network with immunomodulatory properties, is a potential therapeutic target in multiple sclerosis (MS). Dimethyl fumarate (DMF) is an approved drug for MS whose mechanism of action has not been fully elucidated; the possibility exists that its therapeutic effects could imply the ECS. With the aim of studying if DMF can modulate the ECS, the endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) were determined by liquid chromatography-mass spectrometry in peripheral blood mononuclear cells from 21 healthy donors (HD) and 32 MS patients at baseline and after 12 and 24 months of DMF treatment. MS patients presented lower levels of 2-AG and PEA compared to HD. 2-AG increased at 24 months, reaching HD levels. AEA and PEA remained stable at 12 and 24 months. OEA increased at 12 months and returned to initial levels at 24 months. Patients who achieved no evidence of disease activity (NEDA3) presented the same modulation over time as EDA3 patients. PEA was modulated differentially between females and males. Our results show that the ECS is dysregulated in MS patients. The increase in 2-AG and OEA during DMF treatment suggests a possible role of DMF in ECS modulation.

Simple Summary Lymphoma research is a paradigm of integrating basic and applied research within the fields of molecular marker-based diagnosis and therapy. In recent years, major advances in next-generation sequencing have substantially improved the understanding of the genomics underlying diffuse large B-cell lymphoma (DLBCL), the most frequent type of B-cell lymphoma. This review addresses the various approaches that have helped unveil the biology and intricate alterations in this pathology, from cell lines to more sophisticated last-generation experimental models, such as organoids. We also provide an overview of the most recent findings in the field, their potential relevance for designing targeted therapies and the corresponding applicability to personalized medicine. Abstract Lymphoma research is a paradigm of the integration of basic and clinical research within the fields of diagnosis and therapy. Clinical, phenotypic, and genetic data are currently used to predict which patients could benefit from standard treatment. However, alternative therapies for patients at higher risk from refractoriness or relapse are usually empirically proposed, based on trial and error, without considering the genetic complexity of aggressive B-cell lymphomas. This is primarily due to the intricate mosaic of genetic and epigenetic alterations in lymphomas, which are an obstacle to the prediction of which drug will work for any given patient. Matching a patient’s genes to drug sensitivity by directly testing live tissues comprises the “precision medicine” concept. However, in the case of lymphomas, this concept should be expanded beyond genomics, eventually providing better treatment options for patients in need of alternative therapeutic approaches. We provide an overview of the most recent findings in diffuse large B-cell lymphomas genomics, from the classic functional models used to study tumor biology and the response to experimental treatments using cell lines and mouse models, to the most recent approaches with spheroid/organoid models. We also discuss their potential relevance and applicability to daily clinical practice.

7569 Background: Follicular lymphoma (FL) is the most frequently occurring indolent non-Hodgkin lymphoma, with generally favorable outcomes but a variable clinical course. Around 20% of patients suffer progression of disease within 24 months (POD24) of chemoimmunotherapy. In this prospective study, we examined the prognostic value of circulating tumor DNA (ctDNA), before and during treatment, to predict response and POD24 in FL patients. Methods: We collected 110 plasma samples from 39 patients diagnosed with FL, prospectively enrolled in 8 Spanish hospitals and treated with Chemotherapy-Rituximab regimen from April 2017 to November 2020 with a median follow-up of 41 months (m). Samples were collected before treatment (basal), mid-treatment (3m), at the end of treatment (EOT) (6m) and relapse or follow-up. We performed targeted deep sequencing in cell-free DNA and paired genomic DNA from 30 formalin-fixed paraffin-embedded tumoral (FFPET) samples with the same gene panel. Results: Before treatment, ctDNA levels, measured as haploid genome equivalents per milliliter of plasma (hGE/mL), were detected in 17/30 patients. Basal ctDNA levels were higher in patients without complete response (CR) than in CR patients [18 vs 14 log2(hGE/mL); Mann-Whitney P = 0.007] and in patients with POD24 compared to those not-POD24 [18 vs 13 log2(hGE/mL); P = 0.005]. None of the 13 patients with zero basal ctDNA levels, experienced POD24. Patients with at least one mutation detected in basal ctDNA had an inferior 24-months PFS than patients without alterations (64 vs 100%, log-rank test P = 0.027). In addition, basal ctDNA levels were detected in one patient with low FLIPI, but partial response and POD24, and by contrast, no ctDNA was detected in another patient with CR and no progression event but high FLIPI. From the alterations detected in FFPET samples, 66% were also identified in basal ctDNA. No alterations were detected in basal ctDNA from 7 patients with CR and 3 patients with watch-and-wait strategy. Dynamic analysis showed that ctDNA levels decreased after treatment for every patient (3 mo), but the reduction at EOT was higher in patients achieving CR than in non-CR patients (12 vs 3 log-reduction) and in not-POD24 patients compared to those with POD24 (10 vs 8). Furthermore, patients with at least one mutation detected in ctDNA at EOT had a shorter 24-months PFS than those without alterations (50 vs 90%). The sensitivity to detect alterations in ctDNA at EOT was 94% in patients not achieving a CR and no mutation was detected in patients with CR. Conclusions: In a real-life, prospective-based population, we showed that pre-treatment and dynamic molecular response measured by ctDNA, could be useful to stratify patients and predict response to treatment and early relapse in FL patients.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease whose prognosis is associated with clinical features, cell-of-origin and genetic aberrations. Recent integrative, multi-omic analyses had led to identifying overlapping genetic DLBCL subtypes. We used targeted massive sequencing to analyze 84 diagnostic samples from a multicenter cohort of patients with DLBCL treated with rituximab-containing therapies and a median follow-up of 6 years. The most frequently mutated genes were IGLL5 (43%), KMT2D (33.3%), CREBBP (28.6%), PIM1 (26.2%), and CARD11 (22.6%). Mutations in CD79B were associated with a higher risk of relapse after treatment, whereas patients with mutations in CD79B, ETS1, and CD58 had a significantly shorter survival. Based on the new genetic DLBCL classifications, we tested and validated a simplified method to classify samples in five genetic subtypes analyzing the mutational status of 26 genes and BCL2 and BCL6 translocations. We propose a two-step genetic DLBCL classifier (2-S), integrating the most significant features from previous algorithms, to classify the samples as N12-S, EZB2-S, MCD2-S, BN22-S, and ST22-S groups. We determined its sensitivity and specificity, compared with the other established algorithms, and evaluated its clinical impact. The results showed that ST22-S is the group with the best clinical outcome and N12-S, the more aggressive one. EZB2-S identified a subgroup with a worse prognosis among GCB-DLBLC cases.

Key Points Gene expression and mutational analysis confirm the differences among the 3 peripheral TCL subclasses: AITL, PTCL-NOS, and PTCL-TFH.The expression of a gene set, including B-cell genes, is an IPI-independent prognostic factor for AITL cases., Visual Abstract, Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL.

Introduction: Nodal peripheral T-cell lymphoma (PTCL) subclassification (Angioimmunoblastic TCL, PTCL-with T Follicular Helper phenotype and PTCL-NOS) and therapeutic targeting is still controversial. Overall survival (OS) is only around 30% after 5 years. Methods: We performed targeted next-generation sequencing of 61 selected genes in 76 PTCL patients. Among the 76cases, 44 were classified as AITL, 18 as PTCL-TFH and 14 as PTCL-NOS. Results: Our analysis revealed a wide variety of genetic variants that possibly drive the development of AITL, PTCL-TFH and/or PTCL-NOS. We identified an average of 4 variants per patient, mainly clustered in genes regulating chromatin conformation (TET2, DNMT3A) and T-cell differentiation (RHOA, MTOR, IDH2, VAV1 and NOTCH1). More frequently mutated genes were TET2 (76,31%) and DNMT3A (27,63%). Multiple mutations were found in TET2, VAV1, RHOA, NOTCH1, MTOR, JAK1, ZEB1, ATM, DNMT3A and ARID1B. Main difference among the three Nodal PTCL classes was the higher frequency of RHOAG17V mutations (p Conclusions: There is a common molecular basis for the three Nodal PTCL with very frequent mutations in genes regulating chromatin conformation associated with mutations in genes governing T-cell differentiation. RHOA G17V mutations were highly significant enriched in AITL. Findings suggesting Clonal Hematopoiesis were found in the three types of nodal PTCL.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease with variable prognosis associated with clinical features, cell-of-origin and genetic aberrations. The main problems for DLBCL patients are that a substantial percentage of them (30-40%) are refractory to treatment or relapse and the lack of accurate predictive markers that adequately determine which patients will benefit from immunochemotherapy. Several recent deep-sequencing studies have proposed new genetic subtypes based on the DLBCL genomic profile (Wright et al., 2020; Lacy et al. 2020) and associated with clinical outcome. However, a consensus and validated classification is still needed. The aim was to determine whether genetic alterations of individual genes or genes clustered in pathways are associated with clinical outcome in immunochemotherapy-treated patients. We used targeted massive sequencing to analyze 84 diagnostic samples from a multicentric cohort of patients with DLBCL treated with rituximab-containing therapies with a median follow up of 6 years. A two-step genetic classifier was built with mutation information from 27 genes and BCL2/BCL6 translocations, based on recently proposed genetic subtypes (Wright et al., 2020; Lacy et al. 2020). Logistic regression and Kaplan-Meier analyses for survival and risk of relapse were performed to assess the potential clinical value of the classifier. Moreover, this two-step classifier was validated in an external cohort and its specificity and sensitivity were tested to determine its accuracy in classifying patients compared to the previous approaches. We found that the most frequently mutated genes were IGLL5 (43%), KMT2D (33.3%), CREBBP (28.6%), PIM1 (26.2%), and CARD11 (22.6%). Mutations in CD79B, PRDM1, and NOTCH2 were associated with a higher risk of relapse after treatment, whereas patients with mutations in CD79B, ETS1, PRDM1, and TNFAIP3 had significantly shorter survival. Analyzing the impact of gene mutations on predefined gene sets related to lymphomagenesis revealed that mutations in genes involved in B-cell development, and BCR-PI3K and MAPK-ERK pathways were significantly associated with a higher risk of relapse and/or shorter overall survival. These results confirmed the current landscape of genetic alterations in DLBCL. According to the two-step classifier, we categorized the samples into MCD, BN2, EZB, ST2, and N1 genetic subgroups (Figure). We tested the accuracy of our classification in an external series (UK population-based Haematological Malignancy Research Network cohort [HMRN]) to determine its specificity and sensitivity compared with their own classification (Lacy et al. 2020) and the LymphGen algorithm (Wright et al., 2020). The comparison demonstrated (Wright et al., 2020; Lacy et al. 2020), a specificity and sensitivity higher than 85% for each subtype, except for BN2. This controversy may be explained by the fact that BN2 is less strongly defined than the other subtypes. We then tested their clinical impact and found the EZB and ST2 subtypes to have a better clinical course and a lower risk of relapse. The BN2 group had the worst clinical outcome of our series, unlike other published studies (Figure). These results were validated in the HMRN cohort, in which N1 showed a higher risk of relapse and shorter overall survival, whereas ST2 is the group with the most favorable outcome. Although N1 was not included for clinical outcome analysis in our cohort due to the small number of cases, the validation of our classifier defined N1 as the most aggressive subtype. In summary, we propose and validate a feasible genetic DLBCL classifier based on an optimized panel of genes that unifies previous genetic classification algorithms in such a way as to facilitate its implementation as part of pathology laboratories for routine patient management. This genetic classifier, combined with clinical data and other molecular characteristics, should eventually help develop improved risk models for DLBCL patients, and guide precision therapy. Figure Disclosures Perez Callejo: F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company.