Multi-omic profiling of follicular lymphoma reveals changes in tissue architecture and enhanced stromal remodeling in high-risk patients.

Follicular lymphoma (FL) is a generally incurable malignancy that evolves from developmentally blocked germinal center (GC) B cells. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Dynamic interactions between tumor B cells and the tumor microenvironment (TME) are hypothesized to contribute to the broad spectrum of clinical behaviors observed among FL patients. Despite the urgent need, existing clinical tools do not reliably predict disease behavior. Using a multi-modal strategy, we examined cell-intrinsic and -extrinsic factors governing progression and therapeutic outcomes in FL patients enrolled onto a prospective clinical trial. By leveraging the strengths of each platform, we identify several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk FL patients. These features include stromal desmoplasia and changes to the follicular growth pattern present 20 months before first progression and first relapse.

Complex Analysis of Single-Cell RNA Sequencing Data.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell-cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

Femtosecond laser-assisted anterior capsulotomy in children undergoing cataract surgery: a large case series.

OBJECTIVE: To evaluate safety and postoperative visual outcomes of low-energy femtosecond laser-assisted (FLA) anterior capsulotomy in paediatric cataract surgery. METHODS AND ANALYSIS: We report a retrospective single-centre consecutive case series of 51 eyes of 33 paediatric cataract patients with a mean age of 3.22 years (range: 2 months to 13 years) who underwent cataract surgery with FLA anterior capsulotomy, using FEMTO LDV Z8 femtosecond laser (Ziemer Ophthalmic Systems). Anterior laser capsulotomy, phacoaspiration and intraocular lens implantation were performed in all eyes. Both intraoperative and long-term postoperative complications, along with long-term monocular corrected distance visual acuity (CDVA), were assessed during average follow-up period of 32.96 months (range: 13-69 months). RESULTS: In 48 out of 51 eyes, a well-sized and well-located anterior capsulotomy was achieved without intraoperative complications. Anterior capsule tears occurred in three eyes; however, they did not extend posteriorly and the intraocular lens were placed in the bags without any further complications. Posterior capsular opacification developed in 45.10% of eyes (23 out of 51) and was subsequently treated with neodymium:yttrium-aluminum-garnet (Nd:YAG) laser capsulotomy. Mean CDVA at final follow-up was 20/40 (range: 20/63 to 20/32) in unilateral cases younger than 12 months, 20/40 (range: 20/1000 to 20/25) in unilateral cases older than 12 months, 20/66 (range: 20/400 to 20/32) in bilateral cases younger than 12 months and 20/40 (range: 20/200 to 20/20) in bilateral cases older than 12 months. CONCLUSIONS: Paediatric cataract surgery with low-energy FLA anterior capsulotomy is a procedure with long-term safety, resulting in significant improvement in CDVA.

Multiregional single-cell proteogenomic analysis of ccRCC reveals cytokine drivers of intratumor spatial heterogeneity.

Intratumor heterogeneity (ITH) represents a major challenge for anticancer therapies. An integrated, multidimensional, multiregional approach dissecting ITH of the clear cell renal cell carcinoma (ccRCC) tumor microenvironment (TME) is employed at the single-cell level with mass cytometry (CyTOF), multiplex immunofluorescence (MxIF), and single-nucleus RNA sequencing (snRNA-seq) and at the bulk level with whole-exome sequencing (WES), RNA-seq, and methylation profiling. Multiregional analyses reveal unexpected conservation of immune composition within each individual patient, with profound differences among patients, presenting patient-specific tumor immune microenvironment signatures despite underlying genetic heterogeneity from clonal evolution. Spatial proteogenomic TME analysis using MxIF identifies 14 distinct cellular neighborhoods and, conversely, demonstrated architectural heterogeneity among different tumor regions. Tumor-expressed cytokines are identified as key determinants of the TME and correlate with clinical outcome. Overall, this work signifies that spatial ITH occurs in ccRCC, which may drive clinical heterogeneity and warrants further interrogation to improve patient outcomes.

Follicular Lymphoma Microenvironment Characteristics Associated with Tumor Cell Mutations and MHC Class II Expression.

Follicular lymphoma (FL) is a B-cell malignancy with a complex tumor microenvironment that is rich in nonmalignant immune cells. We applied single-cell RNA sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T cells, including a cytotoxic CD4 T-cell population. We characterized four major FL subtypes with differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced MHC expression on FL cells. In turn, expression of MHCII genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics of T cells. This provides a classification framework of the FL microenvironment in association with FL genotypes and MHC expression, and informs different potential immunotherapeutic strategies based upon tumor cell MHCII expression. SIGNIFICANCE: We have characterized the FL-infiltrating T cells, identified cytotoxic CD4 T cells as an important component that is associated with tumor cell-intrinsic characteristics, and identified sets of targetable immune checkpoints on T cells that differed from FLs with normal versus low MHC expression. See related commentary by Melnick, p. 374. This article is highlighted in the In This Issue feature, p. 369.

Dicyemida and Orthonectida: Two Stories of Body Plan Simplification.

Two enigmatic groups of morphologically simple parasites of invertebrates, the Dicyemida (syn. Rhombozoa) and the Orthonectida, since the 19th century have been usually considered as two classes of the phylum Mesozoa. Early molecular evidence suggested their relationship within the Spiralia (=Lophotrochozoa), however, high rates of dicyemid and orthonectid sequence evolution led to contradicting phylogeny reconstructions. Genomic data for orthonectids revealed that they are highly simplified spiralians and possess a reduced set of genes involved in metazoan development and body patterning. Acquiring genomic data for dicyemids, however, remains a challenge due to complex genome rearrangements including chromatin diminution and generation of extrachromosomal circular DNAs, which are reported to occur during the development of somatic cells. We performed genomic sequencing of one species of Dicyema, and obtained transcriptomic data for two Dicyema spp. Homeodomain (homeobox) transcription factors, G-protein-coupled receptors, and many other protein families have undergone a massive reduction in dicyemids compared to other animals. There is also apparent reduction of the bilaterian gene complements encoding components of the neuromuscular systems. We constructed and analyzed a large dataset of predicted orthologous proteins from three species of Dicyema and a set of spiralian animals including the newly sequenced genome of the orthonectid Intoshia linei. Bayesian analyses recovered the orthonectid lineage within the Annelida. In contrast, dicyemids form a separate clade with weak affinity to the Rouphozoa (Platyhelminthes plus Gastrotricha) or (Entoprocta plus Cycliophora) suggesting that the historically proposed Mesozoa is a polyphyletic taxon. Thus, dramatic simplification of body plans in dicyemids and orthonectids, as well as their intricate life cycles that combine metagenesis and heterogony, evolved independently in these two lineages.

H-coupled electron transfer in alkane C-h activations with halogen electrophiles.

The mechanisms for the reactions of isobutane and adamantane with polyhalogen electrophiles (HHal(2)(+), Hal(3)(+), Hal(5)(+), and Hal(7)(+), Hal = Cl, Br, or I) were studied computationally at the MP2 and B3LYP levels of theory with the 6-31G (C, H, Cl, Br) and 3-21G (I) basis sets, as well as experimentally for adamantane halogenations in Br(2), Br(2)/HBr, and I(+)Cl(-)/CCl(4). The transition structures for the activation step display almost linear C...H...Hal interactions and are characterized by significant charge transfer to the electrophile; the hydrocarbon moieties resemble the respective radical cation structures. The regiospecificities for polar halogenations of the 3-degree C-H bonds of adamantane, the high experimental kinetic isotope effects (k(H)/k(D) = 3-4), the rate accelerations in the presence of Lewis and proton (HBr) acids, and the high kinetic orders for halogen (7.5 for Br(2)) can only be understood in terms of an H-coupled electron-transfer mechanism. The three centered-two electron (3c-2e) electrophilic mechanistic concept based on the attack of the electrophile on a C-H bond does not apply; electrophilic 3c-2e interactions dominate the C-H activations only with nonoxidizing electrophiles such as carbocations. This was shown by a comparative computational analysis of the electrophilic and H-coupled electron-transfer activation mechanisms for the isobutane reaction with an ambident electrophile, the allyl cation, at the above levels of theory.