Severe lympho-depletion, abrogated thymopoiesis and systemic EBV positive T-cell lymphoma of childhood, a case.

Epstein-Barr virus (EBV) associated T-cell and NK-cell lymphoproliferative diseases are lethal and extremely rare in Caucasians. We expand on the clinical, immunological and histogenetic characteristics associated with this second European case (19 years old, previously healthy, Caucasian boy) of systemic EBV positive T-cell lymphoma of childhood. We report, as novel findings, severe lympho-depletion and abrogation of thymopoiesis secondary to severe EBV activation and excessive immune activation. Similar to the first European case, we also detected a somatic missense variant in the proto-oncogene FYN. In the first European patient however, the FYN variant allele frequency (VAF) was 10% and the patient only experienced moderate leukopenia, whereas in our case, the VAF was 48% and the patient experienced severe leukopenia and lymphopenia. This could suggest a pathogenic role of these FYN variants in driving excessive T cell activation. If confirmed, FYN might become target in future treatments of this fatal disorder.

The multidisciplinary approach to eosinophilia.

Eosinophilic granulocytes are normally present in low numbers in the bloodstream. Patients with an increased number of eosinophilic granulocytes in the differential count (eosinophilia) are common and can pose a clinical challenge because conditions with eosinophilia occur in all medical specialties. The diagnostic approach must be guided by a thorough medical history, supported by specific tests to guide individualized treatment. Neoplastic (primary) eosinophilia is identified by one of several unique acquired genetic causes. In contrast, reactive (secondary) eosinophilia is associated with a cytokine stimulus in a specific disease, while idiopathic eosinophilia is a diagnosis by exclusion. Rational treatment is disease-directed in secondary cases and has paved the way for targeted treatment against the driver in primary eosinophilia, whereas idiopathic cases are treated as needed by principles in eosinophilia originating from clonal drivers. The vast majority of patients are diagnosed with secondary eosinophilia and are managed by the relevant specialty-e.g., rheumatology, allergy, dermatology, gastroenterology, pulmonary medicine, hematology, or infectious disease. The overlap in symptoms and the risk of irreversible organ involvement in eosinophilia, irrespective of the cause, warrants that patients without a diagnostic clarification or who do not respond to adequate treatment should be referred to a multidisciplinary function anchored in a hematology department for evaluation. This review presents the pathophysiology, manifestations, differential diagnosis, diagnostic workup, and management of (adult) patients with eosinophilia. The purpose is to place eosinophilia in a clinical context, and therefore justify and inspire the establishment of a multidisciplinary team of experts from diagnostic and clinical specialties at the regional level to support the second opinion. The target patient population requires highly specialized laboratory analysis and therapy and occasionally has severe eosinophil-induced organ dysfunction. An added value of a centralized, clinical function is to serve as a platform for education and research to further improve the management of patients with eosinophilia. Primary and idiopathic eosinophilia are key topics in the review, which also address current research and discusses outstanding issues in the field.

Cell-free DNA for detection of clonal B cells in diffuse large B cell lymphoma by sequencing.

INTRODUCTION: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma in the western world. It is highly heterogeneous with a variable clinical course, but curable with chemo-immunotherapy in up to 70% of all cases. The lymphoma presents in lymph nodes and/or extranodal lymphoid tissue, and the diagnosis is based on invasive procedures for histopathologic evaluation. METHODS: In this technical study, we evaluated cell-free DNA (cfDNA) from blood plasma to detect clonal B cells in patients with DLBCL using rearranged immunoglobulin heavy chain gene as targets by next-generation sequencing. Clonal B cell sequences and frequencies were determined from blood plasma cfDNA and cellular DNA from matched excised lymphoma tissues and mononuclear cells isolated from diagnostic bone marrow and blood samples from 15 patients. RESULTS: We showed that identical clonal rearrangements could be detected in blood plasma and excised lymphoma tissue and that plasma cfDNA was superior in detecting clonal rearrangements compared to blood or bone marrow-derived cellular DNA. CONCLUSION: These findings consolidate the role of blood plasma as a reliable and easily accessible source for detecting neoplastic cells in DLBCL.

Methylation microarray-based detection of clinical copy-number aberrations in CLL benchmarked to standard FISH analysis.

Copy-number aberrations (CNAs) are assessed using FISH analysis in diagnostics of chronic lymphocytic leukemia (CLL), but CNAs can also be extrapolated from Illumina BeadChips developed for genome-wide methylation microarray screening. Increasing numbers of microarray data-sets are available from diagnostic samples, making it useful to assess the potential in CNA diagnostics. We benchmarked the limitations of CNA testing from two Illumina BeadChips (EPIC and 450k) and using two common packages for analysis (conumee and ChAMP) to FISH-based assessment of 11q, 13q, and 17p deletions in 202 CLL samples. Overall, the two packages predicted CNAs with similar accuracy regardless of the microarray type, but lower than FISH-based assessment. We showed that the bioinformatics analysis needs to be adjusted to the specific CNA, as no general settings were identified. Altogether, we were able to predict CNAs using methylation microarray data, however, with limited accuracy, making FISH-based assessment of deletions the superior diagnostic choice.

Exploration of residual disease in stem cell products from mantle cell lymphoma using next-generation sequencing.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) has become a treatment option for fit patients with mantle cell lymphoma (MCL). However, these patients often relapse within few years, potentially caused by contaminating lymphoma cells within the reinfused stem cell product (SCP). Studies have shown that measurable residual disease, also termed minimal residual disease (MRD), following ASCT predicts shorter survival. Using next-generation sequencing, we explore whether the diagnostic MCL clonotype is present within the infused SCP. MRD was detected in 4/17 of the SCPs, ranging 4-568 clonal cells/100,000 cells. With a median survival of 17 months, 3/4 of patients with MRD+ graft succumbed from MCL relapse versus 2/13 in the MRD- fraction. Patients receiving MRD+ grafts had increased risk of mortality, and thus screening of SCPs may be important for clinical decision-making.

A retrospective cohort study of patients with eosinophilia referred to a tertiary centre.

INTRODUCTION: Patients with eosinophilia (an increased number of eosinophilic granulocytes > 0.5 × 108/l in the blood) are encountered in all medical specialties and frequently need thorough workup to identify the eliciting causes and decide whether treatment is indicated. In Denmark, highly specialised centres for eosinophilic diseases or conditions have been established to provide a foundation for the management of complicated cases. Here, we present experiences from such a multidisciplinary centre. METHODS: This was a retrospective study of all patients seen in our tertiary centre for eosinophilia in the 2016-2019 period. RESULTS: Referrals mainly derived from specialised secondary care and to a lesser degree from primary care physicians. Patients were either asymptomatic or exhibited symptoms from up to three organ systems and presented a median eosinophil count of 1.7 × 108/l. Up to eight new clonality analyses or imaging studies per patient were performed after referral. One of these, T-cell receptor analysis, was performed frequently but provided limited information, whereas, e.g., flow cytometry proved more clinically applicable owing to its broader diagnostic range. In total, 51 patients were evaluated and classified as secondary (59%), myeloid neoplasm with PDGFRA rearrangement (2%), idiopathic hypereosinophilic syndrome (31%) and idiopathic hypereosinophilia (8%). CONCLUSION: The value of a multidisciplinary and versatile approach in a highly specialised centre has a positive impact on diagnostic processes as well as on the evaluation of treatment need. FUNDING: none. TRIAL REGISTRATION: not relevant.

Diagnosis, treatment, and outcome of primary CNS lymphoma-a single-center experience.

BACKGROUND: Primary CNS lymphoma (PCNSL) is a highly aggressive non-Hodgkin lymphoma (NHL) that occurs in the CNS (e.g. brain, meninges, spinal cord, cerebrospinal fluid, or intraocular involvement) in the absence of systemic NHL. Tumor resection does not improve survival, and neurosurgical intervention is generally limited to stereotactic biopsy to provide a histopathological diagnosis. OBJECTIVE: The objective of this single-center study was to evaluate the management and outcome of PCNSL patients diagnosed by biopsy, using overall survival and progression-free survival as endpoints. METHODS: At our department of neurosurgery, 140 patients were diagnosed with PCNSL by biopsy between January 1, 2009, and December 31, 2018. Of these, 37 patients were included in the study and were divided into three groups according to their postoperative therapy. RESULTS: Median OS was 35.7 months for the intensive treatment group, 29.5 months for the moderate treatment group, and 8.6 months for the palliative treatment group. The intensive and moderate treatment groups had similar progression-free survival, while the palliative treatment group had poor overall and progression-free survival. Six patients were long-term survivors (> 80 months). Age under 65 years was the main significant parameter affecting overall survival. CONCLUSION: In this cohort, patients with PCNSL had an overall fair prognosis if they (1) were under 65 years old, (2) had a performance score < 2 at the time of diagnosis, and (3) received either intensive or moderate chemotherapeutic treatment. Biopsy is still the primary diagnostic tool; other methods have been investigated but are not yet recommended.

IGHV-associated methylation signatures more accurately predict clinical outcomes of chronic lymphocytic leukemia patients than IGHV mutation load.

Currently, no molecular biomarker indices are used in standard care to make treatment decisions at diagnosis of chronic lymphocytic leukemia (CLL). We used Infinium MethylationEPIC array data from diagnostic blood samples of 114 CLL patients and developed a procedure to stratify patients based on methylation signatures associated with mutation load of the IGHV gene. This procedure allowed us to predict the time to treatment with a hazard ratio (HR) of 8.34 (95% confidence interval [CI]: 4.54-15.30), as opposed to a HR of 4.35 (95% CI: 2.60-7.28) using IGHV mutation status. Detailed evaluation of 17 cases for which the two classification procedures gave discrepant results showed that these cases were incorrectly classified using IGHV status. Moreover, methylation-based classification stratified patients with different overall survival (HR=1.82; 95% CI: 1.07-3.09), which was not possible using IGHV status. Furthermore, we assessed the performance of the developed classification procedure using published HumanMethylation450 array data for 159 patients for whom information on time to treatment, overall survival and relapse was available. Despite 450K array methylation data not containing all the biomarkers used in our classification procedure, methylation signatures again stratified patients with significantly better accuracy than did IGHV mutation load regarding all available clinical outcomes. Thus, stratification using IGHV-associated methylation signatures may provide better prognostic power than IGHV mutation status.

Proteomic Profiling Differentiates Lymphoma Patients with and without Concurrent Myeloproliferative Neoplasia.

Myeloproliferative neoplasia (MPN) and lymphoma are regarded as distinct diseases with different pathogeneses. However, patients that are diagnosed with both malignancies occur more frequently in the population than expected. This has led to the hypothesis that the two malignancies may, in some cases, be pathogenetically related. Using a mass spectrometry-based proteomic approach, we show that pre-treatment lymphoma samples from patients with both MPN and lymphoma, either angioimmunoblastic T-cell lymphoma (MPN-AITL) or diffuse large B-cell lymphoma (MPN-DLBCL), show differences in protein expression compared with reference AITL or DLBCL samples from patients without MPN. A distinct clustering of samples from patients with and without MPN was evident for both AITL and DLBCL. Regarding MPN-AITL, a pathway analysis revealed disturbances of cellular respiration as well as oxidative metabolism, and an immunohistochemical evaluation further demonstrated the differential expression of citrate synthase and DNAJA2 protein (p = 0.007 and p = 0.015). Interestingly, IDH2 protein also showed differential expression in the MPN-AITL patients, which contributes to the growing evidence of this protein's role in both myeloid neoplasia and AITL. In MPN-DLBCL, the disturbed pathways included a significant downregulation of protein synthesis as well as a perturbation of signal transduction. These results imply an underlying disturbance of tumor molecular biology, and in turn an alternative pathogenesis for tumors in these patients with both myeloid and lymphoid malignancies.

Detailed characterization of the transcriptome of single B cells in mantle cell lymphoma suggesting a potential use for SOX4.

Mantle cell lymphoma (MCL) is a malignancy arising from naive B lymphocytes with common bone marrow (BM) involvement. Although t(11;14) is a primary event in MCL development, the highly diverse molecular etiology and causal genomic events are still being explored. We investigated the transcriptome of CD19+ BM cells from eight MCL patients at single-cell level. The transcriptomes revealed marked heterogeneity across patients, while general homogeneity and clonal continuity was observed within the patients with no clear evidence of subclonal involvement. All patients were SOX11+CCND1+CD20+. Despite monotypic surface immunoglobulin (Ig) κ or λ protein expression in MCL, 10.9% of the SOX11 + malignant cells expressed both light chain transcripts. The early lymphocyte transcription factor SOX4 was expressed in a fraction of SOX11 + cells in two patients and co-expressed with the precursor lymphoblastic marker, FAT1, in a blastoid case, suggesting a potential prognostic role. Additionally, SOX4 was found to identify non-malignant SOX11- pro-/pre-B cell populations. Altogether, the observed expression of markers such as SOX4, CD27, IgA and IgG in the SOX11+ MCL cells, may suggest that the malignant cells are not fixed in the differentiation state of naïve mature B cells, but instead the patients carry B lymphocytes of different differentiation stages.

Distal chromosome 1q aberrations and initial response to ibrutinib in central nervous system relapsed mantle cell lymphoma.

Relapse involving the central nervous system (CNS) is an infrequent event in the progression of mantle cell lymphoma (MCL) with an incidence of approximately four percent. We report four cases of MCL with CNS relapse. In three of the four patients a large chromosomal copy-number alteration (CNA) of 1q was demonstrated together with TP53 mutation/deletion. These patients experienced brief response to ibrutinib, whereas a fourth patient harboring mutated ATM demonstrated a long-term effect to ibrutinib and no CNA. Although it is unclear whether chromosome 1q CNA contribute to specific phenotypes these reports may be of value as such lesions are uncommon features of MCL.

Secondary cytogenetic abnormalities in core-binding factor AML harboring inv(16) vs t(8;21).

Patients with core-binding factor (CBF) acute myeloid leukemia (AML), caused by either t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22), have higher complete remission rates and longer survival than patients with other subtypes of AML. However, ∼40% of patients relapse, and the literature suggests that patients with inv(16) fare differently from those with t(8;21). We retrospectively analyzed 537 patients with CBF-AML, focusing on additional cytogenetic aberrations to examine their impact on clinical outcomes. Trisomies of chromosomes 8, 21, or 22 were significantly more common in patients with inv(16)/t(16;16): 16% vs 7%, 6% vs 0%, and 17% vs 0%, respectively. In contrast, del(9q) and loss of a sex chromosome were more frequent in patients with t(8;21): 15% vs 0.4% for del(9q), 37% vs 0% for loss of X in females, and 44% vs 5% for loss of Y in males. Hyperdiploidy was more frequent in patients with inv(16) (25% vs 9%, whereas hypodiploidy was more frequent in patients with t(8;21) (37% vs 3%. In multivariable analyses (adjusted for age, white blood counts at diagnosis, and KIT mutation status), trisomy 8 was associated with improved overall survival (OS) in inv(16), whereas the presence of other chromosomal abnormalities (not trisomy 8) was associated with decreased OS. In patients with t(8;21), hypodiploidy was associated with improved disease-free survival; hyperdiploidy and del(9q) were associated with improved OS. KIT mutation (either positive or not tested, compared with negative) conferred poor prognoses in univariate analysis only in patients with t(8;21).

Genome-wide association study identifies novel susceptibility loci for KIT D816V positive mastocytosis.

Mastocytosis is a rare myeloid neoplasm characterized by uncontrolled expansion of mast cells, driven in >80% of affected individuals by acquisition of the KIT D816V mutation. To explore the hypothesis that inherited variation predisposes to mastocytosis, we performed a two-stage genome-wide association study, analyzing 1,035 individuals with KIT D816V positive disease and 17,960 healthy control individuals from five European populations. After quality control, we tested 592,007 SNPs at stage 1 and 75 SNPs at stage 2 for association by using logistic regression and performed a fixed effects meta-analysis to combine evidence across the two stages. From the meta-analysis, we identified three intergenic SNPs associated with mastocytosis that achieved genome-wide significance without heterogeneity between cohorts: rs4616402 (pmeta = 1.37 × 10-15, OR = 1.52), rs4662380 (pmeta = 2.11 × 10-12, OR = 1.46), and rs13077541 (pmeta = 2.10 × 10-9, OR = 1.33). Expression quantitative trait analyses demonstrated that rs4616402 is associated with the expression of CEBPA (peQTL = 2.3 × 10-14), a gene encoding a transcription factor known to play a critical role in myelopoiesis. The role of the other two SNPs is less clear: rs4662380 is associated with expression of the long non-coding RNA gene TEX41 (peQTL = 2.55 × 10-11), whereas rs13077541 is associated with the expression of TBL1XR1, which encodes transducin (ß)-like 1 X-linked receptor 1 (peQTL = 5.70 × 10-8). In individuals with available data and non-advanced disease, rs4616402 was associated with age at presentation (p = 0.009; beta = 4.41; n = 422). Additional focused analysis identified suggestive associations between mastocytosis and genetic variation at TERT, TPSAB1/TPSB2, and IL13. These findings demonstrate that multiple germline variants predispose to KIT D816V positive mastocytosis and provide novel avenues for functional investigation.

[Primary central nervous system lymphomas].

In this review, we discuss primary central nervous system lymphomas: a rare type of tumours confined to the central nervous system. The disease is associated with a poor prognosis, which, however, generally has seen steady improvement over the last four decades, particularly in the younger population. Modern surgical techniques are reserved for diagnosis and has no place in the treatment, which mainly relies on high-dose polychemotherapy treatment regimes with methotrexate as the backbone.

Proteomic profiling identifies outcome-predictive markers in patients with peripheral T-cell lymphoma, not otherwise specified.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) constitutes a heterogeneous category of lymphomas, which do not fit into any of the specifically defined T-cell lymphoma entities. Both the pathogenesis and tumor biology in PTCL-NOS are poorly understood. Protein expression in pretherapeutic PTCL-NOS tumors was analyzed by proteomics. Differentially expressed proteins were compared in 3 distinct scenarios: (A) PTCL-NOS tumor tissue (n = 18) vs benign lymphoid tissue (n = 8), (B) clusters defined by principal component analysis (PCA), and (C) tumors from patients with chemosensitive vs refractory PTCL-NOS. Selected differentially expressed proteins identified by proteomics were correlated with clinico-pathological features and outcome in a larger cohort of patients with PTCL-NOS (n = 87) by immunohistochemistry (IHC). Most proteins with altered expression were identified comparing PTCL-NOS vs benign lymphoid tissue. PCA of the protein profile defined 3 distinct clusters. All benign samples clustered together, whereas PTCL-NOS tumors separated into 2 clusters with different patient overall survival rates (P = .001). Differentially expressed proteins reflected large biological diversity among PTCL-NOS, particularly associated with alterations of "immunological" pathways. The 2 PTCL-NOS subclusters defined by PCA showed disturbance of "stress-related" and "protein metabolic" pathways. α-Enolase 1 (ENO1) was found differentially expressed in all 3 analyses, and high intratumoral ENO1 expression evaluated by IHC correlated with poor outcome (hazard ratio, 2.09; 95% confidence interval, 1.17-3.73; P = .013). High expression of triosephosphate isomerase (TPI1) also showed a tendency to correlate with poor survival (P = .057). In conclusion, proteomic profiling of PTCL-NOS provided evidence of markedly altered protein expression and identified ENO1 as a novel potential prognostic marker.