Multiple parameters shape the 3D chromatin structure of single nuclei

The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei displayed strong insulation even before TADs emerged. Moreover, active transcription within a TAD led to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affected insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

Molecular Characterization and Prognostication of Large Cell Neuroendocrine Carcinoma and Large Cell Carcinoma

PurposeLarge cell neuroendocrine carcinoma (LCNEC) and classic large cell carcinoma (LCC) are two distinct entities with different histological and biological characteristics. However, the mutational profiles and the clinical behavior of the two subtypes of lung cancer remain to be explored.Patients and MethodsPathological diagnoses of all screened patients were finally confirmed by three or four experienced pathologists. Patients with uncertain pathological diagnoses were excluded. Finally, we genetically profiled ten patients with LCNEC and seven with LCC. ALL patients were subjected to next-generation sequencing (NGS) test, which included nine patients sequenced with a 139-gene panel and eight patients with a 425-gene panel. Including only intersected mutations from these two panels, survival analysis was further conducted.ResultsBoth LCNEC and LCC showed high prevalence in male patients, with no clear association with smoking history. Potential targetable mutations in KRAS and RET were detected in the study cohort. However, LCNEC and LCC showed distinct mutational profiles with an enrichment of RB1/TP53 co-mutations in a subset of LCNEC patients. SMARCA4 and KEAP1 mutations were exclusively found in LCC patients, and RICTOR, BRAF, ROS1 and TET2 mutations were only detected in LCNEC. LCC patients in the cohort had shorter survival compared to LCNEC patients (p=0.006). Survival analysis revealed an association between SMARCA4 mutations and poor outcome in the study cohort and in the LCC subset. Mutations in BRAF were associated with a trend of increased survival in the study cohort, as well as in the LCNEC subset. Finally, TET2 mutations were associated with poor outcome in the LCNEC cohort.ConclusionLCC and LCNEC were both heterogeneous diseases with limited treatment options. Our study identified potential targetable mutations and prognostic biomarkers that might provide more therapeutic options and improve individualized patient care.

Cell-of-origin classification using the Hans and Lymph2Cx algorithms in primary cutaneous large B-cell lymphomas

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) and primary cutaneous follicle center lymphoma with a diffuse population of large cells (PCFCL-LC) are both primary cutaneous B-cell lymphomas with large-cell morphology (CLBCL) but with different clinical characteristics and behavior. In systemic diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), gene-expression profiling (GEP) revealed two molecular subgroups based on their cell-of-origin (COO) with prognostic significance: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. This study investigated whether COO classification is a useful tool for classification of CLBCL. For this retrospective study, 51 patients with PCDLBCL-LT and 15 patients with PCFCL-LC were analyzed for their COO according to the immunohistochemistry-based Hans algorithm and the NanoString GEP-based Lymph2Cx algorithm. In PCFCL-LC, all cases (100%) classified as GCB by both Hans and Lymph2Cx. In contrast, COO classification in PCDLBCL-LT was heterogeneous. Using Hans, 75% of the PCDLBCL-LT patients classified as non-GCB and 25% as GCB, while Lymph2Cx classified only 18% as ABC, 43% as unclassified/intermediate, and 39% as GCB. These COO subgroups did not differ in the expression of BCL2 and IgM, mutations in MYD88 and/or CD79B, loss of CDKN2A, or survival. In conclusion, PCFCL-LC uniformly classified as GCB, while PCDLBCL-LT classified along the COO spectrum of DLBCL-NOS using the Hans and Lymph2Cx algorithms. In contrast to DLBCL-NOS, the clinical relevance of COO classification in CLBCL using these algorithms has limitations and cannot be used as an alternative for the current multiparameter approach in differentiation of PCDLBCL-LT and PCFCL-LC.

Comparative Outcomes for Mature T and NK/T-cell Lymphomas in People with and without HIV and to AIDS-Defining Lymphomas

There are no studies comparing the prognosis for mature T-cell lymphoma (TCL) in people with human immunodeficiency virus (PWH) to people without HIV (PWoH) and to AIDS-defining B-cell lymphomas (A-BCL) in the modern antiretroviral therapy (ART) era. NA-ACCORD and COMPLETE are cohorts that enroll patients diagnosed with HIV and TCL, respectively. In our study 52, 64, 101, 500 and 246 PWH with histological confirmation of TCL, primary CNS, Burkitt's, diffuse large B-cell lymphoma (DLBCL) and Hodgkin's lymphoma (HL) respectively and 450 TCL without HIV were eligible for analysis. At the time of TCL diagnosis, Anaplastic large-cell lymphoma (ALCL) was the most common TCL subtype within PWH. While PWH with TCL diagnosed between 1996-2009, experienced a low 5-year survival probability at 0.23 (95% CI: 0.13, 0.41), we observed a marked improvement in their survival when diagnosed between 2010-2016 (0.69; 95% CI: 0.48, 1; p=0.04) in contrast to TCL among PWoH (0.45; 95% CI: 0.41, 0.51; p=0.53). Similarly, PWH with ALCL diagnosed between 1996-2009 were associated with a conspicuously inferior 5-year survival probability (0.17; 95% CI: 0.07, 0.42) and consistently lagged behind A-BCL subtypes such as Burkitt's (0.43; 95% CI:0.33, 0.57; p=0.09) and DLBCL (0.17; 95% CI: 0.06, 0.46; p=0.11) and behind HL (0.57; 95% CI: 0.50, 0.65; p <0.0001). Despite a small number, those diagnosed between 2010-2016, experienced a remarkable improvement in survival (0.67; 95% CI: 0.3, 1) in comparison to PWoH (0.76; 95% CI: 0.66, 0.87; p=0.58). Thus, our analysis confirms improved overall survival for aggressive B and T-cell malignancies among PWH in the last decade.

Clonotype pattern in T-cell lymphomas map the cell of origin to immature lymphoid precursors

Mature T-cell lymphomas (TCLs) are rare, clinically heterogeneous hematologic cancers of high medical need. TCLs have inferior prognosis which is attributed to poor understanding of their pathogenesis. Based on phenotypic similarities between normal and neoplastic lymphocytes it has been assumed that TCLs develop in the periphery, directly from various subtypes of normal T-cells. To address the debated question of the cell of origin in TCLs we analyzed to identify the highly variable complementarity determining regions (CDR3) regions of T-cell receptor (TCR) to trace the clonal history of the T-cells. We have collected previously published whole genome -exome, and -transcriptome sequencing data from 574 TCL patients. TCR clonotypes were identified by de novo assembly of CDR3 regions of TCR γ, β and α. We have found that the vast majority of TCLs are clonotypically oligoclonal, although the pattern oligoclonality varied. Anaplastic large cell lymphoma was most diverse comprising multiple clonotypes of TCRγ, β and α whereas adult T-cell lymphoma/leukemia and peripheral T-cell lymphomas often showed monoclonality for TCRγ and β but had diverse TCRα clonotypes. These patterns of rearrangements indicated that TCLs are initiated at the level of the lymphoid precursor. In keeping with this hypothesis, TCR rearrangements in TCLs resembled the pattern seen in the human thymus showing biased usage of V and J segments of high combinatorial probability resulting in recurrent, "public" CDR3 sequences shared across unrelated patients and different clinical TCL entities. Clonotypically diverse initiating cells may seed target tissues being responsible for disease relapses after therapy.

Specific features of the pathology of the respiratory system in SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus: Sarbecovirus) infected Syrian hamsters (Mesocricetus auratus)

Introduction. Verification of histological changes in respiratory system using Syrian (golden) hamsters (Mesocricetus auratus) as experimental model is an important task for preclinical studies of drugs intended for prevention and treatment of the novel coronavirus infection COVID-19.The aim of this work was to study pathological changes of pulmonary tissue in SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) experimental infection in Syrian hamsters. Material and methods. Male Syrian hamsters weighting 80–100 g were infected by intranasal administration of culture SARS-CoV-2 at dose 4 × 104 TCID50/ml (TCID is tissue culture infectious dose). Animals were euthanatized on 3, 7 and 14 days after infection, with gravimetric registration. The viral load in lungs was measured using the polymerase chain reaction (PCR). Right lung and trachea tissues were stained with hematoxylin-eosin and according to Mallory.Results and discussion. The highest viral replicative activity in lungs was determined 3 days after the infection. After 7 days, on a background of the decrease of the viral load in lungs, a pathologically significant increase of the organ’s gravimetric parameters was observed. Within 3 to 14 days post-infection, the lung histologic pattern had been showing the development of inflammation with a succession of infiltrative-proliferative, edematousmacrophagal and fibroblastic changes. It was found that initial changes in respiratory epithelium can proceed without paranecrotic interstitial inflammation, while in the formation of multiple lung parenchyma lesions, damage to the epithelium of bronchioles and acinar ducts can be secondary. The appearance of epithelioid large-cell metaplastic epithelium, forming pseudoacinar structures, was noted as a pathomorphological feature specific to SARS-CoV-2 infection in Syrian hamsters.Conclusion. As a result of the study, the specific features of the pathology of the respiratory system in SARSCoV-2 infected Syrian hamsters were described. These findings are of practical importance as reference data that can be used for preclinical studies to assess the effectiveness of vaccines and potential drugs.

SOX11 is a sensitive and specific marker for pulmonary high-grade neuroendocrine tumors

Background Synaptophysin (SYN), chromogranin A (CGA), CD56 and insulinoma-associated protein 1 (INSM1) are proposed neuroendocrine (NE) markers used for diagnosis of pulmonary NE tumors. These NE markers have been identified in subsets of non-NE tumors requiring differential diagnosis, thus we sought to explore new NE markers. Methods We evaluated the immunohistochemical expression of SOX11, a transcription factor involved in neurogenesis, in pulmonary NE tumors and large cell carcinomas (LCCs). Results We found that SOX11 showed a sensitivity similar to INSM1 and CGA, and less than SYN and CD56 in small cell lung carcinomas (SCLCs) and large cell neuroendocrine carcinomas (LCNECs). While SOX11 is more specific than the other four markers for diagnosis of high-grade neuroendocrine carcinomas (HG-NECs) because 1) None of LCCs (0/63), the most challenging non-NE tumor type for differential diagnosis due to overlapped morphology with LCNECs displayed SOX11 positivity. While expression of at least one of SYN, CGA, CD56 or INSM1 was identified in approximately 60% (18/30) of LCCs. 2) SOX11 was only expressed in 1 of 37 carcinoid tumors in contrast to diffuse expression of SYN, CGA, CD56 and INSM1. In HG-NECs, we noticed that SOX11 was a good complementary marker for SCLC diagnosis as it was positive in 7 of 18 SYN−/CGA−/CD56− SCLCs and 3 of 8 SYN−/CGA−/CD56−/INSM1− SCLCs, and SOX11 positivity in 4 of 6 SYN−/CGA−/CD56− cases previously diagnosed as LCCs with NE morphology provides additional evidence of NE differentiation for reclassification into LCNECs, which was further confirmed by electromicroscopical identification of neurosecretory granules. We also found SOX11 expression cannot predict the prognosis in patients with HG-NECs. Conclusions Therefore, SOX11 is a useful complementary transcriptional NE marker for diagnosis and differential diagnosis of SCLC and LCNEC.

Current Progress in Breast Implant-Associated Anaplastic Large Cell Lymphoma

Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is an uncommon type of T-cell lymphoma. Although with a low incidence, the epidemiological data raised the biosafety and health concerns of breast reconstruction and breast augmentation for BIA-ALCL. Emerging evidence confirms that genetic features, bacterial contamination, chronic inflammation, and textured breast implant are the relevant factors leading to the development of BIA-ALCL. Almost all reported cases with a medical history involve breast implants with a textured surface, which reflects the role of implant surface characteristics in BIA-ALCL. With this review, we expect to highlight the most significant features on etiology, pathogenesis, diagnosis, and therapy of BIA-ALCL, as well as we review the physical characteristics of breast implants and their potential pathogenic effect and hopefully provide a foundation for optimal choice of type of implant with minimal morbidity.