SOX11 expression is restricted to EBV-negative Burkitt lymphoma and associates with molecular genetic features.

SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B-cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV- BL was associated with an IG∷MYC translocation generated by aberrant class switch recombination, while in EBV-/SOX11- tumors the IG∷MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11 expressing BL showed higher frequency of SMARCA4 and ID3 mutations compared to EBV-/SOX11- cases. By RNA-sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or BCR signaling were found between SOX11- and SOX11+ BL cells. However, SOX11+ BL showed higher adhesion to VCAM-1 than SOX11- BL cell lines. Here we demonstrate that EBV- BL comprises two subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.

Variant ALK-fusion positive anaplastic large cell lymphoma (ALCL): A population-based paediatric study of the NHL-BFM study group.

Frequency, distribution and prognostic meaning of ALK-partner genes other than NPM1 in ALK-positive anaplastic large-cell lymphoma (ALCL) are unknown. Forty-nine of 316 ALCL diagnosed in the NHL-BFM study group showed no nuclear ALK expression suggestive of a variant ALK-partner; 41 were analysed by genomic capture high-throughput sequencing or specific RT-PCRs. NPM1::ALK was detected in 13 cases. Among the 28 patients with a non-NPM1::ALK-fusion partner, ATIC (n = 8; 29%) and TPM3 (n = 9; 32%) were the most common. Five of eight patients with ATIC::ALK-positive ALCL relapsed, none of nine with TPM3::ALK. Variant ALK-partners are rare and potentially associated with different prognoses.

CD27/CD70 pathway activation in primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder.

Primary cutaneous CD4+ small or medium T-cell lymphoproliferative disorder (PCSM-LPD) is a clonal T-cell proliferation disease confined to the skin. PCSM-LPD shares expression of T follicular helper (Tfh) cell markers with various mature T-cell lymphomas. However, the benign presentation of PCSM-LPD contrasts the clinical behavior of other Tfh-lymphomas. The aim of our study was to delineate the molecular similarities and differences between PCSM-LPD and other Tfh-derived lymphomas to explain the clinical behavior and unravel possible pathological mechanisms. We performed targeted next-generation sequencing of 19 genes recurrently mutated in T-cell neoplasms in n = 17 PCSM-LPD with high and in n = 21 PCSM-LPD with low tumor cell content. Furthermore, gene expression profiling was used to identify genes potentially expressed in the PD1-positive (PD1+) neoplastic cells. Expression of some of these genes was confirmed in situ using multistain immunofluorescence. We found that PCSM-LPD rarely harbored mutations recurrently detected in other T-cell neoplasms. PCSM-LPD is characterized by the invariable expression of the T-cell-receptor-associated LCK protein. CD70 and its ligand CD27 are co-expressed on PD1+ PCSM-LPD cells, suggestive of autoactivation of the CD70 pathway. In conclusion, PCSM-LPD differs from disseminated lymphomas of Tfh origin by their mutation profile. Activation of CD70 signaling also found in cutaneous T-cell lymphoma represents a potential driver of neoplastic proliferation of this benign neoplasia of Tfh. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Climbing Technique Evaluation by Means of Skeleton Video Stream Analysis.

Due to the growing interest in climbing, increasing importance has been given to research in the field of non-invasive, camera-based motion analysis. While existing work uses invasive technologies such as wearables or modified walls and holds, or focuses on competitive sports, we for the first time present a system that uses video analysis to automatically recognize six movement errors that are typical for novices with limited climbing experience. Climbing a complete route consists of three repetitive climbing phases. Therefore, a characteristic joint arrangement may be detected as an error in a specific climbing phase, while this exact arrangement may not considered to be an error in another climbing phase. That is why we introduced a finite state machine to determine the current phase and to check for errors that commonly occur in the current phase. The transition between the phases depends on which joints are being used. To capture joint movements, we use a fourth-generation iPad Pro with LiDAR to record climbing sequences in which we convert the climber's 2-D skeleton provided by the Vision framework from Apple into 3-D joints using the LiDAR depth information. Thereupon, we introduced a method that derives whether a joint moves or not, determining the current phase. Finally, the 3-D joints are analyzed with respect to defined characteristic joint arrangements to identify possible motion errors. To present the feedback to the climber, we imitate a virtual mentor by realizing an application on the iPad that creates an analysis immediately after the climber has finished the route by pointing out the detected errors and by giving suggestions for improvement. Quantitative tests with three experienced climbers that were able to climb reference routes without any errors and intentionally with errors resulted in precision-recall curves evaluating the error detection performance. The results demonstrate that while the number of false positives is still in an acceptable range, the number of detected errors is sufficient to provide climbing novices with adequate suggestions for improvement. Moreover, our study reveals limitations that mainly originate from incorrect joint localizations caused by the LiDAR sensor range. With human pose estimation becoming increasingly reliable and with the advance of sensor capabilities, these limitations will have a decreasing impact on our system performance.

Outcome prediction by interim positron emission tomography and IgM monoclonal gammopathy in diffuse large B-cell lymphoma.

In diffuse large B-cell lymphoma (DLBCL), a positive interim positron emission tomography (PET) scan predicts treatment failure, but the proportion of high-risk patients thus identified is small. To improve prediction, we combined the interim PET result with the presence or absence of an associated IgM gammopathy. Of 108 DLBCL patients participating in a prospective trial, nine (8%) were interim PET positive and 19 (18%) had an IgM gammopathy. The monoclonal protein was not associated with distinguishing genetic features, and its light chain restriction was not always concordant with the light chain restriction of the lymphoma. The information provided by interim PET and IgM gammopathy was combined to dichotomize the population into sizeable high-risk (1-2 adverse factors) and low-risk groups (no adverse factor) with widely different outcomes (population size, 25% vs. 75%; 3-year risk of progression, 51% vs. 10%; 3-year overall survival, 64% vs. 95%). Multivariable analyses including established risk factors revealed the interim PET result and the IgM gammopathy status to be the only factors significantly associated with outcome. Information about interim PET response and IgM gammopathy may be useful in studies testing risk-adapted treatment strategies.

The spectrum of nodular lymphocyte predominant Hodgkin lymphoma: a report of the lymphoma workshop of the 20th meeting of the European Association for Haematopathology.

Session 4 of the 2021 European Association of Haematopathology/Society for Hematopathology Workshop focused on nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). First, the spectrum of immunophenotypic variations in NLPHL and the defining criteria for classic Hodgkin Lymphoma (CHL) were discussed. The added value of further immunophenotypic characterization of both tumor cells and microenvironment to support the differential diagnosis was presented. Next, unusual cases with combined growth patterns and evolution of morphological features over time were presented to explore the clinicopathological impact of presumed high-risk patterns. Based on a large collection of cases, the defining morphological, immunophenotypical, and gene expression features of T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) and THRLBCL-like NLPHL (pattern E) were reviewed to explore this challenging differential diagnosis and critically evaluate whether aggressive behavior and transformation of NLPHL can be predicted in practice.

Conserved reduction of m6A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts.

N6-methyladenosine (m6A) regulates mRNA metabolism. While it has been implicated in the development of the mammalian brain and in cognition, the role of m6A in synaptic plasticity, especially during cognitive decline, is not fully understood. In this study, we employed methylated RNA immunoprecipitation sequencing to obtain the m6A epitranscriptome of the hippocampal subregions CA1, CA3, and the dentate gyrus and the anterior cingulate cortex (ACC) in young and aged mice. We observed a decrease in m6A levels in aged animals. Comparative analysis of cingulate cortex (CC) brain tissue from cognitively intact human subjects and Alzheimer's disease (AD) patients showed decreased m6A RNA methylation in AD patients. m6A changes common to brains of aged mice and AD patients were found in transcripts linked to synaptic function including calcium/calmodulin-dependent protein kinase 2 (CAMKII) and AMPA-selective glutamate receptor 1 (Glua1). We used proximity ligation assays to show that reduced m6A levels result in decreased synaptic protein synthesis as exemplified by CAMKII and GLUA1. Moreover, reduced m6A levels impaired synaptic function. Our results suggest that m6A RNA methylation controls synaptic protein synthesis and may play a role in cognitive decline associated with aging and AD.

Molecular profiling of EBV associated diffuse large B-cell lymphoma.

Epstein-Barr virus (EBV) associated diffuse large B-cell lymphoma (DLBCL) represents a rare aggressive B-cell lymphoma subtype characterized by an adverse clinical outcome. EBV infection of lymphoma cells has been associated with different lymphoma subtypes while the precise role of EBV in lymphomagenesis and specific molecular characteristics of these lymphomas remain elusive. To further unravel the biology of EBV associated DLBCL, we present a comprehensive molecular analysis of overall 60 primary EBV positive (EBV+) DLBCLs using targeted sequencing of cancer candidate genes (CCGs) and genome-wide determination of recurrent somatic copy number alterations (SCNAs) in 46 cases, respectively. Applying the LymphGen classifier 2.0, we found that less than 20% of primary EBV + DLBCLs correspond to one of the established molecular DLBCL subtypes underscoring the unique biology of this entity. We have identified recurrent mutations activating the oncogenic JAK-STAT and NOTCH pathways as well as frequent amplifications of 9p24.1 contributing to immune escape by PD-L1 overexpression. Our findings enable further functional preclinical and clinical studies exploring the therapeutic potential of targeting these aberrations in patients with EBV + DLBCL to improve outcome.

Lack of a protective effect of the Tmem106b "protective SNP" in the Grn knockout mouse model for frontotemporal lobar degeneration.

Genetic variants in TMEM106B are a common risk factor for frontotemporal lobar degeneration and the most important modifier of disease risk in patients with progranulin (GRN) mutations (FTLD-GRN). TMEM106B is encoding a lysosomal transmembrane protein of unknown molecular function. How it mediates its disease-modifying function remains enigmatic. Several TMEM106B single nucleotide polymorphisms (SNPs) are significantly associated with disease risk in FTLD-GRN carriers, of which all except one are within intronic sequences of TMEM106B. Of note, the non-coding SNPs are in high linkage disequilibrium with the coding SNP rs3173615 located in exon six of TMEM106B, resulting in a threonine to serine change at amino acid 185 in the minor allele, which is protective in FTLD-GRN carriers. To investigate the functional consequences of this variant in vivo, we generated and characterized a knockin mouse model harboring the Tmem106bT186S variant. We analyzed the effect of this protective variant on FTLD pathology by crossing Tmem106bT186S mice with Grn-/- knockout mice, a model for GRN-mediated FTLD. We did not observe the amelioration of any of the investigated Grn-/- knockout phenotypes, including transcriptomic changes, lipid alterations, or microgliosis in Tmem106bT186S/T186S × Grn-/- mice, indicating that the Tmem106bT186S variant is not protective in the Grn-/- knockout mouse model. These data suggest that effects of the associated SNPs not directly linked to the amino acid exchange in TMEM106B are critical for the modifying effect.

Cyclin D1 expression, cell proliferation, and clonal persistence characterize primary cutaneous CD4+ small or medium T-cell lymphoproliferative disorder.

AIMS: The aim was to gain insight into the biology of primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (PCSM-LPD). METHODS: We describe the histopathological and clinical characteristics of 177 PCSM-LPD diagnosed at our consultation centre. We performed immunohistochemical multistaining in a subset of cases (n = 46) including PD1, Cyclin D1, and multiple markers of proliferation. We evaluated clonal T-cell-receptor-(TCR) rearrangements and used tissue microdissection to analyse TCR-clonality of PD1(+) cells. RESULTS: The cohort of n = 177 PCSM-LPD included 84 males and 93 females (median age 57, range 13-85). Clinical presentation was as a solitary nodule or plaque (head and neck > trunk > extremities). Most patients were treated by local excision or steroids (96%, 69/72); relapses occurred in 12/65 (18%) of patients with follow up. Histopathology revealed the predominance of a nodular pattern (75%, 134/177) and frequent clustering of PD1(+) large cells (70%, 103/147). We detected Cyclin D1 and PD1 coexpression (>10% of PD1(+)-cells) in 26/46 (57%), which was not associated with CCND1 breaks or amplifications. PD1(+)-cells in PCSM-LPDs showed a significantly higher expression of proliferation-associated proteins compared to PD1(-)-cells. A clonal TCR-rearrangement was present in 176/177 (99%), with a clonal persistence in 7/8 patients at relapse including distant sites. Tissue-microdissection revealed PD1(+)-cells as the source of clonality, whilst PD1(-)-cells remained polyclonal. CONCLUSION: PCSM-LPD is a clinically indolent, albeit neoplastic, disease driven by clonal expansion of PD1(+)-cells. We demonstrate Cyclin D1-expression associated with accelerated proliferation as a surprising new biological feature of the disease.

Baseline radiomics features and MYC rearrangement status predict progression in aggressive B-cell lymphoma.

We investigated whether the outcome prediction of patients with aggressive B-cell lymphoma can be improved by combining clinical, molecular genotype, and radiomics features. MYC, BCL2, and BCL6 rearrangements were assessed using fluorescence in situ hybridization. Seventeen radiomics features were extracted from the baseline positron emission tomography-computed tomography of 323 patients, which included maximum standardized uptake value (SUVmax), SUVpeak, SUVmean, metabolic tumor volume (MTV), total lesion glycolysis, and 12 dissemination features pertaining to distance, differences in uptake and volume between lesions, respectively. Logistic regression with backward feature selection was used to predict progression after 2 years. The predictive value of (1) International Prognostic Index (IPI); (2) IPI plus MYC; (3) IPI, MYC, and MTV; (4) radiomics; and (5) MYC plus radiomics models were tested using the cross-validated area under the curve (CV-AUC) and positive predictive values (PPVs). IPI yielded a CV-AUC of 0.65 ± 0.07 with a PPV of 29.6%. The IPI plus MYC model yielded a CV-AUC of 0.68 ± 0.08. IPI, MYC, and MTV yielded a CV-AUC of 0.74 ± 0.08. The highest model performance of the radiomics model was observed for MTV combined with the maximum distance between the largest lesion and another lesion, the maximum difference in SUVpeak between 2 lesions, and the sum of distances between all lesions, yielding an improved CV-AUC of 0.77 ± 0.07. The same radiomics features were retained when adding MYC (CV-AUC, 0.77 ± 0.07). PPV was highest for the MYC plus radiomics model (50.0%) and increased by 20% compared with the IPI (29.6%). Adding radiomics features improved model performance and PPV and can, therefore, aid in identifying poor prognosis patients.

Epigenome-wide analysis of T-cell large granular lymphocytic leukemia identifies BCL11B as a potential biomarker.

BACKGROUND: The molecular pathogenesis of T-cell large granular lymphocytic leukemia (T-LGLL), a mature T-cell leukemia arising commonly from T-cell receptor αß-positive CD8+ memory cytotoxic T cells, is only partly understood. The role of deregulated methylation in T-LGLL is not well known. We analyzed the epigenetic profile of T-LGLL cells of 11 patients compared to their normal counterparts by array-based DNA methylation profiling. For identification of molecular events driving the pathogenesis of T-LGLL, we compared the differentially methylated loci between the T-LGLL cases and normal T cells with chromatin segmentation data of benign T cells from the BLUEPRINT project. Moreover, we analyzed gene expression data of T-LGLL and benign T cells and validated the results by pyrosequencing in an extended cohort of 17 patients, including five patients with sequential samples. RESULTS: We identified dysregulation of DNA methylation associated with altered gene expression in T-LGLL. Since T-LGLL is a rare disease, the samples size is low. But as confirmed for each sample, hypermethylation of T-LGLL cells at various CpG sites located at enhancer regions is a hallmark of this disease. The interaction of BLC11B and C14orf64 as suggested by in silico data analysis could provide a novel pathogenetic mechanism that needs further experimental investigation. CONCLUSIONS: DNA methylation is altered in T-LGLL cells compared to benign T cells. In particular, BCL11B is highly significant differentially methylated in T-LGLL cells. Although our results have to be validated in a larger patient cohort, BCL11B could be considered as a potential biomarker for this leukemia. In addition, altered gene expression and hypermethylation of enhancer regions could serve as potential mechanisms for treatment of this disease. Gene interactions of dysregulated genes, like BLC11B and C14orf64, may play an important role in pathogenic mechanisms and should be further analyzed.

Genetic insights into the social organization of Neanderthals.

Genomic analyses of Neanderthals have previously provided insights into their population history and relationship to modern humans1-8, but the social organization of Neanderthal communities remains poorly understood. Here we present genetic data for 13 Neanderthals from two Middle Palaeolithic sites in the Altai Mountains of southern Siberia: 11 from Chagyrskaya Cave9,10 and 2 from Okladnikov Cave11-making this one of the largest genetic studies of a Neanderthal population to date. We used hybridization capture to obtain genome-wide nuclear data, as well as mitochondrial and Y-chromosome sequences. Some Chagyrskaya individuals were closely related, including a father-daughter pair and a pair of second-degree relatives, indicating that at least some of the individuals lived at the same time. Up to one-third of these individuals' genomes had long segments of homozygosity, suggesting that the Chagyrskaya Neanderthals were part of a small community. In addition, the Y-chromosome diversity is an order of magnitude lower than the mitochondrial diversity, a pattern that we found is best explained by female migration between communities. Thus, the genetic data presented here provide a detailed documentation of the social organization of an isolated Neanderthal community at the easternmost extent of their known range.

Microstructural Constituents and Mechanical Properties of Low-Density Fe-Cr-Ni-Mn-Al-C Stainless Steels.

Metallic material concepts associated with the sustainable and efficient use of resources are currently the subject of intensive research. Al addition to steel offers advantages in view of lightweight, durability, and efficient use of high-Fe scrap from the Al industry. In the present work, Al was added to Fe-12Cr-(9,12)Ni-3Mn-0.3C-xAl (x = 0.1-6) (wt.%) stainless steels to assess its influence on microstructure and mechanical properties. According to density measurements based on Archimedes' principle, densities were between 7.70 and 7.08 g/cm3. High-energy X-ray diffraction estimations of the lattice parameter indicated that nearly 31% of density reduction was caused by the lattice expansion associated with Al addition. Depending on Al concentration, austenitic and duplex matrix microstructures were obtained at room temperature. In the presence of up to 3 wt.% Al, the microstructure remained austenitic. At the same time, strength and hardness were slightly enhanced. Al addition in higher quantities resulted in the formation of duplex matrix microstructures with enhanced yield strength but reduced ductility compared to the austenitic alloys. Due to the ready formation of B2-(Ni,Fe)Al intermetallics in the ferrite phase of the present alloy system, the increase in strength due to the presence of ferrite was more pronounced compared to standard duplex stainless steels. The occurrence of B2 intermetallics was implied by dilatometry measurements and confirmed by electron microscopy examinations and high-energy X-ray diffraction measurements.

PARP14 is a novel target in STAT6 mutant follicular lymphoma.

The variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of tumor cells and complex interactions within the tumor microenvironment (TME). IL-4 producing follicular helper T cells (TFH) are critical components of the FL TME. Binding of IL-4 to IL-4R on FL cells activates JAK/STAT signaling. We identified STAT6 mutations (STAT6MUT) in 13% of FL (N = 33/258), all clustered within the DNA binding domain. Gene expression data and immunohistochemistry showed upregulation of IL-4/STAT6 target genes in STAT6MUT FL, including CCL17, CCL22, and FCER2 (CD23). Functionally, STAT6MUT was gain-of-function by serial replating phenotype in pre-B CFU assays. Expression of STAT6MUT enhanced IL-4 induced FCER2/CD23, CCL17 and CCL22 expression and was associated with nuclear accumulation of pSTAT6. RNA sequencing identified PARP14 -a transcriptional switch and co-activator of STAT6- among the top differentially upregulated genes in IL-4 stimulated STAT6MUT lymphoma cells and in STAT6MUT primary FL cells. Quantitative chromatin immunoprecipitation (qChIP) demonstrated binding of STAT6MUT but not STAT6WT to the PARP14 promotor. Reporter assays showed increased IL-4 induced transactivation activity of STAT6MUT at the PARP14 promotor, suggesting a self-reinforcing regulatory circuit. Knock-down of PARP14 or PARP-inhibition abrogated the STAT6MUT gain-of-function phenotype. Thus, our results identify PARP14 as a novel therapeutic target in STAT6MUT FL.

Clinical relevance of molecular characteristics in Burkitt lymphoma differs according to age.

While survival has improved for Burkitt lymphoma patients, potential differences in outcome between pediatric and adult patients remain unclear. In both age groups, survival remains poor at relapse. Therefore, we conducted a comparative study in a large pediatric cohort, including 191 cases and 97 samples from adults. While TP53 and CCND3 mutation frequencies are not age related, samples from pediatric patients showed a higher frequency of mutations in ID3, DDX3X, ARID1A and SMARCA4, while several genes such as BCL2 and YY1AP1 are almost exclusively mutated in adult patients. An unbiased analysis reveals a transition of the mutational profile between 25 and 40 years of age. Survival analysis in the pediatric cohort confirms that TP53 mutations are significantly associated with higher incidence of relapse (25 ± 4% versus 6 ± 2%, p-value 0.0002). This identifies a promising molecular marker for relapse incidence in pediatric BL which will be used in future clinical trials.

Quantifying and reducing cross-contamination in single- and multiplex hybridization capture of ancient DNA.

The use of hybridization capture has enabled a massive upscaling in sample sizes for ancient DNA studies, allowing the analysis of hundreds of skeletal remains or sediments in single studies. Nevertheless, demands in throughput continue to grow, and hybridization capture has become a limiting step in sample preparation due to the large consumption of reagents, consumables and time. Here, we explored the possibility of improving the economics of sample preparation via multiplex capture, that is, the hybridization capture of pools of double-indexed ancient DNA libraries. We demonstrate that this strategy is feasible, at least for small genomic targets such as mitochondrial DNA, if the annealing temperature is increased and PCR cycles are limited in post-capture amplification to avoid index swapping by jumping PCR, which manifests as cross-contamination in resulting sequence data. We also show that the reamplification of double-indexed libraries to PCR plateau before or after hybridization capture can sporadically lead to small, but detectable cross-contamination even if libraries are amplified in separate reactions. We provide protocols for both manual capture and automated capture in 384-well format that are compatible with single- and multiplex capture and effectively suppress cross-contamination and artefact formation. Last, we provide a simple computational method for quantifying cross-contamination due to index swapping in double-indexed libraries, which we recommend using for routine quality checks in studies that are sensitive to cross-contamination.

Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns.

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.