Sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection among Kidney Transplant Recipients: A Large Single-Center Experience.

Background: Kidney transplant recipients (KTRs) are a vulnerable immunocompromised population at risk of severe COVID-19 disease and mortality after SARS-CoV-2 infection. We sought to characterize the post-infection sequelae in KTRs at our center. Methods: We studied all adult KTRs (with a functioning allograft) who had their first episode of SARS-CoV-2 infection between 04/2020 and 04/2022. Outcomes of interest included risk factors for hospitalization, all-cause mortality, COVID-19-related mortality, and allograft failure. Results: Of 979 KTRs with SARS-CoV-2 infection, 381 (39%) were hospitalized. In the multivariate analysis, risk factors for hospitalization included advanced age/year (HR: 1.03, 95% CI: 1.02-1.04), male sex (HR: 1.29, 95% CI: 1.04-1.60), non-white race (HR: 1.48, 95% CI: 1.17-1.88), and diabetes as a cause of ESKD (HR: 1.77, 95% CI: 1.41-2.21). SARS-CoV-2 Vaccination was associated with decreased risk of hospitalization (HR: 0.73, 95% CI: 0.59-0.90), all-cause mortality (HR: 0.52, 95% CI: 0.37-0.74), and COVID-19-related mortality (HR: 0.47, 95% CI: 0.31-0.71) in the univariate and multivariate analyses. Risk factors for both all-cause and COVID-19-related mortality in the multivariate analyses included advanced age, hospitalization, and respiratory symptoms for hospital admission. Furthermore, additional risk factors for all-cause mortality in the multivariate analysis included being a non-white recipient and diabetes as a cause of ESKD, with being a recipient of a living donor as protective. Conclusions: Hospitalization due to COVID-19-associated symptoms is associated with increased mortality. Vaccination is a protective factor against hospitalization and mortality.

Incidence, Risk Factors, and Outcomes of Posttransplant Erythrocytosis Among Simultaneous Pancreas-Kidney Transplant Recipients.

Background: Posttransplant erythrocytosis (PTE) is a well-known complication of kidney transplantation. However, the risk and outcomes of PTE among simultaneous pancreas-kidney transplant (SPKT) recipients are poorly described. Methods: We analyzed all SPKT recipients at our center between 1998 and 2021. PTE was defined as at least 2 consecutive hematocrit levels of >51% within the first 2 y of transplant. Controls were selected at a ratio of 3:1 at the time of PTE occurrence using event density sampling. Risk factors for PTE and post-PTE graft survival were identified. Results: Of 887 SPKT recipients, 108 (12%) developed PTE at a median of 273 d (interquartile range, 160-393) after transplantation. The incidence rate of PTE was 7.5 per 100 person-years. Multivariate analysis found pretransplant dialysis (hazard ratio [HR]: 3.15; 95% confidence interval [CI], 1.67-5.92; P < 0.001), non-White donor (HR: 2.14; 95% CI, 1.25-3.66; P = 0.01), female donor (HR: 1.50; 95% CI, 1.0-2.26; P = 0.05), and male recipient (HR: 2.33; 95% CI, 1.43-3.70; P = 0.001) to be associated with increased risk. The 108 cases of PTE were compared with 324 controls. PTE was not associated with subsequent pancreas graft failure (HR: 1.36; 95% CI, 0.51-3.68; P = 0.53) or kidney graft failure (HR: 1.16; 95% CI, 0.40-3.42; P = 0.78). Conclusions: PTE is a common complication among SPKT recipients, even in the modern era of immunosuppression. PTE among SPKT recipients was not associated with adverse graft outcomes, likely due to appropriate management.

Spatially Exploring RNA Biology in Archival Formalin-Fixed Paraffin-Embedded Tissues.

Spatial transcriptomics has emerged as a powerful tool for dissecting spatial cellular heterogeneity but as of today is largely limited to gene expression analysis. Yet, the life of RNA molecules is multifaceted and dynamic, requiring spatial profiling of different RNA species throughout the life cycle to delve into the intricate RNA biology in complex tissues. Human disease-relevant tissues are commonly preserved as formalin-fixed and paraffin-embedded (FFPE) blocks, representing an important resource for human tissue specimens. The capability to spatially explore RNA biology in FFPE tissues holds transformative potential for human biology research and clinical histopathology. Here, we present Patho-DBiT combining in situ polyadenylation and deterministic barcoding for spatial full coverage transcriptome sequencing, tailored for probing the diverse landscape of RNA species even in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for five years. Furthermore, genome-wide single nucleotide RNA variants can be captured to distinguish different malignant clones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA-mRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis trajectory. High resolution Patho-DBiT at the cellular level reveals a spatial neighborhood and traces the spatiotemporal kinetics driving tumor progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to study human tissue biology and aid in clinical pathology evaluation.

Liquid biopsy-based circulating tumour (ct)DNA analysis of a spectrum of myeloid and lymphoid malignancies yields clinically actionable results.

AIMS: Liquid biopsy (LBx)-based next-generation sequencing (NGS) of circulating tumour DNA (ctDNA) can facilitate molecular profiling of haematopoietic neoplasms (HNs), particularly when tissue-based NGS is infeasible. METHODS AND RESULTS: We studied HN LBx samples tested with FoundationOne Liquid CDx, FoundationOne Liquid, or FoundationACT between July 2016 and March 2022. We identified 271 samples: 89 non-Hodgkin lymphoma (NHL), 43 plasma-cell neoplasm (PCN), 41 histiocytoses, 27 myelodysplastic syndrome (MDS), 25 diffuse large B-cell lymphoma (DLBCL), 22 myeloproliferative neoplasm (MPN), 14 Hodgkin lymphoma (HL), and 10 acute myeloid leukaemia (AML). Among 73.4% with detectable pathogenic alterations, median maximum somatic allele frequency (MSAF) was 16.6%, with AML (36.2%), MDS (19.7%), and MPN (44.5%) having higher MSAFs than DLBCL (3.9%), NHL (8.4%), HL (1.5%), PCN (2.8%), and histiocytoses (1.8%) (P = 0.001). LBx detected characteristic alterations across HNs, including in TP53, KRAS, MYD88, and BTK in NHLs; TP53, KRAS, NRAS, and BRAF in PCNs; IGH in DLBCL; TP53, ATM, and PDCD1LG2 in HL; BRAF and MAP2K1 in histiocytoses; TP53, SF3B1, DNMT3A, TET2, and ASXL1 in MDS; JAK2 in MPNs; and FLT3, IDH2, and NPM1 in AML. Among 24 samples, the positive percent agreement by LBx was 75.7% for variants present in paired buffy coat, marrow, or tissues. Also, 75.0% of pairs exhibited alterations only present on LBx. These were predominantly subclonal (clonal fraction of 3.8%), reflecting the analytical sensitivity of LBx. CONCLUSION: These data demonstrate that LBx can detect relevant genomic alterations across HNs, including at low clonal fractions, suggesting a potential clinical utility for identifying residual or emerging therapy-resistant clones that may be undetectable in site-specific tissue biopsies.

PD-1H/VISTA mediates immune evasion in acute myeloid leukemia.

Acute myeloid leukemia (AML) presents a pressing medical need in that it is largely resistant to standard chemotherapy as well as modern therapeutics, such as targeted therapy and immunotherapy, including anti-programmed cell death protein (anti-PD) therapy. We demonstrate that programmed death-1 homolog (PD-1H), an immune coinhibitory molecule, is highly expressed in blasts from the bone marrow of AML patients, while normal myeloid cell subsets and T cells express PD-1H. In studies employing syngeneic and humanized AML mouse models, overexpression of PD-1H promoted the growth of AML cells, mainly by evading T cell-mediated immune responses. Importantly, ablation of AML cell-surface PD-1H by antibody blockade or genetic knockout significantly inhibited AML progression by promoting T cell activity. In addition, the genetic deletion of PD-1H from host normal myeloid cells inhibited AML progression, and the combination of PD-1H blockade with anti-PD therapy conferred a synergistic antileukemia effect. Our findings provide the basis for PD-1H as a potential therapeutic target for treating human AML.

Specific Polo-Like Kinase 1 Expression in Nodular Lymphocyte-Predominant Hodgkin Lymphoma Suggests an Intact Immune Surveillance Program.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n = 76). In contrast, <5% of classic Hodgkin lymphoma cases (n = 70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.

A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells.

Impaired redox metabolism is a key contributor to the etiology of many diseases, including primary mitochondrial disorders, cancer, neurodegeneration and aging. However, mechanistic studies of redox imbalance remain challenging due to limited strategies that can perturb redox metabolism in various cellular or organismal backgrounds. Most studies involving impaired redox metabolism have focused on oxidative stress; consequently, less is known about the settings where there is an overabundance of NADH reducing equivalents, termed reductive stress. Here we introduce a soluble transhydrogenase from Escherichia coli (EcSTH) as a novel genetically encoded tool to promote reductive stress in living cells. When expressed in mammalian cells, EcSTH, and a mitochondrially targeted version (mitoEcSTH), robustly elevated the NADH/NAD+ ratio in a compartment-specific manner. Using this tool, we determined that metabolic and transcriptomic signatures of the NADH reductive stress are cellular background specific. Collectively, our novel genetically encoded tool represents an orthogonal strategy to promote reductive stress.

Classification, risk stratification and response assessment in myelodysplastic syndromes/neoplasms (MDS): A state-of-the-art report on behalf of the International Consortium for MDS (icMDS).

The guidelines for classification, prognostication, and response assessment of myelodysplastic syndromes/neoplasms (MDS) have all recently been updated. In this report on behalf of the International Consortium for MDS (icMDS) we summarize these developments. We first critically examine the updated World Health Organization (WHO) classification and the International Consensus Classification (ICC) of MDS. We then compare traditional and molecularly based risk MDS risk assessment tools. Lastly, we discuss limitations of criteria in measuring therapeutic benefit and highlight how the International Working Group (IWG) 2018 and 2023 response criteria addressed these deficiencies and are endorsed by the icMDS. We also address the importance of patient centered care by discussing the value of quality-of-life assessment. We hope that the reader of this review will have a better understanding of how to classify MDS, predict clinical outcomes and evaluate therapeutic outcomes.

IRF8 in Conjunction With CD123 and CD20 to Distinguish Lupus Erythematosus Panniculitis From Subcutaneous Panniculitis-like T-Cell Lymphoma.

Distinguishing lupus erythematosus panniculitis (LEP) from subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a diagnostic challenge with important clinical implications. Immunohistochemical expression of interferon regulatory factor 8 (IRF8) has been shown to highlight cells with plasmacytoid dendritic cell differentiation. Considering that the presence of plasmacytoid dendritic cells highlighted by CD123 immunolabeling is a well-described feature that supports LEP over SPTCL, we hypothesized that IRF8 immunohistochemistry can be used as a diagnostic test to improve accuracy in differentiating LEP from SPTCL. In this study, we assessed the expression of IRF8, CD123, and CD20 in 35 cutaneous biopsies from 31 distinct patients, which included 22 cases of LEP and 13 cases of SPTCL. We found that clusters of IRF8-positive cells within the dermis, and away from subcutaneous fat, could discriminate LEP from SPTCL ( P =0.005). Similarly, CD123-positive clusters in any location were observed in LEP but absent in all cases of SPTCL. In addition, we found that dermal CD20-predominant lymphoid aggregates could help discriminate LEP from SPTCL ( P =0.022). As individual assays, IRF8, CD123, and CD20 were highly specific (100%, 100%, and 92%, respectively) though poorly sensitive (45%, 29%, and 50%, respectively). However, a panel combining IRF8, CD123, and CD20, with at least 1 positive marker was more accurate than any individual marker by receiver operating characteristic curve analysis. Our study provides a rationale for potentially including IRF8 as part of an immunohistochemical panel composed of other currently available markers used to differentiate LEP from SPTCL.

High-grade B-cell lymphoma, not otherwise specified: a multi-institutional retrospective study.

In this multi-institutional retrospective study, we examined the characteristics and outcomes of 160 patients with high-grade B-cell lymphoma, not otherwise specified (HGBL-NOS)-a rare category defined by high-grade morphologic features and lack of MYC rearrangements with BCL2 and/or BCL6 rearrangements ("double hit"). Our results show that HGBL-NOS tumors are heterogeneous: 83% of patients had a germinal center B-cell immunophenotype, 37% a dual-expressor immunophenotype (MYC and BCL2 expression), 28% MYC rearrangement, 13% BCL2 rearrangement, and 11% BCL6 rearrangement. Most patients presented with stage IV disease, a high serum lactate dehydrogenase, and other high-risk clinical factors. Most frequent first-line regimens included dose-adjusted cyclophosphamide, doxorubicin, vincristine, and etoposide, with rituximab and prednisone (DA-EPOCH-R; 43%); rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP; 33%); or other intensive chemotherapy programs. We found no significant differences in the rates of complete response (CR), progression-free survival (PFS), or overall survival (OS) between these chemotherapy regimens. CR was attained by 69% of patients. PFS at 2 years was 55.2% and OS was 68.1%. In a multivariable model, the main prognostic factors for PFS and OS were poor performance status, lactate dehydrogenase >3 × upper limit of normal, and a dual-expressor immunophenotype. Age >60 years or presence of MYC rearrangement were not prognostic, but patients with TP53 alterations had a dismal PFS. Presence of MYC rearrangement was not predictive of better PFS in patients treated with DA-EPOCH-R vs R-CHOP. Improvements in the diagnostic criteria and therapeutic approaches beyond dose-intense chemotherapy are needed to overcome the unfavorable prognosis of patients with HGBL-NOS.

Updates in Classification of Myelodysplastic Syndrome.

ABSTRACT: Myelodysplastic syndrome includes a broad range of myeloid neoplasms characterized by cytopenia and morphologic dysplasia. Recently, 2 new classification systems emerged to further define how these diseases are diagnosed and risk stratified. This review compares these models, provides detailed approaches, and reveals practical ways to move forward in clinical practice of myelodysplastic syndrome diagnosis.

IRF8 may be a useful marker for blastic plasmacytoid dendritic cell neoplasm, especially with weak CD123 expression.

We highlight the utility of interferon regulatory factor 8 (IRF8), a novel marker of monocytic and dendritic cell lineages, in the diagnosis of a case of blastic plasmacytoid dendritic cell neoplasm (BPDCN) presenting initially in the skin. A 60-year-old male with a previous history of myelodysplastic syndrome presented with cutaneous nodules on chest and scalp. A punch biopsy specimen of a skin nodule showed a diffuse dermal infiltrate of atypical mononuclear cells. The neoplastic cells expressed CD4, CD56, CD43, and TdT but showed minimal reaction for TCL-1 and CD123, and were negative for CD34, CD117, and MPO, confounding the diagnosis. IRF8 performed in retrospect was strongly positive. A new punch biopsy specimen of a chest nodule showed the blastoid tumor cells were positive for TCL-1, CD4, and CD56, but dim CD123. Subsequent bone marrow involvement showed blastoid tumor cells with intense positivity for CD123, CD4, and CD56, which was supportive of the BPDCN diagnosis. BPDCN cases with weak or variable CD123 and TCL-1 expression represent a potential diagnostic pitfall. In a recent study, 15 cases of BPDCN showed uniformly strong staining for IRF8, while CD123 was dim or negative in 4 of these 15 cases. We suggest IRF8 may be a useful marker for BPDCN, especially in cases with weak or variable expression of CD123 and TCL1.

Current landscape of translational and clinical research in myelodysplastic syndromes/neoplasms (MDS): Proceedings from the 1st International Workshop on MDS (iwMDS) Of the International Consortium for MDS (icMDS).

Biological events that contribute to the pathogenesis of myelodysplastic syndromes/neoplasms (MDS) are becoming increasingly characterized and are being translated into rationally designed therapeutic strategies. Herein, we provide updates from the first International Workshop on MDS (iwMDS) of the International Consortium for MDS (icMDS) detailing recent advances in understanding the genetic landscape of MDS, including germline predisposition, epigenetic and immune dysregulation, the complexities of clonal hematopoiesis progression to MDS, as well as novel animal models of the disease. Connected to this progress is the development of novel therapies targeting specific molecular alterations, the innate immune system, and immune checkpoint inhibitors. While some of these agents have entered clinical trials (e.g., splicing modulators, IRAK1/4 inhibitors, anti-CD47 and anti-TIM3 antibodies, and cellular therapies), none have been approved for MDS. Additional preclinical and clinical work is needed to develop a truly individualized approach to the care of MDS patients.

Emerging Immunohistochemical Biomarkers for Myeloid Neoplasms.

CONTEXT.­: Pathologists can greatly improve patient care and advance the understanding of disease progression by adeptly employing relevant biomarkers when diagnosing myeloid neoplasms. Although the molecular era has ushered in countless molecular biomarkers in this field, the necessary techniques can be expensive and time-consuming. Novel immunohistochemical biomarkers can help to quickly and inexpensively render the correct diagnosis and predict response to targeted therapies. Hence, it is critical to continue studying and using new and promising immunohistochemical tools for myeloid neoplasms in our current era. OBJECTIVE.­: To review the emerging biomarkers in myeloid neoplasms that can be identified by immunohistochemistry and to discuss their utility, staining patterns, and pitfalls. DATA SOURCES.­: We conducted a scientific literature search of articles related to either a novel immunohistochemical marker or a new utility of an already known marker to assess myeloid neoplasms in PubMed from 2016 to September 30, 2021. We curated relevant contributing studies from the references and subsequent citations of the original articles. CONCLUSIONS.­: Immunohistochemistry is a powerful tool in analyzing biomarkers that play a significant role in the management of patients with myeloid neoplasms. We reviewed 5 immunohistochemical markers, namely, IDH1R132H, ERG, IRF8, GATA1, and NPM1. These markers, depending on the clinical scenario, can be diagnostic, predictive, and also prognostic. Immunohistochemistry also empowers us to evaluate these markers in archival samples, including pretreatment and posttreatment biopsies.

Best Practices in CD30 Immunohistochemistry Testing, Interpretation, and Reporting: An Expert Panel Consensus.

CONTEXT.­: Although CD30 testing is an established tool in the diagnostic workup of lymphomas, it is also emerging as a predictive biomarker that informs treatment. The current definition of CD30 positivity by immunohistochemistry is descriptive and based on reactivity in lymphomas that are defined by their universal strong expression of CD30, rather than any established threshold. Challenges include inconsistencies with preanalytic variables, tissue processing, pathologist readout, and with the pathologist and oncologist interpretation of reported results. OBJECTIVE.­: To develop and propose general best practice recommendations for reporting CD30 expression by immunohistochemistry in lymphoma biopsies to harmonize practices across institutions and facilitate assessment of its significance in clinical decision-making. DESIGN.­: Following literature review and group discussion, the panel of 14 academic hematopathologists and 2 clinical/academic hematologists/oncologists divided into 3 working groups. Each working group was tasked with assessing CD30 testing by immunohistochemistry, CD30 expression readout, or CD30 expression interpretation. RESULTS.­: Panel recommendations were reviewed and discussed. An online survey was conducted to confirm the consensus recommendations. CONCLUSIONS.­: CD30 immunohistochemistry is required for all patients in whom classic Hodgkin lymphoma and any lymphoma within the spectrum of peripheral T-cell lymphoma are differential diagnostic considerations. The panel reinforced and summarized that immunohistochemistry is the preferred methodology and any degree of CD30 expression should be reported. For diagnostic purposes, the interpretation of CD30 expression should follow published guidelines. To inform therapeutic decisions, report estimated percent positive expression in tumor cells (or total cells where applicable) and record descriptively if nontumor cells are positive.

Acute Epstein-Barr virus infection resembling cutaneous T-cell lymphoma.

Primary, acute Epstein-Barr virus (EBV) infection is associated with a variety of cutaneous eruptions, including the viral exanthem of infectious mononucleosis and erythema multiforme. Latent, chronic EBV infection can rarely result in development of lymphoproliferative disorders with cutaneous manifestations; however, these disorders do not arise from primary infection. In this report, we present a case of primary, acute EBV infection presenting with histopathologic features closely mimicking aggressive cytotoxic cutaneous T-cell lymphoma.