Single-cell landscape of idiopathic multicentric Castleman disease in identical twins.

ABSTRACT: Idiopathic multicentric Castleman disease (iMCD) is a rare cytokine-driven disorder characterized by systemic inflammation, generalized lymphadenopathy, and organ dysfunction. Here, we present an unusual occurrence of iMCD in identical twins and examined the immune milieu within the affected lymphoid organs and the host circulation using multiomic high-dimensional profiling. Using spatial enhanced resolution omics sequencing (Stereo-seq) transcriptomic profiling, we performed unsupervised spatially constrained clustering to identify different anatomic structures, mapping the follicles and interfollicular regions. After a cell segmentation approach, interleukin 6 (IL-6) pathway genes significantly colocalized with endothelial cells and fibroblastic reticular cells, confirming observations using a single-cell sequencing approach (10× Chromium). Furthermore, single-cell sequencing of peripheral blood mononuclear cells revealed an "inflammatory" peripheral monocytosis enriched for the expression of S100A family genes in both twins. In summary, we provided evidence of the putative cell-of-origin of IL-6 signals in iMCD and described a distinct monocytic host immune response phenotype through a unique identical twin model.

Single-Cell Analysis Reveals Malignant Cells Reshape the Cellular Landscape and Foster an Immunosuppressive Microenvironment of Extranodal NK/T-Cell Lymphoma.

Extranodal natural killer/T-cell lymphoma (NKTCL) is an aggressive type of lymphoma associated with Epstein-Barr virus (EBV) and characterized by heterogeneous tumor behaviors. To better understand the origins of the heterogeneity, this study utilizes single-cell RNA sequencing (scRNA-seq) analysis to profile the tumor microenvironment (TME) of NKTCL at the single-cell level. Together with in vitro and in vivo models, the study identifies a subset of LMP1+ malignant NK cells contributing to the tumorigenesis and development of heterogeneous malignant cells in NKTCL. Furthermore, malignant NK cells interact with various immunocytes via chemokines and their receptors, secrete substantial DPP4 that impairs the chemotaxis of immunocytes and regulates their infiltration. They also exhibit an immunosuppressive effect on T cells, which is further boosted by LMP1. Moreover, high transcription of EBV-encoded genes and low infiltration of tumor-associated macrophages (TAMs) are favorable prognostic indicators for NKTCL in multiple patient cohorts. This study for the first time deciphers the heterogeneous composition of NKTCL TME at single-cell resolution, highlighting the crucial role of malignant NK cells with EBV-encoded LMP1 in reshaping the cellular landscape and fostering an immunosuppressive microenvironment. These findings provide insights into understanding the pathogenic mechanisms of NKTCL and developing novel therapeutic strategies against NKTCL.

Clinical features and prognostic outcomes of angioimmunoblastic T cell lymphoma in an Asian multicenter study.

In our Asian multicenter retrospective study, we investigated the clinical prognostic factors affecting the outcomes of AITL patients and identified a novel prognostic index relevant in the Asian context. In our 174-patient cohort, the median PFS and OS was 1.8 years and 5.6 years respectively. Age > 60, bone marrow involvement, total white cell count >12 × 109/L and raised serum lactate dehydrogenase were associated with poorer PFS and OS in multivariate analyses. This allowed for a prognostic index (AITL-PI) differentiating patients into low (0-1 factors, n = 64), moderate (2 factors, n = 59) and high-risk (3-4 factors, n = 49) subgroups with 5-year OS of 84.0%, 44.0% and 28.0% respectively (p < 0.0001). POD24 proved to be strongly prognostic (5-year OS 24% vs 89%, p < 0.0001). Exploratory gene expression studies were performed and disparate immune cell profiles and cell signaling signatures were seen in the low risk group as compared to the intermediate and high risk groups.

A genomic-augmented multivariate prognostic model for the survival of natural-killer/T-cell lymphoma patients from an international cohort.

With lowering costs of sequencing and genetic profiling techniques, genetic drivers can now be detected readily in tumors but current prognostic models for Natural-killer/T cell lymphoma (NKTCL) have yet to fully leverage on them for prognosticating patients. Here, we used next-generation sequencing to sequence 260 NKTCL tumors, and trained a genomic prognostic model (GPM) with the genomic mutations and survival data from this retrospective cohort of patients using LASSO Cox regression. The GPM is defined by the mutational status of 13 prognostic genes and is weakly correlated with the risk-features in International Prognostic Index (IPI), Prognostic Index for Natural-Killer cell lymphoma (PINK), and PINK-Epstein-Barr virus (PINK-E). Cox-proportional hazard multivariate regression also showed that the new GPM is independent and significant for both progression-free survival (PFS, HR: 3.73, 95% CI 2.07-6.73; p < .001) and overall survival (OS, HR: 5.23, 95% CI 2.57-10.65; p = .001) with known risk-features of these indices. When we assign an additional risk-score to samples, which are mutant for the GPM, the Harrell's C-indices of GPM-augmented IPI, PINK, and PINK-E improved significantly (p < .001, χ2 test) for both PFS and OS. Thus, we report on how genomic mutational information could steer toward better prognostication of NKTCL patients.

Artificial intelligence modelling in differentiating core biopsies of fibroadenoma from phyllodes tumor.

Breast fibroepithelial lesions (FEL) are biphasic tumors which consist of benign fibroadenomas (FAs) and the rarer phyllodes tumors (PTs). FAs and PTs have overlapping features, but have different clinical management, which makes correct core biopsy diagnosis important. This study used whole-slide images (WSIs) of 187 FA and 100 PT core biopsies, to investigate the potential role of artificial intelligence (AI) in FEL diagnosis. A total of 9228 FA patches and 6443 PT patches was generated from WSIs of the training subset, with each patch being 224 × 224 pixel in size. Our model employed a two-stage architecture comprising a convolutional neural network (CNN) component for feature extraction from the patches, and a recurrent neural network (RNN) component for whole-slide classification using activation values from the global average pooling layer in the CNN model. It achieved an overall slide-level accuracy of 87.5%, with accuracies of 80% and 95% for FA and PT slides respectively. This affirms the potential role of AI in diagnostic discrimination between FA and PT on core biopsies which may be further refined for use in routine practice.

T-Cell Lymphoma Clonality by Copy Number Variation Analysis of T-Cell Receptor Genes.

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination.

A Clinico-Genotypic Prognostic Index for De Novo Composite Diffuse Large B-Cell Lymphoma Arising from Follicular Lymphoma in Asian patients treated in the Rituximab Era.

Composite follicular lymphoma with diffuse large B-cell lymphoma (FL/DLBCL) is uncommonly found on lymph node biopsy and represents a rare haematological malignancy. We aim to examine clinico-pathological features of patients with FL/DLBCL and investigate predictors of survival outcome. We included in our retrospective study patients with histologically-proven FL/DLBCL at diagnosis (n = 106) and who were subsequently treated with rituximab-based chemoimmunotherapy from 2002-2017 at the National Cancer Centre. The cohort consisted of 34 women and 72 men with a median age of 59 years (range, 24-82). In a multivariate model inclusive of known clinico-pathological parameters at diagnosis, advanced stage (p = 0.0136), presence of MYC and/or BCL6 rearrangement (p = 0.0376) and presence of B symptoms (p = 0.0405) were independently prognostic for worse overall survival (OS). The only remaining independent prognostic variables for worse OS after including first-line treatment data in the model were use of chemotherapy regimens other than R-CHOP (p = 0.0360) and lack of complete response to chemotherapy (p < 0.0001) besides the presence of B symptoms (p = 0.0022). We generated a Clinico-Genotypic Index by point-wise addition of all five adverse parameters (score of 0-1, 2, 3, 4-5) which revealed four prognostic risk groups with a predicted 5-year OS of 100%, 62%, 40% and 0% (p < 0.0001) accounting for 50.0%, 24.5%, 18.9% and 6.6% of the cohort respectively. We propose that R-CHOP should be the recommended first-line regimen for composite FL/DLBCL.

Evaluation of the PIK3 pathway in peripheral T-cell lymphoma and NK/T-cell lymphoma.

Peripheral T-cell lymphomas (PTCL) and natural killer (NK)/T-cell lymphomas (NKTCL) are a heterogeneous group of aggressive malignancies with dismal outcomes and limited treatment options. While the phosphatidylinositol 3-kinase (PIK3) pathway has been shown to be highly activated in many B-cell lymphomas, its therapeutic relevance in PTCL and NKTCL remains unclear. The aim of this study is to investigate the expression of PIK3 and phosphatase and tensin homolog (PTEN) in these subtypes of lymphoma and to identify potential therapeutic targets for clinical testing. Therefore, the expression of PIK3α, PIK3ß, PIK3γ, PIK3δ and PTEN was analyzed in 88 cases of PTCL and NKTCL samples by immunohistochemistry. All PTCL and NKTCL samples demonstrated high expression of PIK3 isoforms. In particular, high PIK3α expression was significantly associated with poor survival, even after adjustment for age, International Prognostic Index (IPI) score and anthracycline-based chemotherapy in first line. Notably, copanlisib, a pan-class I inhibitor with predominant activities towards PIK3α and PIK3δ isoforms, effectively inhibited phosphorylation of AKT, 4E-BP-1 and STAT3, causing G0 /G1 cell cycle arrest and resulting in suppression of tumour cell growth in vitro and in vivo. This study provides evidence that targeting the PIK3 pathway, particularly simultaneous inhibition of PIK3α and δ, could be a promising approach for the treatment of PTCL and NKTCL.

Genetic risk of extranodal natural killer T-cell lymphoma: a genome-wide association study in multiple populations.

BACKGROUND: Extranodal natural killer T-cell lymphoma (NKTCL; nasal type) is an aggressive malignancy with a particularly high prevalence in Asian and Latin American populations. Epstein-Barr virus infection has a role in the pathogenesis of NKTCL, and HLA-DPB1 variants are risk factors for the disease. We aimed to identify additional novel genetic variants affecting risk of NKTCL. METHODS: We did a genome-wide association study of NKTCL in multiple populations from east Asia. We recruited a discovery cohort of 700 cases with NKTCL and 7752 controls without NKTCL of Han Chinese ancestry from 19 centres in southern, central, and northern regions of China, and four independent replication samples including 717 cases and 12 650 controls. Three of these independent samples (451 cases and 5301 controls) were from eight centres in the same regions of southern, central, and northern China, and the fourth (266 cases and 7349 controls) was from 11 centres in Hong Kong, Taiwan, Singapore, and South Korea. All cases had primary NKTCL that was confirmed histopathologically, and matching with controls was based on geographical region and self-reported ancestry. Logistic regression analysis was done independently by geographical regions, followed by fixed-effect meta-analyses, to identify susceptibility loci. Bioinformatic approaches, including expression quantitative trait loci, binding motif and transcriptome analyses, and biological experiments were done to fine-map and explore the functional relevance of genome-wide association loci to the development of NKTCL. FINDINGS: Genetic data were gathered between Jan 1, 2008, and Jan 23, 2019. Meta-analysis of all samples (a total of 1417 cases and 20 402 controls) identified two novel loci significantly associated with NKTCL: IL18RAP on 2q12.1 (rs13015714; p=2·83 × 10-16; odds ratio 1·39 [95% CI 1·28-1·50]) and HLA-DRB1 on 6p21.3 (rs9271588; 9·35 × 10-26 1·53 [1·41-1·65]). Fine-mapping and experimental analyses showed that rs1420106 at the promoter of IL18RAP was highly correlated with rs13015714, and the rs1420106-A risk variant had an upregulatory effect on IL18RAP expression. Cell growth assays in two NKTCL cell lines (YT and SNK-6 cells) showed that knockdown of IL18RAP inhibited cell proliferation by cell cycle arrest in NKTCL cells. Haplotype association analysis showed that haplotype 47F-67I was associated with reduced risk of NKTCL, whereas 47Y-67L was associated with increased risk of NKTCL. These two positions are component parts of the peptide-binding pocket 7 (P7) of the HLA-DR heterodimer, suggesting that these alterations might account for the association at HLA-DRB1, independent of the previously reported HLA-DPB1 variants. INTERPRETATION: Our findings provide new insights into the development of NKTCL by showing the importance of inflammation and immune regulation through the IL18-IL18RAP axis and antigen presentation involving HLA-DRB1, which might help to identify potential therapeutic targets. Taken in combination with additional genetic and other risk factors, our results could potentially be used to stratify people at high risk of NKTCL for targeted prevention. FUNDING: Guangdong Innovative and Entrepreneurial Research Team Program, National Natural Science Foundation of China, National Program for Support of Top-Notch Young Professionals, Chang Jiang Scholars Program, Singapore Ministry of Health's National Medical Research Council, Tanoto Foundation, National Research Foundation Singapore, Chang Gung Memorial Hospital, Recruitment Program for Young Professionals of China, First Affiliated Hospital and Army Medical University, US National Institutes of Health, and US National Cancer Institute.

A clinicohaematological prognostic model for nasal-type natural killer/T-cell lymphoma: A multicenter study.

Extranodal NK/T-cell lymphoma, nasal type (NKTL) is an aggressive type of non-Hodgkin lymphoma closely associated with Epstein-Barr virus and characterized by varying degrees of systemic inflammation. We aim to examine the prognostic significance of peripheral blood neutrophil-lymphocyte ratio (NLR) in patients with NKTL. Therefore, we conducted a retrospective review of 178 patients with biopsy-proven NKTL from the National Cancer Centre Singapore and Samsung Medical Center, South Korea. Using receiver operating curve analysis, an optimal cut-off for high NLR (>3.5) in predicting overall survival (OS) was derived. Survival analysis was performed using the Kaplan-Meier method and multivariable Cox proportional regression. In patients with high NLR, estimated 5-year OS was 25% compared to 53% in those with low NLR. In multivariable analysis, high NLR, in addition to age ≥60 years, presence of B-symptoms and stage III/IV at diagnosis, was independently correlated with worse OS (HR 2.08; 95% CI 1.36 to 3.18; p = 0.0008) and progression-free survival (HR 1.66; 95% CI 1.11 to 2.46; p = 0.0128). A new prognostic index (NABS score) derived from these factors stratified patients into low (0), low-intermediate (1), high-intermediate (2) and high (3-4) risk subgroups, which were associated with 5-year OS of 76.5%, 55.7%, 29.2% and 0% respectively. In conclusion, high NLR is an independent prognostic marker and the NABS model can be used to risk-stratify NKTL patients.

Successful therapeutic rechallenge after a severe episode of high dose methotrexate-induced choreoathetosis: A case report.

Methotrexate (MTX) is an essential chemotherapy drug used in the treatment of malignancies, but it is known to cause complications to the central nervous system. We report a case of severe MTX neurotoxicity in an adult presenting with choreoathetosis despite a normal clearance of MTX. High dose-MTX has been successfully rechallenged without any neurological sequelae. We reviewed the relevant literature of similar manifestations and summarized their clinical data, magnetic resonance imaging features and treatment given. None of them has recurrence of neurotoxicity. We concluded that it is safe to persist with MTX even after a previous episode of toxic leukoencephalopathy.

Using computer assisted image analysis to determine the optimal Ki67 threshold for predicting outcome of invasive breast cancer.

BACKGROUND: Ki67 positivity in invasive breast cancers has an inverse correlation with survival outcomes and serves as an immunohistochemical surrogate for molecular subtyping of breast cancer, particularly ER positive breast cancer. The optimal threshold of Ki67 in both settings, however, remains elusive. We use computer assisted image analysis (CAIA) to determine the optimal threshold for Ki67 in predicting survival outcomes and differentiating luminal B from luminal A breast cancers. METHODS: Quantitative scoring of Ki67 on tissue microarray (TMA) sections of 440 invasive breast cancers was performed using Aperio ePathology ImmunoHistochemistry Nuclear Image Analysis algorithm, with TMA slides digitally scanned via Aperio ScanScope XT System. RESULTS: On multivariate analysis, tumours with Ki67 ≥14% had an increased likelihood of recurrence (HR 1.941, p=0.021) and shorter overall survival (HR 2.201, p=0.016). Similar findings were observed in the subset of 343 ER positive breast cancers (HR 2.409, p=0.012 and HR 2.787, p=0.012 respectively). The value of Ki67 associated with ER+HER2-PR<20% tumours (Luminal B subtype) was found to be <17%. CONCLUSION: Using CAIA, we found optimal thresholds for Ki67 that predict a poorer prognosis and an association with the Luminal B subtype of breast cancer. Further investigation and validation of these thresholds are recommended.

Digital pathology in the diagnostic setting: beyond technology into best practice and service management.

Digital pathology (DP) and whole-slide imaging (WSI) technology have matured substantially over the last few years and there is growing evidence from validation studies that WSI is comparable to glass slides for histopathology diagnosis, although with some limitations, which can be appropriately minimised. Whether the controlled environment of validation studies translates to the same level of robustness when WSI is used in the actual diagnostic setting depends on the technical quality of WSI acquisition and on factors that influence the pre-image acquisition variables including the quality of glass slide inputs, and postimage acquisition variables such as access and use of WSI. The concept of 'DP service management' is introduced to fulfil the holistic needs of a laboratory intending to use the DP solution incorporating WSI for diagnostic purposes. The DP service management team should be an integral part of the diagnostic laboratory as it plays a central role undertaking responsibility to address an extensive range of issues from technical and training to governance and accreditation, hence ensuring a viable and sustainable diagnostic DP integration and usage. The pathologist as a specialist in the field and key decision maker of histopathology diagnoses has the duty and responsibility to acquaint and familiarise with DP and WSI when using the technology, especially on their indications and limitations, so as to take full advantage of these tools to enhance diagnostic quality.

T cell-rich lymphoid infiltrates with large B cells: a review of key entities and diagnostic approach.

Accurate diagnostic interpretation of a lymphoid population composed predominantly of small T cells, together with smaller numbers of large B cells, with or without a nodular architecture, is a common problem faced by the histopathologist. The differential diagnosis of this histological pattern is wide, ranging from reactive conditions such as drug reactions and viral infections, through borderline entities such as immunodeficiency-related lymphoproliferative disorders to lymphomas. The latter includes entities where the large B cells are primarily neoplastic (classical and nodular lymphocyte-predominant Hodgkin lymphomas and T cell/histiocyte-rich large B cell lymphoma) as well as T cell lymphomas such as angioimmunoblastic T cell lymphoma where the large B cells represent an epiphenomenon and may or may not be neoplastic. Several rare variants of these conditions, and the fact that treatment can significantly modify appearances, add to the diagnostic difficulty of these pathological entities. Unlike monomorphic lymphoid infiltrates, the histological pattern of T cell-rich proliferation with large B cells requires close evaluation of the inter-relationship between B cells and T cells, follicular dendritic cells and sometimes other inflammatory cells. Epstein-Barr virus plays a key role in several of these scenarios, and interpreting not only its presence but also its distribution within cellular subgroups is essential to accurate diagnosis and the avoidance of some important diagnostic pitfalls. An understanding of normal immunoarchitecture and lymphoid maturational pathways is also fundamental to resolving these cases, as is a knowledge of their common patterns of spread, which facilitates correlation with clinical and radiological findings.

Enabling digital pathology in the diagnostic setting: navigating through the implementation journey in an academic medical centre.

AIMS: As digital pathology (DP) and whole slide imaging (WSI) technology advance and mature, there is an increasing drive to incorporate DP into the diagnostic environment. However, integration of DP into the diagnostic laboratory is a non-trivial task and filled with unexpected challenges unlike standalone implementations. We share our journey of implementing DP in the diagnostic laboratory setting, highlighting seven key guiding principles that drive the progression through implementation into deployment and beyond. METHODS: The DP implementation with laboratory information system integration was completed in 8 months, including validation of the solution for diagnostic use in accordance with College of American Pathologists guidelines. We also conducted prospective validation via paired delivery of glass slides and WSI to our pathologists postdeployment. RESULTS: Common themes in our guiding principles included emphasis on workflow and being comprehensive in the approach, looking beyond pathologist user champions and expanding into an extended project team involving laboratory technicians, clerical/data room staff and archival staff. Concordance between glass slides and WSI ranged from 93% to 100% among various applications on validation. We also provided equal opportunities for every pathologist throughout the department to be competent and confident with DP through prospective validation, with overall concordance of 96% compared with glass slides, allowing appreciation of the advantages and limitations of WSI, hence enabling the use of DP as a useful diagnostic modality. CONCLUSIONS: Smooth integration of DP into the diagnostic laboratory is possible with careful planning, discipline and a systematic approach adhering to our guiding principles.