An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Evaluation of clinical parameters and biomarkers in older, untreated mantle cell lymphoma patients receiving bendamustine-rituximab.

Mantle cell lymphoma (MCL) is clinically and biologically heterogeneous. While various prognostic features have been proposed, none currently impact therapy selection, particularly in older patients, for whom treatment is primarily dictated by age and comorbidities. Herein, we undertook a comprehensive comparison of clinicopathological features in a cohort of patients 60 years and older, uniformly treated with bendamustine and rituximab, with a median survival of >8 years. The strongest prognostic indicators in this cohort were a high-risk call by a simplified MCL international prognostic index (s-MIPI) (HR: 3.32, 95% CI: 1.65-6.68 compared to low risk), a high-risk call by MCL35 (HR: 10.34, 95% CI: 2.37-45.20 compared to low risk) and blastoid cytology (HR: 4.21, 95% CR: 1.92-9.22 compared to classic). Patients called high risk by both the s-MIPI and MCL35 had the most dismal prognosis (HR: 11.58, 95% CI: 4.10-32.72), while those with high risk by either had a moderate but clinically relevant prognosis (HR: 2.95, 95% CI: 1.49-5.82). A robust assay to assess proliferation, such as MCL35, along with stringent guidelines for cytological evaluation of MCL, in combination with MIPI, may be a strong path to risk-stratify older MCL patients in future clinical trials.

BCL3 rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

Addressing Modern Diagnostic Pathology for Patient-Derived Soft Tissue Sarcosphere Models in the Era of Functional Precision Oncology.

Responses to therapy often cannot be exclusively predicted by molecular markers, thus evidencing a critical need to develop tools for better patient selection based on relations between tumor phenotype and genotype. Patient-derived cell models could help to better refine patient stratification procedures and lead to improved clinical management. So far, such ex vivo cell models have been used for addressing basic research questions and in preclinical studies. As they now enter the era of functional precision oncology, it is of utmost importance that they meet quality standards to fully represent the molecular and phenotypical architecture of patients' tumors. Well-characterized ex vivo models are imperative for rare cancer types with high patient heterogeneity and unknown driver mutations. Soft tissue sarcomas account for a very rare, heterogeneous group of malignancies that are challenging from a diagnostic standpoint and difficult to treat in a metastatic setting because of chemotherapy resistance and a lack of targeted treatment options. Functional drug screening in patient-derived cancer cell models is a more recent approach for discovering novel therapeutic candidate drugs. However, because of the rarity and heterogeneity of soft tissue sarcomas, the number of well-established and characterized sarcoma cell models is extremely limited. Within our hospital-based platform we establish high-fidelity patient-derived ex vivo cancer models from solid tumors for enabling functional precision oncology and addressing research questions to overcome this problem. We here present 5 novel, well-characterized, complex-karyotype ex vivo soft tissue sarcosphere models, which are effective tools to study molecular pathogenesis and identify the novel drug sensitivities of these genetically complex diseases. We addressed the quality standards that should be generally considered for the characterization of such ex vivo models. More broadly, we suggest a scalable platform to provide high-fidelity ex vivo models to the scientific community and enable functional precision oncology.

Towards precision medicine in lymphoid malignancies.

Careful histopathologic examination remains the cornerstone in the diagnosis of the clinically and biologically heterogeneous group of lymphoid malignancies. However, recent advances in genomic and epigenomic characterization using high-throughput technologies have significantly improved our understanding of these tumors. Although no single genomic alteration is completely specific for a lymphoma entity, some alterations are highly recurrent in certain entities and thus can provide complementary diagnostic information when integrated in the hematopathological diagnostic workup. Moreover, other alterations may provide important information regarding the clinical course, that is, prognostic or risk-stratifying markers, or response to treatment, that is, predictive markers, which may allow tailoring of the patient's treatment based on (epi)genetic characteristics. In this review, we will focus on clinically relevant diagnostic, prognostic, and predictive biomarkers identified in more common types of B-cell malignancies, and discuss how diagnostic assays designed for comprehensive molecular profiling may pave the way for the implementation of precision diagnostics/medicine approaches. We will also discuss future directions in this rapidly evolving field, including the application of single-cell sequencing and other omics technologies, to decipher clonal dynamics and evolution in lymphoid malignancies.

Molecular and immunophenotypic characterization of SMARCB1 (INI1) - deficient intrathoracic Neoplasms.

The switch/sucrose-non-fermenting (SWI/SNF) complex is an ATP-dependent chromatin remodeling complex that plays important roles in DNA repair, transcription and cell differentiation. This complex consists of multiple subunits and is of particular interest in thoracic malignancies due to frequent subunit alteration of SMARCA4 (BRG1). Much less is known about SMARCB1 (INI1) deficient intrathoracic neoplasms, which are rare, often misclassified and understudied. In a retrospective analysis of 1479 intrathoracic malignant neoplasms using immunohistochemistry for INI1 (SMARCB1) on tissue micro arrays (TMA) and a search through our hospital sarcoma database, we identified in total nine intrathoracic, INI1 deficient cases (n = 9). We characterized these cases further by additional immunohistochemistry, broad targeted genomic analysis, methylation profiling and correlated them with clinical and radiological data. This showed that genomic SMARCB1 together with tumor suppressor alterations drive tumorigenesis in some of these cases, rather than epigenetic changes such as DNA methylation. A proper diagnostic classification, however, remains challenging. Intrathoracic tumors with loss or alteration of SMARCB1 (INI1) are highly aggressive and remain often underdiagnosed due to their rarity, which leads to false diagnostic interpretations. A better understanding of these tumors and proper diagnosis is important for better patient care as clinical trials and more targeted therapeutic options are emerging.

Impact of dual tasking on gaze behaviour and locomotor strategies adopted while circumventing virtual pedestrians during a collision avoidance task.

We investigated gaze behaviour and collision avoidance strategies in 16 healthy young individuals walking towards a goal while exposed to virtual pedestrians (VRPs) approaching from different directions (left, middle, right). This locomotor task and an auditory-based cognitive task were performed under single and dual-task conditions. Longer gaze fixation durations were observed on the approaching vs. other VRPs, with longer fixations devoted to the upper trunk and head compared to other body segments. Compared to other pedestrian approaches, the middle pedestrian received longer fixations and elicited faster walking speeds, larger onset distances of trajectory devitation and smaller obstacle clearances. Gaze and locomotor behaviours were similar between single and dual-task conditions but dual-task costs were observed for the cognitive task. The longer gaze fixations on approaching vs. other pedestrians suggest that enhanced visual attention is devoted to pedestrians posing a greater risk of collision. Likewise, longer gaze fixations for the middle pedestrians may be due to the greater collision risk entailed by this condition, and/or to the fact that this pedestrian was positioned in front of the end goal. Longer fixations on approaching VRPs' trunk and head may serve the purpose of anticipating their walking trajectory. Finally, the dual-task effects that were limited to the cognitive task suggest that healthy young adults prioritize the locomotor task and associated acquisition of visual information. The healthy patterns of visuomotor behaviour characterized in this study will serve as a basis for comparison to further understand defective collision avoidance strategies in patient populations.

Epiphyseal lymphoid infiltrates of the knee in adolescents.

We present three adolescents with focal lesions of the distal femur that were shown to be benign aggregates of small lymphocytes and plasma cells of unknown origin. The patients were within the age of 12 and 14 years. All lesions presented with similar MRI findings and provided the same histopathological findings in the biopsy. Although all lesions increased in size, only one patient remained symptomatic and underwent subsequent tumor resection. To our best knowledge, tumor or pseudotumoral epiphyseal lymphoid infiltrates as seen in these three patients have not previously been described. We recommend a biopsy to rule out a malignant tumor or an infection. Observation, without further invasive testing in asymptomatic patients, is recommended until the resolution of the lesions. However, if a lesion becomes symptomatic, surgical resection should be considered.

Oral manifestation of Langerhans cell histiocytosis: a case report.

BACKGROUND: Bone necrosis of the jaw is a serious condition with a broad differential diagnosis of pathologies such as cutaneous histiocytosis, bone metastases or malignant tumours. In addition to the most common cause, medication related osteonecrosis of the jaw (MRONJ), one must consider a number of other causes, such as histiocytosis. Langerhans cell histiocytosis (LCH) is a histiocytic disorder with a large spectrum of clinical manifestations and with possible involvement of a variety of organs. This case shows the importance of an early detection of this rare disease in order to prevent further spreading. Even if an initial diagnosis in the oral cavity is rare, dentists should be aware of this disease. CASE PRESENTATION: The presented case describes a patient who was referred for evaluation and treatment due to exposed bone and extensive osteolysis in the region of the upper and lower jaw. After biopsy and diagnosis of LCH, the patient was treated with systemic therapy, achieved remission and is disease free after a 2 year of follow up. CONCLUSIONS: This case report illustrates that when dealing with unclear osteolytic changes of the jawbone, Langerhans cell histiocytosis must be taken into consideration in the differential diagnosis and biopsy must be performed in case of suspicion.

Definition of MYC genetic heteroclonality in diffuse large B-cell lymphoma with 8q24 rearrangement and its impact on protein expression.

MYC rearrangement can be detected in a subgroup of diffuse large B-cell lymphoma characterized by unfavorable prognosis. In contrast to Burkitt lymphoma, the correlation between MYC rearrangement and MYC protein expression in diffuse large B-cell lymphoma is less clear, as approximately one-third of rearranged cases show negative or low expression by immunohistochemistry. To better understand whether specific characteristics of the MYC rearrangement may influence its protein expression, we investigated 43 de novo diffuse large B-cell lymphoma positive for 8q24 rearrangement by FISH, using 14 Burkitt lymphoma for comparison. Different cell populations (clones), breakpoints (classical vs non-classical FISH patterns), partner genes (IGH vs non-IGH) and immunostaining were detected and analyzed using computerized image systems. In a subgroup of diffuse large B-cell lymphoma, we observed different clones within the same tumor distinguishing the founder clone with MYC rearrangement alone from other subclones, carrying MYC rearrangement coupled with loss/extra copies of derivatives/normal alleles. This picture, which we defined MYC genetic heteroclonality, was found in 42% of cases and correlated to negative MYC expression (P=0.026). Non-classical FISH breakpoints were detected in 16% of diffuse large B-cell lymphoma without affecting expression (P=0.040). Non-IGH gene was the preferential partner of rearrangement in those diffuse large B-cell lymphoma showing MYC heteroclonality (P=0.016) and/or non-classical FISH breakpoints (P=0.058). MYC heteroclonality was not observed in Burkitt lymphoma and all cases had positive MYC expression. Non-classical FISH MYC breakpoint and non-IGH partner were found in 29 and 20% of Burkitt lymphoma, respectively. In conclusion, MYC genetic heteroclonality is a frequent event in diffuse large B-cell lymphoma and may have a relevant role in modulating MYC expression.

Mutational patterns in therapy-related acute lymphoblastic leukemia subgroups: one step closer to unveiling the genetic odyssey.

There is increasing evidence that therapy-related acute lymphoblastic leukemia (trALL) resulting from chemo- and/or radiotherapy represents a distinct entity. However, apart from KMT2A rearrangements, which have been repeatedly reported in this subgroup, the relevance of other aberrations remains controversial due to divergent study results and sparse molecular analyses. Within our ALL patient cohort, 15% (n = 19/131) met the criteria for trALL with a high proportion of Ph + and KMT2A rearrangements. On the molecular level, the most frequently observed mutation was KMT2D, followed by CDKN2A, KRAS and DNMT3A. No TP53 mutation was detected. Outcome was particularly poor in Ph + trALL compared to Ph+ de novo ALL, which seemed to be mitigated by allogeneic stem cell transplantation. Our findings further define trALL as a distinct entity but highlight the need for further molecular genome sequencing of somatic and germline variants to advance our understanding of trALL.

Conservative surgical treatment with fertility preservation in a young adult with NTRK rearranged spindle cell neoplasm of the uterine cervix.

In depth molecular studies are constantly expanding our understanding and refining the classification of gynecological neoplasms. NTRK rearranged spindle cell neoplasms of the lower genital tract are an emerging entity, of particular interest due to possible targeted treatment with selective kinase inhibitors. Nonetheless, surgery remains the initial treatment of choice. We present the case of a 24-year-old patient suffering from a NTRK rearranged spindle cell neoplasm of the uterine cervix which was treated with a fertility preserving conservative surgical approach.

The Association of Age and Sex With Joint Angles and Coordination During Unanticipated Cutting in Soccer Players.

Deficits in movement patterns during cutting while running might place soccer players at risk of injury. The objective was to compare joint angles and intersegment coordination between sexes and ages during an unanticipated side-step cutting task in soccer players. This cross-sectional study recruited 11 male (four adolescents and seven adults) and 10 female (six adolescents and four adults) soccer players. Three-dimensional motion capture was used to measure lower-extremity joint and segment angles as participants performed an unanticipated cutting task. Hierarchical linear models examined relationships between joint angle characteristics with age and sex. Continuous relative phase was used to quantify intersegment coordination amplitude and variability. These values were compared between age and sex groups using analysis of covariance. Adult males had greater hip flexion angle excursions than adolescent males, while adult females had smaller excursions than adolescent females (p = .011). Females had smaller changes in hip flexion angles (p = .045), greater hip adduction angles (p = .043), and greater ankle eversion angles (p = .009) than males. Adolescents had greater hip internal rotation (p = .044) and knee flexion (p = .033) angles than adults, but smaller changes in knee flexion angles at precontact compared with stance/foot off (p < .001). For intersegment coordination, females were more out-of-phase than males in the foot/shank segment in the sagittal plane. There were no differences in intersegment coordination variability between groups. Differences in joint motion during an unanticipated cutting task were present between age groups and sexes. Injury prevention programs or training programs may be able target specific deficits to lower injury risk and improve performance.

MYC and TP53 Alterations but Not MAPK Pathway Mutations Are Common Oncogenic Mechanisms in Follicular Dendritic Cell Sarcomas.

CONTEXT.­: Despite their stromal origin, follicular dendritic cells (FDCs) share many functions with hematopoietic system cells. FDC neoplasms are currently classified by the World Health Organization along with those of a histiocytic nature. However, the molecular alterations driving oncogenesis in FDC sarcomas (FDCSs) are beginning to be unveiled and do not seem to concur with those described in histiocytic neoplasms, namely MAPK pathway activation. OBJECTIVE.­: To identify molecular alterations driving tumorigenesis in FDCS. DESIGN.­: We investigated the role of MYC and TP53 in FDC-derived tumor oncogenesis and assessed comprehensively the status of the MAPK pathway in 16 FDCSs, 6 inflammatory pseudotumor (IPT)-like FDCSs, and 8 IPTs. RESULTS.­: MYC structural alterations (both amplifications and rearrangements) were identified in 5 of 14 FDCSs (35.7%), all associated with MYC overexpression. TP53 mutations were identified in 4 of 14 FDCSs (28.6%), all of which displayed intense and diffuse p53 expression. None of these alterations were identified in any IPT-like FDCSs or in IPT cases. No MAPK pathway gene alterations were identified in any of the cases studied. CONCLUSIONS.­: The presence of MYC and TP53 alterations and the lack of association with Epstein-Barr virus segregate classical FDCS from IPT-like FDCS, pointing at different oncogenic mechanisms in both entities. Our results suggest a possible oncogenic role of MYC and TP53 alterations in FDCS. The absence of MAPK pathway alterations confirms the lack of a significant role of this pathway in the oncogenesis of FDC-derived neoplasms.

Development of a virtual reality-based intervention for community walking post stroke: an integrated knowledge translation approach.

PURPOSE: To develop a virtual reality (VR) based intervention targeting community walking requirements. METHODS: Two focus groups each involving 7 clinicians allowed exploring optimal features, needed support and perceived favorable/unfavorable factors associated with the use of the VR-based intervention from the clinicians' perspective. Three stroke survivors and 2 clinicians further interacted with the intervention and filled questionnaires related to acceptability and favorable/unfavorable perceptions on the VR intervention. Stroke participants additionally rated their perceived effort (NASA Tax Load Index), presence (Slater-Usoh-Steed) and cybersickness (Simulator Sickness Questionnaire). RESULTS: Results identified optimal features (patient eligibility criteria, task complexity), needed support (training, human assistance), as well as favorable (cognitive stimulation, engagement, representativeness of therapeutic goals) and unfavorable factors (misalignment with a natural walking pattern, client suitability, generalization to real-life) associated with the intervention. Acceptability scores following the interaction with the tool were 28 and 42 (max 56) for clinicians and ranged from 43 to 52 for stroke participants. Stroke participants reported moderate perceptions of effort (range:20-33/max:60), high levels of presence (29-42/42) and minimal cybersickness (0-3/64). CONCLUSION: Findings collected in the early development phase of the VR intervention will allow addressing favorable/unfavorable factors and incorporating desired optimal features, prior to conducting effectiveness and implementation studies.

This study presents the development process of a new virtual reality (VR) intervention for community walking and participation in stroke survivors.Results from the focus group and hands-on pilot trial suggest that the VR intervention is feasible and accepted by clinicians and stroke survivors.Addressing favorable/unfavorable factors and incorporating features desired by clinicians in the development of the VR tool should promote its eventual implementation in clinical setting.

Coordinating Clearance and Postural Reorientation When Avoiding Physical and Virtual Pedestrians.

Community ambulation requires efficient locomotor adaptations to avoid collisions with other pedestrians. Virtual reality (VR) offers the possibility to experimentally manipulate the environment, allowing researchers to safely assess locomotor responses during pedestrian interactions. In this paper, locomotor adjustments in response to interferers approaching from different directions were examined in both a physical and a virtual environment. Further analysis compared the extent to which locomotor adjustments were influenced by different circumvention strategies. To achieve these goals, twelve healthy young participants were assessed while walking towards a target and avoiding pedestrians approaching from the left, middle, or right, in both VR and the real world. Results showed that in VR, participants walked with slower velocities, maintained larger minimum distances, and reached larger trajectory deviations. Additionally, trajectory deviations and postural reorientations were executed earlier in VR. There were additional differences according to whether participants had to give way to a head-on approaching interferer or choose to pass in front or behind a diagonally approaching interferer. Compared to the other circumvention strategies, passing in front was associated with faster walking speeds as well as smaller and later trajectory deviations and postural reorientations. Lastly, while a cephalocaudal sequence of segment reorientation was observed in both environments, no difference between the onset of head and thorax reorientation was observed in VR. In conclusion, obstacle clearance and postural reorientation are modulated by the environment and circumvention strategies. These modulations should be considered when designing experiments or clinical interventions.

Enhanced engraftment of human myelofibrosis stem and progenitor cells in MISTRG mice.

The engraftment potential of myeloproliferative neoplasms in immunodeficient mice is low. We hypothesized that the physiological expression of human cytokines (macrophage colony-stimulating factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) combined with human signal regulatory protein α expression in Rag2-/-Il2rγ-/- (MISTRG) mice might provide a supportive microenvironment for the development and maintenance of hematopoietic stem and progenitor cells (HSPC) from patients with primary, post-polycythemia or post-essential thrombocythemia myelofibrosis (MF). We show that MISTRG mice, in contrast to standard immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ and Rag2-/-Il2rγ-/- mice, supported engraftment of all patient samples investigated independent of MF disease stage or risk category. Moreover, MISTRG mice exhibited significantly higher human MF engraftment levels in the bone marrow, peripheral blood, and spleen and supported secondary repopulation. Bone marrow fibrosis development was limited to 3 of 14 patient samples investigated in MISTRG mice. Disease-driving mutations were identified in all xenografts, and targeted sequencing revealed maintenance of the primary patient sample clonal composition in 7 of 8 cases. Treatment of engrafted mice with the current standard-of-care Janus kinase inhibitor ruxolitinib led to a reduction in human chimerism. In conclusion, the established MF patient-derived xenograft model supports robust engraftment of MF HSPCs and maintains the genetic complexity observed in patients. The model is suited for further testing of novel therapeutic agents to expedite their transition into clinical trials.