Tabelecleucel for allogeneic haematopoietic stem-cell or solid organ transplant recipients with Epstein-Barr virus-positive post-transplant lymphoproliferative disease after failure of rituximab or rituximab and chemotherapy (ALLELE): a phase 3, multicentre, open-label trial.

BACKGROUND: Survival in Epstein-Barr virus (EBV)-positive post-transplant lymphoproliferative disease following haematopoietic stem-cell transplant (HSCT) or solid organ transplant (SOT) is poor after failure of initial therapy, indicating an urgent need for therapies for this ultra-rare disease. With recent EU marketing authorisation, tabelecleucel is the first off-the-shelf, allogeneic, EBV-specific T-cell immunotherapy to receive approval for treatment of relapsed or refractory EBV-positive post-transplant lymphoproliferative disease. We aimed to determine the clinical benefit of tabelecleucel in patients with relapsed or refractory EBV-positive post-transplant lymphoproliferative disease following HSCT or SOT. METHODS: In this global, multicentre, open-label, phase 3 trial, eligible patients (of any age) had biopsy-proven EBV-positive post-transplant lymphoproliferative disease, disease that was relapsed or refractory to rituximab after HSCT and rituximab with or without chemotherapy after SOT, and partially HLA-matched and appropriately HLA-restricted tabelecleucel available. Patients received tabelecleucel administered intravenously at 2 × 106 cells per kg on days 1, 8, and 15 in 35-day cycles and are assessed for up to 5 years for survival post-treatment initiation. The primary endpoint was objective response rate. All patients who received at least one dose of tabelecleucel were included in safety and efficacy analyses. This trial is registered with ClinicalTrials.gov, NCT03394365, and is ongoing. FINDINGS: From June 27, 2018, to Nov 5, 2021, 63 patients were enrolled, of whom 43 (24 [56%] male and 19 [44%] female) were included, 14 had prior HSCT, 29 had SOT. Seven (50%, 95% CI 23-77) of 14 participants in the HSCT group and 15 (52%, 33-71) of 29 participants in the SOT group had an objective response, with a median follow-up of 14·1 months (IQR 5·7-23·9) and 6·0 months (1·8-18·4), respectively. The most common grade 3 or 4 treatment-emergent adverse events were disease progression (in four [29%] of 14 in HSCT and eight [28%] of 29 in SOT) and decreased neutrophil count (in four [29%] of 14 in HSCT and four [14%] of 29 in SOT). Treatment-emergent serious adverse events were reported in 23 (53%) of 43 patients and fatal treatment-emergent adverse events in five (12%); no fatal treatment-emergent adverse event was treatment-related. There were no reports of tumour flare reaction, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, transmission of infectious diseases, marrow rejection, or infusion reactions. No events of graft-versus-host disease or SOT rejection were reported as related to tabelecleucel. INTERPRETATION: Tabelecleucel provides clinical benefit in patients with relapsed or refractory EBV-positive post-transplant lymphoproliferative disease, for whom there are no other approved therapies, without evidence of safety concerns seen with other adoptive T-cell therapies. These data represent a potentially transformative and accessible treatment advance for patients with relapsed or refractory disease with few treatment options. FUNDING: Atara Biotherapeutics.

Aberrant MYCN expression drives oncogenic hijacking of EZH2 as a transcriptional activator in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of hematological cancers arising from the malignant transformation of mature T cells. In a cohort of 28 PTCL cases, we identified recurrent overexpression of MYCN, a member of the MYC family of oncogenic transcription factors. Approximately half of all PTCL cases was characterized by a MYC expression signature. Inducible expression of MYCN in lymphoid cells in a mouse model caused T-cell lymphoma that recapitulated human PTCL with an MYC expression signature. Integration of mouse and human expression data identified EZH2 as a key downstream target of MYCN. Remarkably, EZH2 was found to be an essential cofactor for the transcriptional activation of the MYCN-driven gene expression program, which was independent of methyltransferase activity but dependent on phosphorylation by CDK1. MYCN-driven T-cell lymphoma was sensitive to EZH2 degradation or CDK1 inhibition, which displayed synergy with US Food and Drug Administration-approved histone deacetylase (HDAC) inhibitors.

Genetic and immunological features of immune deficiency and dysregulation-associated lymphoproliferations and lymphomas as a basis for classification.

Immune deficiency and dysregulation-associated lymphoproliferative disorders and lymphomas (IDD-LPDs) encompass a heterogeneous clinical and pathological spectrum of disorders that range from indolent lymphoproliferations to aggressive lymphomas. They arise in a variety of clinical settings and are associated with oncogenic viruses such as the Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus/human herpes virus (KSHV/HHV8) in some, but not all, cases. The recognition of IDD-LPDs as distinct from LPDs in immune competent patients is essential to tailor clinical management options for affected patients. The 5th edition of the World Health Organisation classification has introduced an integrated classification of IDD-LPDs with the goal of standardising diagnoses among different settings to enhance clinical decision support. In parallel, new knowledge in the field, particularly surrounding the role of oncogenic viruses and the tumour microenvironment, has led to clearer understanding of the complex pathogenesis of IDD-LPDs and how these features can be precisely harnessed for therapeutic purposes. In this perspective, we highlight the need for multidisciplinary decision-making to augment patient care as well as key areas where evolving concepts offer challenges and opportunities for clinical management, research and future iterations of the classification.

Safety and performance of the Spectra Optia apheresis system for white blood cell depletion in patients with elevated white blood cell counts.

BACKGROUND: For the past 30 years, white blood cell depletion (WBCD) or leukocytapheresis has been conducted to rapidly reduce excessive circulating white blood cell (WBC) concentrations in patients at risk for or with symptoms of leukostasis due to hyperleukocytosis. The goal of leukocytapheresis is to prevent or treat acute complications from leukostasis, thereby enabling patients to receive potentially curative chemotherapy. METHODS: This report details the results from a retrospective and a prospective clinical study conducted in the European Union and the People's Republic of China, which assessed the use of the Spectra Optia Apheresis System for leukocytapheresis in patients with hyperleukocytosis. The primary objective of both studies was to the assess the safety and performance of the WBCD procedure in patients with elevated WBC counts. RESULTS: Data were collected from 72 participants completing 87 WBCD procedures. The mean percent change in participant WBC counts post-procedure was 50.3 ± 21.2% and the collection efficiency (CE1) of the WBCD procedures was 53.7 ± 19.8%. Sixty-one participants (95.3%) experienced a total of 279 adverse events (AEs) with the majority of the AEs related to post-procedure changes in laboratory values, which is an anticipated AE in this patient population. CONCLUSION: The data collected within these studies indicate that the WBCD procedure is safe and well tolerated in patients with hyperleukocytosis as evaluated by percent decrease in WBC count, CE1, and AE incidence.

Primary mediastinal large B-cell lymphoma is characterized by large-scale copy-neutral loss of heterozygosity.

Development of primary mediastinal B-cell lymphoma (PMBL) is driven by cumulative genomic aberrations. We discovered a unique copy-neutral loss of heterozygosity (CN-LOH) landscape of PMBL which distinguishes this tumor from other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma. Using single nucleotide polymorphism array analysis we identified large-scale CN-LOH lesions in 91% (30/33) of diagnostic PMBLs and both investigated PMBL-derived cell lines. Altogether, the cohort showed 157 extra-large (25.3-248.4 Mb) CN-LOH lesions affecting up to 14 chromosomes per case (mean of 4.4) and resulting in a reduction of heterozygosity an average of 9.9% (range 1.3-51%) of the genome. Predominant involvement of terminal chromosomal segments suggests the implication of B-cell specific crossover events in the pathogenesis of PMBL. Notably, CN-LOH stretches non-randomly clustered on 6p (60%), 15 (37.2%), and 17q (40%), and frequently co-occurred with homozygous mutations in the MHC I (6p21), B2M (15q15), and GNA13 (17q23) genes, respectively, as shown by preliminary whole-exome/genome sequencing data. Altogether, our findings implicate CN-LOH as a novel and distinct mutational process contributing to the molecular pathogenesis of PMBL. The aberration acting as "second hit" in the Knudson hypothesis, ranks as the major mechanism converting to homozygosity the PMBL-related driver genes. Screening of the cohort of 199 B cell leukemia/lymphoma whole-genomes revealed significant differences in the CN-LOH landscape of PMBL and other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma.

Epstein-Barr virus-associated posttransplant lymphoproliferative disorders: new insights in pathogenesis, classification and treatment.

PURPOSE OF REVIEW: Posttransplant lymphoproliferative disorder (PTLD) is a serious complication following transplantation from an allogeneic donor. Epstein-Barr Virus (EBV) is involved in a substantial number of cases. In this review, we aim to summarize recent knowledge on pathogenesis, classification and treatment of EBV + PTLD. RECENT FINDINGS: New insights in the complex oncogenic properties of EBV antigens noncoding Ribonucleic acids (RNAs), especially EBV MicroRNA (miRNAs), have increased our knowledge of the pathogenesis of EBV + PTLD. In addition the potential influence of EBV on the tumor microenvironment is becoming clearer, paving the way for new types of immunotherapy. Currently PTLD is classified according to the World Health Organization classification together with other lymphoproliferative disorders, based on the specific immunosuppression. However, a new framework integrating all types of lymphoproliferative disorders in all different settings of immune deficiency and dysregulation is needed. Although treatment of EBV + and EBV - PTLD was largely similar in the past, EBV-directed therapies are currently increasingly used. SUMMARY: The use of EBV-directed therapies and new agents, based on better understanding of pathogenesis and classification of PTLD, will change the treatment landscape of EBV + PTLD in the next era.

Characteristics and Outcome of Post-Transplant Lymphoproliferative Disorders After Solid Organ Transplantation: A Single Center Experience of 196 Patients Over 30 Years.

Post-transplant lymphoproliferative disorder (PTLD) is a rare but life-threatening complication after transplantation. In this retrospective, monocentric study we aimed to collect real life data regarding PTLD and determine the role of Epstein Barr Virus (EBV) status and year of diagnosis on prognosis. We identified 196 biopsy-proven PTLD after solid organ transplantation (SOT) diagnosed at the University Hospitals Leuven (Belgium) from 1989 to 2019. EBV status was positive in 61% of PTLD. The median overall survival (OS) was 5.7 years (95% CI: 2.99-11.1). Although EBV positivity was not significantly correlated with OS in multivariate analyses (HR: 1.44 (95% CI: 0.93-2.24); p = 0.10), subgroup analysis showed a significantly better median OS for EBV negative post-transplant diffuse large B-cell lymphoma (DLBCL) compared to EBV positive post-transplant DLBCL (8.8 versus 2.5 years respectively; p = 0.0365). There was a significant relation between year of PTLD diagnosis and OS: the more recent the PTLD diagnosis, the lower the risk for death (adjusted HR: 0.962 (95% CI: 0.931-0.933); p = 0.017). In conclusion, the prognosis of PTLD after SOT has improved in the past decades. Our analysis shows a significant relation between EBV status and OS in post-transplant DLBCL.

Immunotherapy for cancer treatment during pregnancy.

Immunotherapy has greatly improved outcomes for subgroups of patients with cancer. As indications keep expanding, there is an unmet need to gain a better understanding of the effect of these therapies on pregnancy and fertility. During pregnancy, substantial adaptations occur in the maternal immune system to maintain protection against pathogens while avoiding detrimental reactions to the semi-allogeneic fetus. The pathways involved in the establishment of this fetomaternal tolerance can be hijacked by cancers. Immunotherapies that target these inhibitory pathways, or that directly interact with the regulatory immune cells involved in tolerance mechanisms, might therefore result in complications during pregnancy. Similarly, by activating the patient's immune system with immunotherapy, a broad range of immune-related adverse events can occur that could negatively affect the fetus or impede a future desired pregnancy. This Review summarises preclinical and clinical data related to the use of immunotherapy during pregnancy, including all approved immune checkpoint inhibitors, recombinant cytokines, cell therapies, vaccines, and immunomodulatory drugs.

Maternal and neonatal outcomes in 80 patients diagnosed with non-Hodgkin lymphoma during pregnancy: results from the International Network of Cancer, Infertility and Pregnancy.

This cohort study of the International Network on Cancer, Infertility and Pregnancy (INCIP) reports the maternal and neonatal outcomes of 80 pregnant patients diagnosed with non-Hodgkin lymphoma (NHL) between 1986 and 2019, focussing on 57 (71%) patients with diffuse large B-cell lymphoma (DLBCL). Of all 80 patients, 54 (68%) pregnant patients received chemotherapy; mostly (89%) CHOP-like (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimens. Four early pregnancies were terminated. Among 76 ongoing pregnancies, there was one stillbirth (1·3%). Overall, there was a high incidence of small for gestational age neonates (39%), preterm delivery (52%), obstetric (41%) and neonatal complications (12·5%), and this could not exclusively be explained by the receipt of antenatal chemotherapy. Half of preterm deliveries (46%) were planned in order to tailor oncological treatment. The 3-year progression-free and overall survival for patients with DLBCL treated with rituximab-CHOP was 83·4% and 95·7% for limited stage (n = 29) and 60·6% and 73·3% for advanced stage (n = 15). Of 36 pregnant patients who received rituximab, five (13%) cases with neonatal complications and three (8%) with maternal infections were reported. In conclusion, standard treatment for DLBCL can be offered to pregnant patients in obstetric centres that cater for high-risk patients.

Posttransplant Lymphoproliferative Disorder Following Kidney Transplantation: A Review.

Posttransplant lymphoproliferative disorder (PTLD) is one of the most feared complications following kidney transplantation. Over a 10-year period, the risk of PTLD in kidney transplant recipients (KTRs) is 12-fold higher than in a matched nontransplanted population. Given the number of kidney transplants performed, KTRs who experience PTLD outnumber other organ transplant recipients who experience PTLD. Epstein-Barr virus infection is one of the most important risk factors for PTLD, even though 40% of PTLD cases in contemporary series are not Epstein-Barr virus-associated. The overall level of immunosuppression seems to be the most important driver of the increased occurrence of PTLD in solid organ transplant recipients. Reduction in immunosuppression is commonly accepted to prevent and treat PTLD. Although the cornerstone of PTLD treatment had been chemotherapy (typically cyclophosphamide-doxorubicin-vincristinr-prednisone), the availability of rituximab has changed the treatment landscape in the past 2 decades. The outcome of PTLD in KTRs has clearly improved as a result of the introduction of more uniform treatment protocols, improved supportive care, and increased awareness and use of positron emission tomography combined with computed tomography in staging and response monitoring. In this review, we will focus on the most recent data on epidemiology, presentation, risk factors, and management of PTLD in KTRs.

In-depth characterization of the tumor microenvironment in central nervous system lymphoma reveals implications for immune-checkpoint therapy.

Primary central nervous system lymphoma (PCNSL) is a rare type of non-Hodgkin lymphoma with an aggressive clinical course. To investigate the potential of immune-checkpoint therapy, we retrospectively studied the tumor microenvironment (TME) using high-plex immunohistochemistry in 22 PCNSL and compared to 7 secondary CNS lymphomas (SCNSL) and 7 "other" CNSL lymphomas with the presence of the Epstein-Barr virus and/or compromised immunity. The TME in PCNSL was predominantly composed of CD8+ cytotoxic T cells and CD163+ phagocytes. Despite molecular differences between PCNSL and SCNSL, the cellular composition and the functional spectrum of cytotoxic T cells were similar. But cytotoxic T cell activation was significantly influenced by pre-biopsy corticosteroids intake, tumor expression of PD-L1 and the presence of EBV. The presence of low numbers of CD8+ T cells and geographic-type necrosis each predicted inferior outcome in PCNSL. Both M1-like (CD68 + CD163low) and M2-like (CD68 + CD163high) phagocytes were identified, and an increased ratio of M1-like/M2-like phagocytes was associated with a better survival. PD-L1 was expressed in lymphoma cells in 28% of cases, while PD1 was expressed in only 0.4% of all CD8+ T cells. TIM-3, a marker for T cell exhaustion, was significantly more expressed in CD8posPD-1pos T cells compared to CD8posPD-1neg T cells, and a similar increased expression was observed in M2-like pro-tumoral phagocytes. In conclusion, the clinical impact of TME composition supports the use of immune-checkpoint therapies in PCNSL. Based on observed differences in immune-checkpoint expression, combinations that boost cytotoxic T cell activation (by blocking TIM-3 or TGFBR1) prior to the administration of PD-L1 inhibition could be of interest.

Treatment of patients with MYC rearrangement positive large B-cell lymphoma with R-CHOP plus lenalidomide: results of a multicenter HOVON phase II trial.

Patients with MYC-rearrangement positive large B-cell lymphoma (MYC+ LBCL) have an inferior prognosis following standard first-line therapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) as compared to patients without MYC rearrangement. Although intensive chemotherapy regimens yield higher remission rates, toxicity remains a concern. Lenalidomide is an oral immunomodulatory drug which downregulates MYC and its target genes thereby providing support using lenalidomide as additional therapeutic option for MYC+ LBCL. A phase II trial was conducted evaluating the efficacy of lenalidomide (15 mg day 1-14) in combination with R-CHOP (R2CHOP) in newly diagnosed MYC+ LBCL patients identified through a nationwide MYC-FISH screening program. The primary endpoint was complete metabolic response (CMR) on centrally reviewed 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)-computer tomography (CT)-scan at end-of-treatment. Secondary endpoints were overall survival (OS), disease-free survival (DFS) and event-free survival (EFS). Eighty-two patients with stage II-IV MYC+ LBCL were treated with 6 cycles of R2CHOP. At EOT, 67% (confidence interval (CI) 58-75%) of the patients reached CMR. With a median follow-up of 25.4 months, 2-year estimates (95% CI) for OS, DFS, EFS were 73% (62-82%), 75% (63-84%) and 63% (52-73%) respectively. In this prospective trial for newly diagnosed MYC+ LBCL patients, we found that administering R2CHOP was safe, and yields comparable CMR and survival rates as in studies applying more intensive chemotherapy regimens. Hence, these findings offer new prospects for MYC+ LBCL patients and warrant comparison in prospective randomized clinical trials. This trial was registered at www.clinicaltrialsregister.eu (#2014-002654-39).

Bortezomib for autoimmune hemolytic anemia after intestinal transplantation.

AIHA is rare in the general population and associated with a mortality of 8%. In contrast, AIHA occurs in up to 12.2% of cases after intestinal transplantation and is associated with mortality up to 50%. Treatment entails a "step-up" approach including corticosteroids, IvIg, plasmapheresis, and rituximab. However, AIHA after transplantation often is refractory to this strategy, contributing to a poor outcome. We describe a child with microvillous inclusion disease who developed AIHA 1 year after multivisceral transplantation that was refractory to standard therapy and was subsequently treated with bortezomib.We observed remission of AIHA within 1 week after the start of bortezomib. Bortezomib was associated with transient diarrhea, leucopenia, and elevated liver enzymes. Three years later, he remains in remission without important complications. Published data on bortezomib for autoimmune cytopenias outside SOT are discussed. This is the first report to support bortezomib as an important therapeutic alternative for AIHA after SOT. The occurrence and treatment of AIHA after SOT, and specifically intestinal transplantation, should be the subject of future registry studies to collect additional experience and explore the optimal therapeutic approach.

Role of interim and end of treatment positron emission tomography for response assessment and prediction of relapse in posttransplant lymphoproliferative disorder.

Background: Fluorine-18-fluorodeoxyglucose positron emission tomography (PET) has an established and central role in diagnosis, staging and response evaluation of lymphoproliferative diseases. It has shown a high sensitivity and specificity at diagnosis in posttransplant lymphoproliferative disorders (PTLDs). However, little is known about the performance of interim and end of treatment (EOT) PET in PTLD patients with regards to response assessment, relapse prediction and outcome. Methods: We performed a single-center retrospective study in which we analyzed consecutive patients diagnosed with CD20-positive PTLD after solid organ transplantation between 2008 and 2017, who all received risk-stratified sequential treatment according to the PTLD-1 phase II trial. Interim and EOT PET studies were scored according to the Deauville criteria. Results: Forty-one patients were included with median follow-up of 41.5 months (range 1-108). Positive and negative predictive values for disease recurrence were 13% and 85% for interim and 33% and 87% for EOT PET, respectively. There was no significant difference in overall survival, progression-free survival nor time to progression between negative versus positive patients on interim and EOT scans. Conclusions: Negative interim and/or negative end of treatment PET identify PTLD patients with low risk of disease recurrence.

Other immunomodulatory agent-related lymphoproliferative diseases: a single-center series of 72 biopsy-confirmed cases.

Ongoing development of new drugs, as well as novel indications in the treatment of autoimmune diseases leads to the increasing use of immunomodulatory and immunosuppressive drugs. Immunomodulatory agent-related lymphoproliferative disorders are a known and potentially life threatening complication of chronic administration of these drugs, but are less well characterized compared with post-transplant lymphoproliferative disorders. The heterogeneous drug targets, various underlying disease indications, different drug combinations used and relatively low incidence render data collection and interpretation difficult. In this retrospective paper, we describe the clinicopathological characteristics of a larger single-center series of 72 immunomodulatory agent-related lymphoproliferative disorder cases. We divided the cases according to the therapy, administered in the year preceding diagnosis of a lymphoproliferative disorder, in an immunosuppressive drug, an immunomodulatory drug and a combination of immunosuppressive and immunomodulatory drugs group. We observed differences in "time to lymphoproliferative disorder development" with a shorter time for all the immunomodulatory drug-related cases combined (immunomodulatory and immunomodulatory + immunosuppressive = immunomodulatory-all) vs immunosuppressive-only (p = 0.0031). The proportion of malignant cases in patients receiving immunomodulatory therapy was, however, also significantly lower when compared with the immunosuppressive treated cases (43 vs 88%; p = 0.0184). The immunomodulatory/suppressive agent-related lymphoproliferative disorders were less often associated with the Epstein-Barr virus (EBV) (31 vs 66%; p = 1.829e-05) and the lymphoproliferative disorders incidence in the first year after immunomodulatory/immunosuppressive therapy initiation was lower (18 vs 41%; p = 0.04151)-compared with a published series of 140 post-transplant lymphoproliferative disorder cases from the same center. However, a similar histopathological spectrum from nondestructive, to polymorphic and monomorphic lesions as in post-transplant lymphoproliferative disorders is observed. With increasing use of immunosuppressive and especially immunomodulatory therapy, a higher incidence of immunomodulatory/suppressive agent-related lymphoproliferative disorders is to be expected. Life-long awareness for development of immunomodulatory/suppressive agent-related lymphoproliferative disorders with clinical follow-up and timely biopsies of suspicious lesions is required since these lymphoproliferative disorders arise both early after therapy initiation and many years later. Histopathological confirmation and correct classification is necessary to guide therapy and EBV ISH should be a part of routine pathological diagnostics.

Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma.

BACKGROUND: Plasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC. METHODS: By using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science). RESULTS: Our data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit. CONCLUSIONS: We demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.

Improving lymph node characterization in staging malignant lymphoma using first-order ADC texture analysis from whole-body diffusion-weighted MRI.

BACKGROUND: Correct staging and treatment initiation in malignant lymphoma depends on accurate lymph node characterization. However, nodal assessment based on conventional and diffusion-weighted (DWI) MRI remains challenging, particularly in smaller nodes. PURPOSE: To evaluate first-order apparent diffusion coefficient (ADC) texture parameters compared to mean ADC for lymph node characterization in non-Hodgkin lymphoma (NHL) using whole-body DWI (WB-DWI). STUDY TYPE: Retrospective. POPULATION: Twenty-eight patients with NHL. FIELD STRENGTH/SEQUENCE: 3T whole-body DWI using two b-values (0-1000 s/mm2 ). ASSESSMENT: Regions of interest were drawn on the three most hyperintense lymph nodes on b1000-images, irrespective of size, in all nodal body regions. Diagnostic performance of mean ADC (ADCmean ) was compared with first-order ADC texture parameters: standard deviation (ADCstdev ), kurtosis (ADCkurt ), and skewness (ADCskew ). Additional subanalyses focused on the accuracy of ADCmean and ADC texture parameters in different lymph node volumes and nodal regions. STATISTICAL TESTS: Benign and malignant nodes were compared using Mann-Whitney U-tests with 18-Fluoro-deoxyglucose positron emission tomography computed tomography and bone marrow biopsy as reference standard. Receiver operating characteristic analyses were performed to determine cutoff values and calculate sensitivity, specificity, accuracy, and positive and negative predictive value (PPV, NPV). RESULTS: ADCmean (P = 0.008), ADCskew and ADCkurt differed significantly between benign and malignant nodes (P < 0.001), while ADCstdev didn't (P = 0.21). ADCskew was the best discriminating parameter, with 79% sensitivity, 86% specificity, 83% accuracy, 85% PPV, and 81% NPV. In every volume category, ADCskew yielded the highest accuracy (88% in 0-25th percentile volume, 75% in 25th -75th percentile, 93% in 75-100th percentile). On a per-region basis, ADCskew accuracy varied 13.6% between nodal regions, while ADCmean , ADCkurt , and ADCstdev showed interregional variation of 17.4%, 20.3%, and 14.9%, respectively. DATA CONCLUSION: First-order ADC texture analysis with WB-DWI improved lymph node characterization compared to ADCmean . ADCskew was the most accurate and robust discriminatory parameter over all lymph node volumes and nodal body regions. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;48:897-906.

Clinicopathological characteristics of de novo and secondary myeloid sarcoma: A monocentric retrospective study.

OBJECTIVE: Diagnosing myeloid sarcoma remains challenging, and we aimed to provide clinicopathological features to facilitate diagnosis. METHOD: Clinicopathological data from 41 patients with de novo and 31 with secondary myeloid sarcoma were reviewed. RESULTS: Most de novo cases presented with isolated myeloid sarcoma (n = 19) or myeloid sarcoma with concurrent acute myeloid leukemia (n = 15). Most secondary cases presented after acute myeloid leukemia (n = 11), myeloproliferative neoplasm (n = 9), or myelodysplastic syndrome (n = 8). Most frequent localizations were skin and lymph nodes. Immunohistochemistry showed immature and/or aberrant antigenic expression in 29% of de novo and 39% of secondary cases. Most genetic abnormalities were RUNX1-RUNX1T1 (n = 4), CBFB-MYH11 (n = 2), KMT2A-MLLT3 (n = 2), and JAK2 V617F (n = 2) mutations in de novo myeloid sarcoma, and BCR-ABL1 (n = 5) and KMT2A rearrangements (n = 2) in secondary cases. A complex karyotype was seen in 17% of de novo and 39% of secondary cases. Most prevalent treatment was induction chemotherapy followed by consolidation chemotherapy (n = 10) or allogeneic stem cell transplantation (n = 9) for de novo and radiotherapy (n = 11) for secondary cases. CONCLUSION: De novo myeloid sarcoma mostly presented isolated. Lesions were often localized at skin and lymph nodes. Genetic aberrations frequently involved core-binding factor rearrangements in de novo cases and a complex karyotype in secondary cases.

Identification of distinct subgroups of EBV-positive post-transplant diffuse large B-cell lymphoma.

Post-transplantation lymphoproliferative disorder is an aggressive complication of transplantation, most frequently of diffuse large B-cell lymphoma morphology and associated with Epstein-Barr virus (EBV) infection/reactivation. In this study the microenvironment of EBV+ (n=23) and EBV- (n=9) post-transplant non-germinal center B-cell diffuse large B-cell lymphoma was characterized. Of EBV+ cases somatic hypermutation analysis, gene expression profiling, and extensive phenotyping were performed. Our results demonstrated variable cytotoxic T-cell infiltration and significantly increased CD163+ M2 macrophage infiltration in EBV+ compared with EBV- post-transplant diffuse large B-cell lymphoma. On the basis of IgM staining and hypermutation analysis, two EBV+ post-transplant diffuse large B-cell lymphoma subgroups were identified: IgM+ tumors lacking somatic hypermutations and IgM- tumors harboring somatic hypermutations. IgM- tumors arose late following transplantation (median interval: 16 months), mainly in kidney recipients. IgM+ tumors on the other hand arose early (median interval: 3 months, P-value=0.0032), almost exclusively following stem cell transplantation and were associated with worse outcome (median survival 1 month for IgM+ versus 41 months for IgM- tumors, log-rank/Wilcoxon P-value 0.07/0.04). Notably, IgM+ tumors were characterized by plasma cell features (monotypic kappa/lambda expression, high MUM1 expression, and partial CD138 expression) and a high proliferation index. Consistent with the plasma cell phenotype, unfolded protein response signaling was upregulated. In contrast, IgM- EBV+ post-transplant diffuse large B-cell lymphoma did not express kappa, lambda, IgD, or CD138 and expressed limited MUM1. In these tumors T-cell signaling was enhanced associated with increased T-cell infiltration compared with IgM+ cases. Overall, our results allow further molecular classification of EBV+ post-transplant diffuse large B-cell lymphoma and provide a rationale for the use of subtype-specific-targeted therapies (eg, bortezomib in IgM+ tumors). Our findings also provide a biological basis for the clinical differences between post-transplant lymphoproliferative disorder following solid organ and stem cell transplantation, which are regarded as different disorders.