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Lorea Manterola,1 Marta Fernandez-Mercado,1 Erika Larrea,1 Ibai Goicoechea,1 María Arestin,1 María Armesto,1 Luiza Hernandez,1 Charles H Lawrie1,2,3 1Oncology Area, Biodonostia Research Institute, San Sebastián, Spain; 2Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, UK; 3Ikerbasque, Basque Foundation for Science, Bilbao, Spain Abstract: B-cell lymphomas represent a group of more than 35 recognized mature B-cell neoplasms differentiated largely on the basis of immunohistochemical staining patterns that are often challenging to accurately diagnose. Despite having been only formally recognized just over 10 years ago, microRNAs (miRNAs) have become one of the trendiest topics in biology. Dysregulation of miRNAs is a ubiquitous feature of cancer in general, including B-cell lymphomas. Many of the miRNAs aberrantly expressed in B-cell lymphomas also play a crucial regulatory role in normal hematopoietic function. MiRNAs show great potential as novel biomarkers of cancer, as they can differentiate cancers according to diagnosis and developmental stage, even discriminating between cancers that are poorly separated histologically. Furthermore, they can be robustly measured from routinely prepared formalin-fixed paraffin-embedded biopsy material and biological fluids such as blood. Here, we consider the identity, function, and biomarker potential of miRNAs in B-cell lymphomas and, most importantly, the hurdles that remain to be overcome if they are really to become part of future clinical practice. Keywords: microRNA, lymphoma, biomarker, B-cell

Water treatment plants consist in systems where water is treated and subjected to diverse processes based on the intended industrial use of the water. In the case of treatment plants for water from the cooling of continuous rolling-mill trains, the elimination and separation of oils, filtration, cooling or the thickening or dehydrating of sludge are some of the processes to which the water may be subjected. In order to design the process we use a series of pre-established technical requirements, such as, for example, thermal differences needed to cool the fluid, speed, flow, and pressure at specific points and the different concentrations of materials permitted in the water. In addition to complying with said requirements we attempt to optimise the process applying the following criteria: - Recirculation of the total flow which passes through the lines. - Reduced water consumption. - Improving, as far as possible, the quality of the water recirculated through the train. - Reducing maintenance costs, installation project costs and equipment costs. [ABSTRACT FROM AUTHOR]

Leucocytes are said to be the agent mainly responsible for intestinal reperfusion damage. The article reports on a study conducted to check the effect of reperfusion and anitoxidant drugs (AO) on the intestinal mucosa. For the sudy Male Wag rats have been used. The superior mesenteric artery and the portal vein were canulated, and both the jejunum and ileum were excised, cleansed with heparinized Ringer solution, and perfused with 10 mL of either Ringer or Ringer supplemented with AO. The study concludes that the absence of leucocytes during reperfusion of ischemic intestines does not prevent mucosal injury. This damage may be partially reduced by pretreatment of the organs with antioxidants. The addition of antioxidants during reperfusion has no effect on reperfusion injury, in our experimental model.

Large-scale genomic studies have significantly increased our knowledge of genetic variability across populations. Regional genetic profiling is essential for distinguishing common benign variants from disease-causing ones. To this end, we conducted a comprehensive characterization of exonic variants in the population of Navarre (Spain), utilizing whole genome sequencing data from 358 unrelated individuals of Spanish origin. Our analysis revealed 61,410 biallelic single nucleotide variants (SNV) within the Navarrese cohort, with 35% classified as common (MAF > 1%). By comparing allele frequency data from 1000 Genome Project (excluding the Iberian cohort of Spain, IBS), Genome Aggregation Database, and a Spanish cohort (including IBS individuals and data from Medical Genome Project), we identified 1069 SNVs common in Navarre but rare (MAF ≤ 1%) in all other populations. We further corroborated this observation with a second regional cohort of 239 unrelated exomes, which confirmed 676 of the 1069 SNVs as common in Navarre. In conclusion, this study highlights the importance of population-specific characterization of genetic variation to improve allele frequency filtering in sequencing data analysis to identify disease-causing variants.

Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (n = 4) and patients with precursor (n = 8) and full-blown MM (n = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27 - and CD27 + T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations., (© 2023. Springer Nature Limited.)

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8 + T cells with reduced immunosuppressive regulatory T (T reg ) cells, while late MYC acquisition in slow progressors was associated with lower CD8 + T cell infiltration and more abundant T reg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8 + T cells versus T reg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8 + T/T reg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8 + T cell cytotoxicity or depleting T reg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials., (© 2023. The Author(s).)

Immunoglobulin light chain (AL) amyloidosis is caused by a small, minimally proliferating B-cell/plasma-cell clone secreting a patient-unique, aggregation-prone, toxic light chain (LC). The pathogenicity of LCs is encrypted in their sequence, yet molecular determinants of amyloidogenesis are poorly understood. Higher rates of N-glycosylation among clonal κ LCs from patients with AL amyloidosis compared to other monoclonal gammopathies indicate that this post-translational modification is associated with a higher risk of developing AL amyloidosis. Here, we exploited LC sequence information from previously published amyloidogenic and control clonal LCs and from a series of 220 patients with AL amyloidosis or multiple myeloma followed at our Institutions to define sequence and spatial features of N-glycosylation, combining bioinformatics, biochemical, proteomics, structural and genetic analyses. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic κ LCs, with most of the N-glycosylation sites laying in the framework region 3, particularly within the E strand, and consisting mainly of the NFT sequon, setting them apart with respect to non-amyloidogenic clonal LCs. Our data further support a potential role of N-glycosylation in determining the pathogenic behavior of a subset of amyloidogenic LCs and may help refine current N-glycosylation-based prognostic assessments for patients with monoclonal gammopathies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Undetectable measurable residual disease (MRD) is a surrogate of prolonged survival in multiple myeloma. Thus, treatment individualization based on the probability of a patient achieving undetectable MRD with a singular regimen could represent a new concept toward personalized treatment, with fast assessment of its success. This has never been investigated; therefore, we sought to define a machine learning model to predict undetectable MRD at the onset of multiple myeloma., Experimental Design: This study included 487 newly diagnosed patients with multiple myeloma. The training (n = 152) and internal validation cohorts (n = 149) consisted of 301 transplant-eligible patients with active multiple myeloma enrolled in the GEM2012MENOS65 trial. Two external validation cohorts were defined by 76 high-risk transplant-eligible patients with smoldering multiple myeloma enrolled in the Grupo Español de Mieloma(GEM)-CESAR trial, and 110 transplant-ineligible elderly patients enrolled in the GEM-CLARIDEX trial., Results: The most effective model to predict MRD status resulted from integrating cytogenetic [t(4;14) and/or del(17p13)], tumor burden (bone marrow plasma cell clonality and circulating tumor cells), and immune-related biomarkers. Accurate predictions of MRD outcomes were achieved in 71% of cases in the GEM2012MENOS65 trial (n = 214/301) and 72% in the external validation cohorts (n = 134/186). The model also predicted sustained MRD negativity from consolidation onto 2 years maintenance (GEM2014MAIN). High-confidence prediction of undetectable MRD at diagnosis identified a subgroup of patients with active multiple myeloma with 80% and 93% progression-free and overall survival rates at 5 years., Conclusions: It is possible to accurately predict MRD outcomes using an integrative, weighted model defined by machine learning algorithms. This is a new concept toward individualized treatment in multiple myeloma. See related commentary by Pawlyn and Davies, p. 2482., (©2022 American Association for Cancer Research.)

Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88 . We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88 L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88 L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation-related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies., (© 2021 by The American Society of Hematology.)

Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252., (© 2021 by The American Society of Hematology.)

Follicular lymphoma (FL) is a common indolent B-cell lymphoma that can transform into the more aggressive transformed FL (tFL). However, the molecular process driving this transformation is uncertain. In this work, we aimed to identify microRNA (miRNA)-binding sites recurrently mutated in follicular lymphoma patients, as well as in transformed FL patients. Using whole-genome sequencing data from FL tumors, we discovered 544 mutations located in bioinformatically predicted microRNA-binding sites. We then studied these specific regions using targeted sequencing in a cohort of 55 FL patients, found 16 recurrent mutations, and identified a further 69 variants. After filtering for QC, we identified 21 genes with mutated miRNA-binding sites that were also enriched for B-cell-associated genes by Gene Ontology. Over 40% of mutations identified in these genes were present exclusively in tFL patients. We validated the predicted miRNA-binding sites of five of the genes by luciferase assay and demonstrated that the identified mutations in BCL2 and EZH2 genes impaired the binding efficiency of miR-5008 and miR-144 and regulated the endogenous levels of messenger RNA (mRNA).

Multiple myeloma (MM) patients undergo repetitive bone marrow (BM) aspirates for genetic characterization. Circulating tumor cells (CTCs) are detectable in peripheral blood (PB) of virtually all MM cases and are prognostic, but their applicability for noninvasive screening has been poorly investigated. Here, we used next-generation flow (NGF) cytometry to isolate matched CTCs and BM tumor cells from 53 patients and compared their genetic profile. In eight cases, tumor cells from extramedullary (EM) plasmacytomas were also sorted and whole-exome sequencing was performed in the three spatially distributed tumor samples. CTCs were detectable by NGF in the PB of all patients with MM. Based on the cancer cell fraction of clonal and subclonal mutations, we found that ~22% of CTCs egressed from a BM (or EM) site distant from the matched BM aspirate. Concordance between BM tumor cells and CTCs was high for chromosome arm-level copy number alterations (≥95%) though not for translocations (39%). All high-risk genetic abnormalities except one t(4;14) were detected in CTCs whenever present in BM tumor cells. Noteworthy, ≥82% mutations present in BM and EM clones were detectable in CTCs. Altogether, these results support CTCs for noninvasive risk-stratification of MM patients based on their numbers and genetic profile.

Prostate cancer (PCa) is the second most common cancer of men and is typically slow-growing and asymptomatic. The use of blood PSA as a screening method has greatly improved PCa diagnosis, but high levels of false positives has raised much interest in alternative biomarkers. We used next-generation sequencing (NGS) to elucidate the urinary transcriptome of whole urine collected from high-stage and low-stage PCa patients as well as from patients with the confounding diagnosis of benign hyperplasia (BPH). We identified and validated five differentially expressed protein-coding genes ( FTH1 BRPF1 , OSBP , PHC3 , and UACA ) in an independent validation cohort of small-volume (1 mL) centrifuged urine ( n = 94) and non-centrifuged urine ( n = 84) by droplet digital (dd)PCR. These biomarkers were able to discriminate between BPH and PCa patients and healthy controls using either centrifuged or non-centrifuged whole urine samples, suggesting that the urinary transcriptome is a valuable source of non-invasive biomarkers for PCa that warrants further investigation.

The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10 -16 ), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.

Glioblastoma is the most common primary central nervous system malignancy and has a poor prognosis. Standard first-line treatment, which includes surgery followed by adjuvant radio-chemotherapy, produces only modest benefits to survival 1,2 . Here, to explore the feasibility, safety and immunobiological effects of PD-1 blockade in patients undergoing surgery for glioblastoma, we conducted a single-arm phase II clinical trial (NCT02550249) in which we tested a presurgical dose of nivolumab followed by postsurgical nivolumab until disease progression or unacceptable toxicity in 30 patients (27 salvage surgeries for recurrent cases and 3 cases of primary surgery for newly diagnosed patients). Availability of tumor tissue pre- and post-nivolumab dosing and from additional patients who did not receive nivolumab allowed the evaluation of changes in the tumor immune microenvironment using multiple molecular and cellular analyses. Neoadjuvant nivolumab resulted in enhanced expression of chemokine transcripts, higher immune cell infiltration and augmented TCR clonal diversity among tumor-infiltrating T lymphocytes, supporting a local immunomodulatory effect of treatment. Although no obvious clinical benefit was substantiated following salvage surgery, two of the three patients treated with nivolumab before and after primary surgery remain alive 33 and 28 months later.

The circulating transcriptome is a valuable source of cancer biomarkers, which, with the exception of microRNAs (miRNAs), remains relatively unexplored. To elucidate which RNAs are present in plasma from melanoma patients and which could be used to distinguish cancer patients from healthy individuals, we used next generation sequencing (NGS), and validation was carried out by qPCR and/or ddPCR. We identified 442 different microRNAs in samples, eleven of which were differentially expressed ( p < 0.05). Levels of miR-134-5p and miR-320a-3p were significantly down-regulated ( p < 0.001) in melanoma samples ( n = 96) compared to healthy controls ( n = 28). Differentially expressed protein-encoding mRNA 5'-fragments were enriched for the angiopoietin, p21-activated kinase (PAK), and EIF2 pathways. Levels of ATM1 , AMFR , SOS1 , and CD109 gene fragments were up-regulated ( p < 0.001) in melanoma samples ( n = 144) compared to healthy controls ( n = 41) (AUC = 0.825). Over 40% of mapped reads were YRNAs, a class of non-coding RNAs that to date has been little explored. Expression levels of RNY3P1 , RNY4P1 , and RNY4P25 were significantly higher in patients with stage 0 disease than either healthy controls or more advanced stage disease ( p < 0.001). In conclusion, we have identified a number of novel RNA biomarkers, which, most importantly, we validated in multi-center retrospective and prospective cohorts, suggesting potential diagnostic use of these RNA species.

Early diagnosis of laryngeal squamous cell carcinoma (LSCC) at the stage of dysplasia could greatly improve the outcome of affected patients. For the first time we compared the mutational landscape of non-progressing dysplasia (NPD; n = 42) with progressing dysplasia (PD; n = 24), along with patient-matched LSCC biopsies; a total of 90 samples. Using targeted next-generation sequencing identified non-synonymous mutations in six genes (PIK3CA, FGFR3, TP53, JAK3, MET, FBXW7), and mutations were validated by Sanger sequencing and/or qPCR. Analysis was extended in silico to 530 head and neck (HNSCC) cases using TCGA data. Mutations in PIK3CA and FGFR3 were detected in PD and LSCC cases, as well as other HNSCC cases, but absent in NPD cases. In contrast, mutations in JAK3, MET and FBXW7 were found in NPD cases but not PD, LSCC or other HNSCC cases. TP53 was the most frequently mutated gene in both PD and NPD cases. With the exception of R248W, mutations were mutually exclusive. Moreover, five of seven PD mutations were located in motif H2 of p53, whereas none of the NPD mutations were. In summary, we propose that the mutational profile of laryngeal dysplasia has utility for the early detection of patients at risk of progression.

Tubulocystic renal cell carcinoma (TC-RCC) is a rare recently described renal neoplasm characterized by gross, microscopic, and immunohistochemical differences from other renal tumor types and was recently classified as a distinct entity. However, this distinction remains controversial particularly because some genetic studies suggest a close relationship with papillary RCC (PRCC). The molecular basis of this disease remains largely unexplored. We therefore performed noncoding (nc) RNA/miRNA expression analysis and targeted next-generation sequencing mutational profiling on 13 TC-RCC cases (11 pure, two mixed TC-RCC/PRCC) and compared with other renal neoplasms. The expression profile of miRNAs and other ncRNAs in TC-RCC was distinct and validated 10 differentially expressed miRNAs by quantitative RT-PCR, including miR-155 and miR-34a, that were significantly down-regulated compared with PRCC cases (n = 22). With the use of targeted next-generation sequencing we identified mutations in 14 different genes, most frequently (>60% of TC-RCC cases) in ABL1 and PDFGRA genes. These mutations were present in <5% of clear cell RCC, PRCC, or chromophobe RCC cases (n > 600) of The Cancer Genome Atlas database. In summary, this study is by far the largest molecular study of TC-RCC cases and the first to investigate either ncRNA expression or their genomic profile. These results add molecular evidence that TC-RCC is indeed a distinct entity from PRCC and other renal neoplasms., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)