Publications by authors named "Stefania Pittaluga"

269 Publications

Neutrophil and Eosinophil Extracellular Traps in Hodgkin Lymphoma.

Hemasphere 2021 Sep 1;5(9):e633. Epub 2021 Sep 1.

Hematopathology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, USA.

Classic Hodgkin lymphoma (cHL), nodular sclerosis (NS) subtype, is characterized by the presence of Hodgkin/Reed-Sternberg (HRS) cells in an inflammatory background containing neutrophils and/or eosinophils. Both types of granulocytes release extracellular traps (ETs), web-like DNA structures decorated with histones, enzymes, and coagulation factors that promote inflammation, thrombosis, and tumor growth. We investigated whether ETs from neutrophils (NETs) or eosinophils (EETs) are detected in cHL, and evaluated their association with fibrosis. We also studied expression of protease-activated receptor-2 (PAR-2) and phospho-extracellular signal-related kinase (p-ERK), potential targets/effectors of ETs-associated elastase, in HRS cells. Expression of tissue factor (TF) was evaluated, given the procoagulant properties of ETs. We analyzed 32 HL cases, subclassified as 12 NS, 5 mixed-cellularity, 5 lymphocyte-rich, 1 lymphocyte-depleted, 4 nodular lymphocyte-predominant HL (NLPHL), and 5 reactive nodes. Notably, a majority of NS cHL cases exhibited NET formation by immunohistochemistry for citrullinated histones, with 1 case revealing abundant EETs. All other cHL subtypes as well as NLPHL were negative. Immunofluorescence microscopy confirmed NETs with filamentous/delobulated morphology. Moreover, ETs formation correlates with concurrent fibrosis ( = 0.7999; 95% CI, 0.6192-0.9002; ≤ 0.0001). Results also showed that HRS cells in NS cHL expressed PAR-2 with nuclear p-ERK staining, indicating a neoplastic or inflammatory phenotype. Remarkably, TF was consistently detected in the endothelium of NS cHL cases compared with other subtypes, in keeping with a procoagulant status. A picture emerges whereby the release of ETs and resultant immunothrombosis contribute to the inflammatory tumor microenvironment of NS cHL. This is the first description of NETs in cHL.
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http://dx.doi.org/10.1097/HS9.0000000000000633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410234PMC
September 2021

Next-Generation Sequencing-Based Monitoring of Circulating Tumor DNA Reveals Clonotypic Heterogeneity in Untreated PTCL.

Blood Adv 2021 Aug 25. Epub 2021 Aug 25.

National Cancer Institute, NIH, Bethesda, Maryland, United States.

Peripheral T-cell lymphomas (PTCL) have marked biologic and clinical heterogeneity, which confounds treatment decisions. Advances in circulating tumor DNA (ctDNA) assays employing next generation sequencing (NGS) has improved the detection of molecular relapse and driver mutations in diffuse large B-cell lymphoma, and highlight the potential utility of ctDNA across lymphomas. We investigated NGS-based monitoring of T-cell receptor (TCR) sequences in PTCL patients undergoing frontline treatment (NCT00001337). Of 45 patients, 34 (76%) had tumor-specific clonotypes of the TCR β or ɣ genes identified, which included 18 (86%) from baseline tissue and 16 (67%) from baseline serum. Twenty-five (74%) patients had both TCRβ and TCRɣ clonotypes, 23 (68%) patients had more than one TCRɣ clonotype, and 4 (9%) had multiple TCRβ or TCRɣ clonotypes, demonstrating significant intra-patient clonotypic heterogeneity. Among 24 patients with available serial serum samples during treatment, 9 (38%) cleared ctDNA after 2 cycles of therapy, and 11 (46%) patients had detectable ctDNA at the end of treatment. Patients with detectable ctDNA after therapy showed a trend towards worse survival. Notably, two patients with persistently detectable ctDNA after therapy remain in remission with 10-years of follow-up. Clonotypic heterogeneity in tumors and persistence despite long-term remission suggests variability in oncological potential.
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http://dx.doi.org/10.1182/bloodadvances.2020003679DOI Listing
August 2021

IFNγR1 deficiency presenting with visceral leishmaniasis and Mycobacterium Avium infections mimicking HLH.

Pediatr Allergy Immunol 2021 Aug 18. Epub 2021 Aug 18.

Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

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http://dx.doi.org/10.1111/pai.13653DOI Listing
August 2021

Short-course IL-15 given as a continuous infusion led to a massive expansion of effective NK cells: implications for combination therapy with antitumor antibodies.

J Immunother Cancer 2021 Apr;9(4)

Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.

Background: Full application of cytokines as oncoimmunotherapeutics requires identification of optimal regimens. Our initial effort with intravenous bolus recombinant human interleukin-15 (rhIL-15) was limited by postinfusional reactions. Subcutaneous injection and continuous intravenous infusion for 10 days (CIV-10) provided rhIL-15 with less toxicity with CIV-10 giving the best increases in CD8 lymphocytes and natural killer (NK) cells. To ease rhIL-15 administration, we shortened time of infusion. Treatment with rhIL-15 at a dose of 3-5 µg/kg as a 5-day continuous intravenous infusion (CIV-5) had no dose-limiting toxicities while effector cell stimulation was comparable to the CIV-10 regimen.

Methods: Eleven patients with metastatic cancers were treated with rhIL-15 CIV-5, 3 µg (n=4), 4 µg (n=3), and 5 µg/kg/day (n=4) in a phase I dose-escalation study (April 6, 2012).

Results: Impressive expansions of NK cells were seen at all dose levels (mean 34-fold), including CD56 NK cells (mean 144-fold for 4 µg/kg), as well as an increase in CD8 T cells (mean 3.38-fold). At 5 µg/kg/day, there were no dose-limiting toxicities but pulmonary capillary leak and slower patient recovery. This led to our choice of the 4 µg/kg as CIV-5 dose for further testing. Cytolytic capacity of CD56 and CD56 NK cells was increased by interleukin-15 assayed by antibody-dependent cellular cytotoxicity (ADCC), natural cytotoxicity and natural killer group 2D-mediated cytotoxicity. The best response was stable disease.

Conclusions: IL-15 administered as CIV-5 substantially expanded NK cells with increased cytotoxic functions. Tumor-targeting monoclonal antibodies dependent on ADCC as their mechanism of action including alemtuzumab, obinutuzumab, avelumab, and mogamulizumab could benefit from those NK cell expansions and provide a promising therapeutic strategy.

Trial Registration Numbers: NCT01572493, NCT03759184, NCT03905135, NCT04185220 and NCT02689453.
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http://dx.doi.org/10.1136/jitc-2020-002193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061813PMC
April 2021

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation.

Sci Immunol 2021 04;6(58)

Department of Biochemistry, Purdue University, West Lafayette, IN, USA.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.
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http://dx.doi.org/10.1126/sciimmunol.abg0833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139422PMC
April 2021

SARS-CoV-2 infection of the oral cavity and saliva.

Nat Med 2021 05 25;27(5):892-903. Epub 2021 Mar 25.

Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.
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http://dx.doi.org/10.1038/s41591-021-01296-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240394PMC
May 2021

Characteristics and outcomes of KSHV-associated multicentric Castleman disease with or without other KSHV diseases.

Blood Adv 2021 03;5(6):1660-1670

HIV & AIDS Malignancy Branch and.

Kaposi sarcoma (KS)-associated herpesvirus (KSHV)-associated multicentric Castleman disease (MCD) is a relapsing and remitting systemic lymphoproliferative disorder characterized by severe inflammatory symptoms most common among people living with HIV (PLWH). Patients with KSHV-MCD may present with concurrent KSHV-associated diseases, such as KS and/or primary effusion lymphoma (PEL). We evaluated clinical and immunologic characteristics, the effects of concurrent KSHV malignancies, and treatments from the largest prospective natural history study of participants with KSHV-MCD within the United States. Treatment options administered at investigator discretion included high-dose zidovudine with valganciclovir (AZT/VGC), rituximab, or rituximab with liposomal doxorubicin (R-Dox) during KSHV-MCD flares. Survival analyses and prognostic factors were explored for all participants. Sixty-two participants with HIV were enrolled, including 20 with KSHV-MCD alone, 34 with KSHV-MCD and KS, 1 with KSHV-MCD and PEL, and 7 with all KSHV-associated diseases. Forty-four percent of KSHV-MCD diagnoses were made at our institution. Forty-four participants received rituximab-based therapies, 20 of whom had maintenance AZT/VGC or interferon. Participants receiving R-Dox and then maintenance AZT/VGC had the highest 5-year progression-free survival (89%). Cytokine profiles during KSHV-MCD flares did not differ by the presence of concurrent KSHV-associated diseases. The 10-year survival was 71% (95% confidence interval [CI], 56% to 82%) for all participants. A concurrent diagnosis of PEL negatively impacted survival (PEL hazard ratio, 5.4; 95% CI, 1.8 to 16.8). KSHV-MCD is an underdiagnosed condition among PLWH, including those with KS. KSHV-MCD has an excellent prognosis with appropriate treatment. Physicians should be alert for patients with multiple KSHV diseases, which impact optimal treatment and survival outcomes. This study was registered at www.clinicaltrials.gov as #NCT00099073.
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http://dx.doi.org/10.1182/bloodadvances.2020004058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993110PMC
March 2021

Siglec-6 is a target for chimeric antigen receptor T-cell treatment of chronic lymphocytic leukemia.

Leukemia 2021 09 25;35(9):2581-2591. Epub 2021 Feb 25.

Experimental Transplantation and Immunotherapy Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, 20892, USA.

The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.
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http://dx.doi.org/10.1038/s41375-021-01188-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384967PMC
September 2021

Lost in Translation: Lack of CD4 Expression due to a Novel Genetic Defect.

J Infect Dis 2021 Feb;223(4):645-654

National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

CD4 expression identifies a subset of mature T cells primarily assisting the germinal center reaction and contributing to CD8+ T-cell and B-cell activation, functions, and longevity. Herein, we present a family in which a novel variant disrupting the translation-initiation codon of the CD4 gene resulted in complete loss of membrane and plasma soluble CD4 in peripheral blood, lymph node, bone marrow, skin, and ileum of a homozygous proband. This inherited CD4 knockout disease illustrates the clinical and immunological features of a complete deficiency of any functional component of CD4 and its similarities and differences with other clinical models of primary or acquired loss of CD4+ T cells. The first inherited loss of any functional component of CD4, including soluble CD4, is clinically distinct from any other congenital or acquired CD4 T-cell defect and characterized by compensatory changes in T-cell subsets and functional impairment of B cells, monocytes, and natural killer cells.
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http://dx.doi.org/10.1093/infdis/jiab025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904290PMC
February 2021

Aberrant type 1 immunity drives susceptibility to mucosal fungal infections.

Science 2021 01;371(6526)

Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA.

Human monogenic disorders have revealed the critical contribution of type 17 responses in mucosal fungal surveillance. We unexpectedly found that in certain settings, enhanced type 1 immunity rather than defective type 17 responses can promote mucosal fungal infection susceptibility. Notably, in mice and humans with deficiency, an autoimmune disease characterized by selective susceptibility to mucosal but not systemic fungal infection, mucosal type 17 responses are intact while type 1 responses are exacerbated. These responses promote aberrant interferon-γ (IFN-γ)- and signal transducer and activator of transcription 1 (STAT1)-dependent epithelial barrier defects as well as mucosal fungal infection susceptibility. Concordantly, genetic and pharmacologic inhibition of IFN-γ or Janus kinase (JAK)-STAT signaling ameliorates mucosal fungal disease. Thus, we identify aberrant T cell-dependent, type 1 mucosal inflammation as a critical tissue-specific pathogenic mechanism that promotes mucosal fungal infection susceptibility in mice and humans.
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http://dx.doi.org/10.1126/science.aay5731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8326743PMC
January 2021

Genomic Rearrangement as a Secondary Event in High Grade B-Cell Lymphoma.

Hemasphere 2021 Jan 9;5(1):e505. Epub 2020 Dec 9.

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

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http://dx.doi.org/10.1097/HS9.0000000000000505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732336PMC
January 2021

Compromised counterselection by FAS creates an aggressive subtype of germinal center lymphoma.

J Exp Med 2021 03;218(3)

Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.

Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B cell lymphoma (DLBCL). Although GC-derived DLBCL has better overall outcomes than other DLBCL types, some cases are refractory, and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes cell death in the light zone, likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. FAS alterations occurred most commonly in GC-derived DLBCL, were associated with inferior outcomes and an enrichment of Tfh cells, and co-occurred with deficiency in HVEM and PD-L1 that regulate the Tfh-B cell interaction. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in GC-derived lymphoma.
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http://dx.doi.org/10.1084/jem.20201173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694576PMC
March 2021

Metastatic and recurrent adrenocortical cancer is not defined by its genomic landscape.

BMC Med Genomics 2020 11 4;13(1):165. Epub 2020 Nov 4.

Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, 20892, USA.

Background: Adrenocortical carcinoma (ACC) is a rare, often-aggressive neoplasm of the adrenal cortex, with a 14-17 month median overall survival. We asked whether tumors from patients with advanced or metastatic ACC would offer clues as to putative genes that might have critical roles in disease progression or in more aggressive disease biology.

Methods: We conducted comprehensive genomic and expression analyses of ACCs from 43 patients, 30 female, and 42 from metastatic sites, including deep sequencing, copy number analysis, mRNA expression and microRNA arrays.

Results: Copy number gains and losses were similar to that previously reported for ACC. We identified a median mutation rate of 3.38 per megabase (Mb). The mutational signature was characterized by a predominance of C > T, C > A and T > C transitions. Only cancer genes TP53 (26%) and beta-catenin (CTNNB1, 14%) were mutated in more than 10% of samples. The TCGA-identified putative cancer genes MEN1 and PRKAR1A were found in low frequency-4.7 and 2.3%, respectively. The majority of the mutations were in genes not implicated in the etiology or maintenance of cancer. Specifically, amongst the 38 genes that were mutated in more than 9% of samples, only four were represented in Tier 1 of the 576 COSMIC Cancer Gene Census (CCGC). Thus, 82% of genes found to have mutations likely have no role in the etiology or biology of ACC; while the role of the other 18%, if any, remains to be proven. Finally, the transcript length for the 38 most frequently mutated genes in ACC is statistically longer than the average of all coding genes, raising the question of whether transcript length in part determined mutation probability.

Conclusions: We conclude that the mutational and expression profiles of advanced and metastatic tumors are very similar to those from newly diagnosed patients-with very little in the way of genomic aberration to explain differences in biology. With relatively low mutation rates, few major oncogenic drivers, and loss of function mutations in several epigenetic regulators, an epigenetic basis for ACC may be postulated and serve as the basis for future studies.
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http://dx.doi.org/10.1186/s12920-020-00809-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640690PMC
November 2020

Integrated Single-Cell Atlases Reveal an Oral SARS-CoV-2 Infection and Transmission Axis.

medRxiv 2020 Oct 27. Epub 2020 Oct 27.

Despite signs of infection, the involvement of the oral cavity in COVID-19 is poorly understood. To address this, single-cell RNA sequencing data-sets were integrated from human minor salivary glands and gingiva to identify 11 epithelial, 7 mesenchymal, and 15 immune cell clusters. Analysis of SARS-CoV-2 viral entry factor expression showed enrichment in epithelia including the ducts and acini of the salivary glands and the suprabasal cells of the mucosae. COVID-19 autopsy tissues confirmed in vivo SARS-CoV-2 infection in the salivary glands and mucosa. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting expression and SARS-CoV-2 RNA. Matched nasopharyngeal and saliva samples found distinct viral shedding dynamics and viral burden in saliva correlated with COVID-19 symptoms including taste loss. Upon recovery, this cohort exhibited salivary antibodies against SARS-CoV-2 proteins. Collectively, the oral cavity represents a robust site for COVID-19 infection and implicates saliva in viral transmission.
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http://dx.doi.org/10.1101/2020.10.26.20219089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605572PMC
October 2020

Haploinsufficiency of immune checkpoint receptor CTLA4 induces a distinct neuroinflammatory disorder.

J Clin Invest 2020 10;130(10):5551-5561

Immunopathogenesis Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease (NIAID), NIH, Bethesda, Maryland, USA.

BACKGROUNDCytotoxic T lymphocyte antigen 4 (CTLA4) is essential for immune homeostasis. Genetic mutations causing haploinsufficiency (CTLA4h) lead to a phenotypically heterogenous, immune-mediated disease that can include neuroinflammation. The neurological manifestations of CTLA4h are poorly characterized.METHODSWe performed an observational natural history study of 50 patients with CTLA4h who were followed at the NIH. We analyzed clinical, radiological, immunological, and histopathological data.RESULTSEvidence for neuroinflammation was observed in 32% (n = 16 of 50) of patients in this cohort by magnetic resonance imaging (MRI) and/or by cerebrospinal fluid analysis. Clinical symptoms were commonly absent or mild in severity, with headaches as the leading complaint (n = 13 of 16). The most striking findings were relapsing, large, contrast-enhancing focal lesions in the brain and spinal cord observed on MRI. We detected inflammation in the cerebrospinal fluid and leptomeninges before the parenchyma. Brain biopsies of inflammatory lesions from 10 patients showed perivascular and intraparenchymal mixed cellular infiltrates with little accompanying demyelination or neuronal injury.CONCLUSIONSNeuroinflammation due to CTLA4h is mediated primarily by an infiltrative process with a distinct and striking dissociation between clinical symptoms and radiological findings in the majority of patients.FUNDINGNIAID, NIH, Division of Intramural Research, NINDS, NIH, Division of Intramural Research, and the National Multiple Sclerosis Society-American Brain Foundation.TRIAL REGISTRATIONClinicalTrials.gov NCT00001355.
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http://dx.doi.org/10.1172/JCI135947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524497PMC
October 2020

The mutational landscape of histiocytic sarcoma associated with lymphoid malignancy.

Mod Pathol 2021 02 14;34(2):336-347. Epub 2020 Sep 14.

Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Histiocytic sarcoma and tumors with dendritic cell differentiation (HDT) are uncommon neoplasms often with an aggressive clinical course that may occur in association with another hematologic malignancy or mediastinal germ cell tumor (secondary HDT, sHDT). Previous studies have shown mutations in the RAS/MAPK pathway in HDT and have demonstrated a clonal relationship between HDT and associated lymphoid malignancies through common translocations or identical immunoglobulin or T-cell receptor gene rearrangements. We performed whole exome sequencing on 16 cases of sHDT to further evaluate the spectrum of mutations that occur in sHDT in the context of an associated lymphoid malignancy, including cases associated with follicular lymphoma (FL), chronic lymphocytic leukemia/small lymphocytic lymphoma, B- and T-cell acute lymphoblastic leukemia/lymphoma and peripheral T-cell lymphoma, NOS. In addition, we assessed the clonal relationship between the HDT and the associated lymphoid malignancy in three cases for which matched samples were available. We found mutations in RAS/MAPK pathway genes in 14/16 cases of sHDT associated with diverse mature and precursor B-cell and T-cell neoplasms, involving KRAS (8/16), BRAF (2/16), NRAS (2/16), MAP2K1 (1/16), and NF1 (1/16). In addition, we note that FL-associated sHDT frequently shares a similar mutational profile to the associated malignancy, identifying mutations in CREBBP or KMT2D in all cases and "aberrant" somatic hypermutation in 5/6 cases. Our study confirms the role of the RAS/MAPK pathway in the pathogenesis of sHDT, provides further evidence of a common neoplastic precursor and, in the case of FL, gives additional insight into the stage in lymphomagenesis at which transdifferentiation may occur.
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http://dx.doi.org/10.1038/s41379-020-00673-xDOI Listing
February 2021

Gene Expression Profiling of Mediastinal Gray Zone Lymphoma and Its Relationship to Primary Mediastinal B-cell Lymphoma and Classical Hodgkin Lymphoma.

Blood Cancer Discov 2020 Sep 30;1(2):155-161. Epub 2020 Mar 30.

Lymphoid Malignancies Branch, NCI, Bethesda, Maryland.

Mediastinal gray zone lymphoma (MGZL) has immunopathologic features between classical Hodgkin lymphoma (cHL) and primary mediastinal thymic B-cell lymphoma (PMBL), leading to uncertainty regarding its biological relationship to these entities. We performed gene expression profiling from patients with MGZL (20), cHL (18), and PMBL (17) and show MGZL clusters between cHL and PMBL. Expression signatures reveal germinal B-cell and IFN regulatory factor 4 (IRF4) signatures were relatively low in MGZL and cHL compared with PMBL, indicating downregulation of the B-cell program in MGZL, a hallmark of cHL. T-cell and macrophage signatures were higher in MGZL and cHL compared with PMBL, consistent with infiltrating immune cells, which are found in cHL. The NFκB signature was higher in MGZL than PMBL, and like cHL, MGZL and PMBL express NFκB inducing kinase (NIK), indicating noncanonical signaling. These findings indicate that while MGZL has distinctive clustering, it is biologically closer to cHL.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478163PMC
September 2020

Megakaryocytes and platelet-fibrin thrombi characterize multi-organ thrombosis at autopsy in COVID-19: A case series.

EClinicalMedicine 2020 Jul 25;24:100434. Epub 2020 Jun 25.

New York University Grossman School of Medicine, New York, NY, United States.

Background: There is increasing recognition of a prothrombotic state in COVID-19. Post-mortem examination can provide important mechanistic insights.

Methods: We present a COVID-19 autopsy series including findings in lungs, heart, kidneys, liver, and bone, from a New York academic medical center.

Findings: In seven patients (four female), regardless of anticoagulation status, all autopsies demonstrated platelet-rich thrombi in the pulmonary, hepatic, renal, and cardiac microvasculature. Megakaryocytes were seen in higher than usual numbers in the lungs and heart. Two cases had thrombi in the large pulmonary arteries, where casts conformed to the anatomic location. Thrombi in the IVC were not found, but the deep leg veins were not dissected. Two cases had cardiac venous thrombosis with one case exhibiting septal myocardial infarction associated with intramyocardial venous thrombosis, without atherosclerosis. One case had focal acute lymphocyte-predominant inflammation in the myocardium with no virions found in cardiomyocytes. Otherwise, cardiac histopathological changes were limited to minimal epicardial inflammation ( = 1), early ischemic injury ( = 3), and mural fibrin thrombi ( = 2). Platelet-rich peri‑tubular fibrin microthrombi were a prominent renal feature. Acute tubular necrosis, and red blood cell and granular casts were seen in multiple cases. Significant glomerular pathology was notably absent. Numerous platelet-fibrin microthrombi were identified in hepatic sinusoids. All lungs exhibited diffuse alveolar damage (DAD) with a spectrum of exudative and proliferative phases including hyaline membranes, and pneumocyte hyperplasia, with viral inclusions in epithelial cells and macrophages. Three cases had superimposed acute bronchopneumonia, focally necrotizing.

Interpretation: In this series of seven COVID-19 autopsies, thrombosis was a prominent feature in multiple organs, in some cases despite full anticoagulation and regardless of timing of the disease course, suggesting that thrombosis plays a role very early in the disease process. The finding of megakaryocytes and platelet-rich thrombi in the lungs, heart and kidneys suggests a role in thrombosis.

Funding: None.
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http://dx.doi.org/10.1016/j.eclinm.2020.100434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316051PMC
July 2020

CRISPR/Cas9-mediated introduction of the sodium/iodide symporter gene enables noninvasive in vivo tracking of induced pluripotent stem cell-derived cardiomyocytes.

Stem Cells Transl Med 2020 10 23;9(10):1203-1217. Epub 2020 Jul 23.

Translational Stem Cell Biology Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, Maryland, USA.

Techniques that enable longitudinal tracking of cell fate after myocardial delivery are imperative for optimizing the efficacy of cell-based cardiac therapies. However, these approaches have been underutilized in preclinical models and clinical trials, and there is considerable demand for site-specific strategies achieving long-term expression of reporter genes compatible with safe noninvasive imaging. In this study, the rhesus sodium/iodide symporter (NIS) gene was incorporated into rhesus macaque induced pluripotent stem cells (RhiPSCs) via CRISPR/Cas9. Cardiomyocytes derived from NIS-RhiPSCs (NIS-RhiPSC-CMs) exhibited overall similar morphological and electrophysiological characteristics compared to parental control RhiPSC-CMs at baseline and with exposure to physiological levels of sodium iodide. Mice were injected intramyocardially with 2 million NIS-RhiPSC-CMs immediately following myocardial infarction, and serial positron emission tomography/computed tomography was performed with F-tetrafluoroborate to monitor transplanted cells in vivo. NIS-RhiPSC-CMs could be detected until study conclusion at 8 to 10 weeks postinjection. This NIS-based molecular imaging platform, with optimal safety and sensitivity characteristics, is primed for translation into large-animal preclinical models and clinical trials.
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http://dx.doi.org/10.1002/sctm.20-0019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519772PMC
October 2020

High level MYCN amplification and distinct methylation signature define an aggressive subtype of spinal cord ependymoma.

Acta Neuropathol Commun 2020 07 8;8(1):101. Epub 2020 Jul 8.

Neuro-Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

We report a novel group of clinically aggressive spinal cord ependymomas characterized by Grade III histology, MYCN amplification, an absence of NF2 alterations or other recurrent pathogenic mutations, and a unique methylation classifier profile. Seven cases were found to have MYCN amplification in the course of routine mutational profiling of 552 patients with central nervous system tumors between December 2016 and July of 2019 and an eighth patient was identified from an unrelated set of cases. Methylation array analysis revealed that none of the 8 cases clustered with any of the nine previously described ependymoma methylation subgroups, and 7 of 8 formed their own tight unique cluster. Histologically all cases showed grade III features, and all demonstrated aggressive clinical behavior. These findings are presented in the context of data from three other studies describing similar cases. Therefore, a combined total of 27 MYCN amplified spinal cord ependymoma cases have now been reported in the literature, warranting their consideration as a distinctive subtype of spinal cord ependymoma (SP-EPN-MYCN) with their unique molecular characteristics and aggressive clinical behavior.
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http://dx.doi.org/10.1186/s40478-020-00973-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346356PMC
July 2020

Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies.

Histopathology 2020 Dec 16;77(6):915-925. Epub 2020 Oct 16.

Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Introduction: We describe post-mortem pulmonary histopathologic findings of COVID-19 pneumonia in patients with a spectrum of disease course, from rapid demise to prolonged hospitalisation.

Methods And Results: Histopathologic findings in post-mortem lung tissue from eight patients who died from COVID-19 pneumonia were reviewed. Immunohistochemistry (IHC) and next-generation sequencing (NGS) were performed to detect virus. Diffuse alveolar damage (DAD) was seen in all cases with a spectrum of acute phase and/or organising phase. IHC with monoclonal antibodies against SARS-CoV-2 viral nucleoprotein and spike protein detected virus in areas of acute but not organising DAD, with intracellular viral antigen and RNA expression seen predominantly in patients with duration of illness less than 10 days. Major vascular findings included thrombi in medium- and large-calibre vessels, platelet microthrombi detected by CD61 IHC and fibrin microthrombi.

Conclusions: Presence of SARS-CoV-2 viral RNA by NGS early in the disease course and expression of viral antigen by IHC exclusively in the acute, but not in the organising phase of DAD, suggests that the virus may play a major role in initiating the acute lung injury of DAD, but when DAD progresses to the organising phase the virus may have been cleared from the lung by the patient's immune response. These findings suggest the possibility of a major change during the disease course of COVID-19 pneumonia that may have therapeutic implications. Frequent thrombi and microthrombi may also present potential targets for therapeutic intervention.
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http://dx.doi.org/10.1111/his.14201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361244PMC
December 2020

CREBBP and STAT6 co-mutation and 16p13 and 1p36 loss define the t(14;18)-negative diffuse variant of follicular lymphoma.

Blood Cancer J 2020 06 17;10(6):69. Epub 2020 Jun 17.

Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA.

The diffuse variant of follicular lymphoma (dFL) is a rare variant of FL lacking t(14;18) that was first described in 2009. In this study, we use a comprehensive approach to define unifying pathologic and genetic features through gold-standard pathologic review, FISH, SNP-microarray, and next-generation sequencing of 16 cases of dFL. We found unique morphologic features, including interstitial sclerosis, microfollicle formation, and rounded nuclear cytology, confirmed absence of t(14;18) and recurrent deletion of 1p36, and showed a novel association with deletion/CN-LOH of 16p13 (inclusive of CREBBP, CIITA, and SOCS1). Mutational profiling demonstrated near-uniform mutations in CREBBP and STAT6, with clonal dominance of CREBBP, among other mutations typical of germinal-center B-cell lymphomas. Frequent CREBBP and CIITA codeletion/mutation suggested a mechanism for immune evasion, while subclonal STAT6 activating mutations with concurrent SOCS1 loss suggested a mechanism of BCL-xL/BCL2L1 upregulation in the absence of BCL2 rearrangements. A review of the literature showed significant enrichment for 16p13 and 1p36 loss/CN-LOH, STAT6 mutation, and CREBBP and STAT6 comutation in dFL, as compared with conventional FL. With this comprehensive approach, our study demonstrates confirmatory and novel genetic associations that can aid in the diagnosis and subclassification of this rare type of lymphoma.
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http://dx.doi.org/10.1038/s41408-020-0335-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299932PMC
June 2020

Epstein-Barr Virus-negative Marginal Zone Lymphoma as an Uncommon Form of Monomorphic Posttransplant Lymphoproliferative Disorder.

Am J Surg Pathol 2020 10;44(10):1340-1352

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD.

Monomorphic posttransplant lymphoproliferative disorders have been defined as lymphoid or plasmacytic proliferations that fulfill criteria for one of the B-cell or T/NK-cell neoplasms recognized in immunocompetent hosts in the current WHO Classification. Low-grade B-cell neoplasms have historically been excluded from this category, although rare reports of marginal zone lymphoma (MZL) have been described. We report 9 cases of posttransplant Epstein-Barr virus-negative MZL, all arising in solid organ transplant recipients (4 renal, 3 liver, 1 cardiac, and 1 liver, pancreas, and small bowel). Seven were extranodal MZL of mucosa-associated lymphoid tissue type, all of which had gastrointestinal involvement (4 colon, 1 duodenum, 1 stomach, and 1 oropharynx/base of tongue). Notably, the preferential involvement of intestine distinguishes posttransplant extranodal MZL from sporadic cases. Immunoglobulin light-chain restriction was seen in all cases, with polymerase chain reaction showing a monoclonal pattern in 7 of 8 cases with successful amplification of polymerase chain reaction products. A clonally unrelated recurrence was seen in one case. Next-generation sequencing identified recurrent mutations previously reported in MZL in 3/5 cases. MZL was diagnosed at least 1 year after solid organ transplant (median time to presentation, 84 mo; range, 13 to 108 mo). The median age was 44 (range, 9 to 73 y); the male: female ratio was 5:4. The mean follow-up was 33.4 months, with an indolent clinical course observed. A subset responded to reduction in immunosuppression and anti-CD20 therapy alone. These data support the designation of Epstein-Barr virus-negative MZL as an uncommon form of monomorphic posttransplant lymphoproliferative disorders.
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http://dx.doi.org/10.1097/PAS.0000000000001514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492423PMC
October 2020

Multicenter Study of Risk-Adapted Therapy With Dose-Adjusted EPOCH-R in Adults With Untreated Burkitt Lymphoma.

J Clin Oncol 2020 08 26;38(22):2519-2529. Epub 2020 May 26.

Lymphoid Malignancies Branch, National Cancer Institute, Bethesda, MD.

Purpose: Burkitt lymphoma is an aggressive B-cell lymphoma curable with dose-intensive chemotherapy derived from pediatric leukemia regimens. Treatment is acutely toxic with late sequelae. We hypothesized that dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and rituximab (DA-EPOCH-R) may obviate the need for highly dose-intensive chemotherapy in adults with Burkitt lymphoma.

Methods: We conducted a multicenter risk-adapted study of DA-EPOCH-R in untreated adult Burkitt lymphoma. Low-risk patients received three cycles without CNS prophylaxis, and high-risk patients received six cycles with intrathecal CNS prophylaxis or extended intrathecal treatment if leptomeninges were involved. The primary endpoint was event-free survival (EFS), and secondary endpoints were toxicity and predictors of EFS and overall survival (OS).

Results: Between 2010 and 2017, 113 patients were enrolled across 22 centers, and 98 (87%) were high risk. The median age was 49 (range, 18-86) years, and 62% were ≥ 40 years. Bone marrow and/or CSF was involved in 29 (26%) of patients, and 28 (25%) were HIV positive. At a median follow-up of 58.7 months, EFS and OS were 84.5% and 87.0%, respectively, and EFS was 100% and 82.1% in low- and high-risk patients. Therapy was equally effective across age groups, HIV status, and International Prognostic Index risk groups. Involvement of the CSF identified the group at greatest risk for early toxicity-related death or treatment failure. Five treatment-related deaths (4%) occurred during therapy. Febrile neutropenia occurred in 16% of cycles, and tumor lysis syndrome was rare.

Conclusion: Risk-adapted DA-EPOCH-R therapy is effective in adult Burkitt lymphoma regardless of age or HIV status and was well tolerated. Improved therapeutic strategies for adults with CSF involvement are needed (funded by the National Cancer Institute; ClinicalTrials.gov identifier: NCT01092182).
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http://dx.doi.org/10.1200/JCO.20.00303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392744PMC
August 2020
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