EBV-associated primary CNS lymphoma occurring after immunosuppression is a distinct immunobiological entity.

Primary central nervous system lymphoma (PCNSL) is confined to the brain, eyes, and cerebrospinal fluid without evidence of systemic spread. Rarely, PCNSL occurs in the context of immunosuppression (eg, posttransplant lymphoproliferative disorders or HIV [AIDS-related PCNSL]). These cases are poorly characterized, have dismal outcome, and are typically Epstein-Barr virus (EBV)-associated (ie, tissue-positive). We used targeted sequencing and digital multiplex gene expression to compare the genetic landscape and tumor microenvironment (TME) of 91 PCNSL tissues all with diffuse large B-cell lymphoma histology. Forty-seven were EBV tissue-negative: 45 EBV- HIV- PCNSL and 2 EBV- HIV+ PCNSL; and 44 were EBV tissue-positive: 23 EBV+ HIV+ PCNSL and 21 EBV+ HIV- PCNSL. As with prior studies, EBV- HIV- PCNSL had frequent MYD88, CD79B, and PIM1 mutations, and enrichment for the activated B-cell (ABC) cell-of-origin subtype. In contrast, these mutations were absent in all EBV tissue-positive cases and ABC frequency was low. Furthermore, copy number loss in HLA class I/II and antigen-presenting/processing genes were rarely observed, indicating retained antigen presentation. To counter this, EBV+ HIV- PCNSL had a tolerogenic TME with elevated macrophage and immune-checkpoint gene expression, whereas AIDS-related PCNSL had low CD4 gene counts. EBV-associated PCNSL in the immunosuppressed is immunobiologically distinct from EBV- HIV- PCNSL, and, despite expressing an immunogenic virus, retains the ability to present EBV antigens. Results provide a framework for targeted treatment.

Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma.

Follicular lymphoma (FL) is typically an indolent disease, but 30-40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies and identified 45 recurrently mutated genes in the FL-tFL data set and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D (MLL2), CREBBP, EZH2, BCL2 and MEF2B. Many recurrently mutated genes are involved in epigenetic regulation, the Janus-activated kinase-signal transducer and activator of transcription (STAT) or the nuclear factor-κB pathways, immune surveillance and cell cycle regulation or are TFs involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.

Synergistic induction of apoptosis in high-risk DLBCL by BCL2 inhibition with ABT-199 combined with pharmacologic loss of MCL1.

Better treatments are needed for patients with diffuse large B-cell lymphoma (DLBCL) at high risk of failing standard therapy. Avoiding apoptosis is a hallmark of cancer, and in DLBCL the redundantly functioning antiapoptotic proteins BCL2 and MCL1 are frequently expressed. Here we explore drugs that cause loss of MCL1, particularly the potent new cyclin-dependent kinase inhibitor dinaciclib, which knocks down MCL1 by inhibiting CDK9. Dinaciclib induces apoptosis in DLBCL cells but is completely overcome by increased activity of BCL2. We find that clinical samples have frequent co-expression of MCL1 and BCL2, suggesting that therapeutic strategies targeting only one will lead to treatment failures owing to activity of the other. The BH3 mimetic ABT-199 potently and specifically targets BCL2. Single-agent ABT-199 had modest antitumor activity against most DLBCL lines and resulted in compensatory upregulation of MCL1 expression. ABT-199 synergized strongly, however, when combined with dinaciclib and with other drugs affecting MCL1, including standard DLBCL chemotherapy drugs. We show potent antitumor activities of these combinations in xenografts and in a genetically accurate murine model of MYC-BCL2 double-hit lymphoma. In sum, we reveal a rational treatment paradigm to strip DLBCL of its protection from apoptosis and improve outcomes for high-risk patients.

Prognostic value of regulatory T cells, lymphoma-associated macrophages, and MUM-1 expression in follicular lymphoma treated before and after the introduction of monoclonal antibody therapy: a Southwest Oncology Group Study.

BACKGROUND: The purpose was to examine the prognostic impact of features of tumor cells and immune microenvironment in patients with follicular lymphoma treated with and without anti-CD20 monoclonal antibody therapy. PATIENTS AND METHODS: Tissue microarrays were constructed from archived tissue obtained from patients on three sequential Southwest Oncology Group (SWOG) trials for FL. All three trials included anthracycline-based chemotherapy. Anti-CD20 monoclonal antibodies were included for patients in the latter two trials. Immunohistochemistry was used to study the number and distribution of cells staining for forkhead box protein P3 (FOXP3) and lymphoma-associated macrophages (LAMs) and the number of lymphoma cells staining for myeloma-associated antigen-1 (MUM-1). Cox proportional hazards regression was used to evaluate the association between marker expression and overall survival (OS). RESULTS: The number or pattern of infiltrating FOXP3 cells and LAMs did not correlate with OS in sequential SWOG studies for FL. The presence of MUM-1 correlated with lower OS for patients who received monoclonal antibody but not for those treated with chemotherapy alone. CONCLUSIONS: Immune cell composition of lymph nodes did not correlate with OS in this analysis of trials in FL. The mechanism of the observed correlation between MUM-1 expression and adverse prognosis in patients receiving monoclonal antibody therapy requires confirmation.

Array-based DNA methylation profiling in follicular lymphoma.

Quantitative methylation profiling was performed using the Illumina GoldenGate Assay in untreated follicular lymphoma (FL) (164), paired pre- and post-transformation FL (20), benign haematopoietic (24) samples and purified B and T cells from two FL cases. Methylation values allowed separation of untreated FL samples from controls with one exception, based primarily on tumour-specific gains of methylation typically occurring within CpG islands. Genes that are targets for epigenetic repression in stem cells by Polycomb Repressor Complex 2 were significantly over-represented among hypermethylated genes. Methylation profiles were conserved in sequential FL and t-FL biopsies, suggesting that widespread methylation represents an early event in lymphomagenesis and may not contribute substantially to transformation. A significant (P<0.05) correlation between FL methylation values and reduced gene expression was shown for up to 28% of loci. Methylation changes occurred predominantly in B cells with variability in the amount of non-malignant tissue between samples preventing conclusive correlation with survival. This represents an important caveat in attributing prognostic relevance to methylation and future studies in cancer will optimally require purified tumour populations to address the impact of methylation on clinical outcome.

Stromal gene signatures in large-B-cell lymphomas.

BACKGROUND: The addition of rituximab to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or R-CHOP, has significantly improved the survival of patients with diffuse large-B-cell lymphoma. Whether gene-expression signatures correlate with survival after treatment of diffuse large-B-cell lymphoma is unclear. METHODS: We profiled gene expression in pretreatment biopsy specimens from 181 patients with diffuse large-B-cell lymphoma who received CHOP and 233 patients with this disease who received R-CHOP. A multivariate gene-expression-based survival-predictor model derived from a training group was tested in a validation group. RESULTS: A multivariate model created from three gene-expression signatures--termed "germinal-center B-cell," "stromal-1," and "stromal-2"--predicted survival both in patients who received CHOP and patients who received R-CHOP. The prognostically favorable stromal-1 signature reflected extracellular-matrix deposition and histiocytic infiltration. By contrast, the prognostically unfavorable stromal-2 signature reflected tumor blood-vessel density. CONCLUSIONS: Survival after treatment of diffuse large-B-cell lymphoma is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment.

Mechanisms underlying thrombocytopenia in the neonatal intensive care unit.

UNLABELLED: Thrombocytopenia is one of the most common hematological problems among neonates in the neonatal intensive care unit (NICU), but in the majority of cases the kinetic mechanism responsible is unclear. This review focuses on both traditional and innovative methods used to evaluate the mechanisms responsible for thrombocytopenia in neonates, and analyzes the data generated from those methods. CONCLUSION: Results of studies using new methods for evaluating thrombocytopenia, coupled with recent descriptions of marrow megakaryocyte mass, suggest that decreased platelet production complicates most cases of thrombocytopenia among neonates in the NICU.

Treatment of refractory acute leukemia with timed sequential chemotherapy using topotecan followed by etoposide + mitoxantrone (T-EM) and correlation with topoisomerase II levels.

A phase I/II clinical study evaluated 17 patients with refractory/recurrent acute leukemia treated with 1.5 mg/m2/day topotecan on days 1-3 followed by etoposide (100 mg/m2/day)+mitoxantrone (10 mg/m2/day) on days 4, 5 and 9, 10. Timed sequential chemotherapy using the topoisomerase I-inhibitor topotecan before the topoisomerase II-inhibitors, etoposide+mitoxantrone (T-EM) treatment is proposed to induce topoisomerase II protein levels and potentiate the cytotoxic activity of the topoisomerase II-directed drugs. Fourteen patients had refractory and three had recurrent acute leukemia. The majority of patients were heavily pre-treated with greater than three re-induction chemotherapy regimens. Ten patients responded to T-EM treatment (59%). Four of seventeen (24%) had a complete remission and one had a partial remission. Four additional patients (24%) who scored complete leukemia clearance had no evidence of disease with complete white and red blood cell recovery but with platelet counts less than 100,000. The lack of platelet recovery in one patient having a partial response was scored as a partial leukemia clearance. The toxicity profile included major non-hematological toxicity including grade 3 mucositis (29%) and neutropenic fever (65%). Paired measurements of intracellular levels of topoisomerase II isoforms alpha and beta in leukemia blast cells (bone marrow) collected before (day 0) and after topotecan treatment (day 4) showed that a relative increase of topoisomerase IIalpha (Topo IIalpha) > or = 40% strongly correlated with response after T-EM treatment. Increased Topo IIalpha levels also corresponded to increased DNA fragmentation. Two patients who had an increase of Topo IIalpha of 20-25% had either a PR or PLC while patients with a < 10% increase showed no response to T-EM treatment. We conclude that timed sequential chemotherapy using topotecan followed by etoposide+mitoxantrone is an effective regimen for patients with refractory acute leukemia, and demonstrate Topo IIalpha protein level increases after topotecan treatment.

The diagnosis of hairy cell leukemia can be established by flow cytometric analysis of peripheral blood, even in patients with low levels of circulating malignant cells.

The diagnosis of hairy cell leukemia (HCL) has traditionally been based on microscopic means. Immunophenotypic analysis of peripheral blood by flow cytometry is not widely recognized as a method for diagnosing HCL, perhaps due to the expectation of low yield of neoplastic cells in patients who are characteristically leukopenic. The abnormal coexpression of CD103, CD25, and intense CD11c and CD20 on monotypic, slightly large B-lymphocytes has previously been shown to be highly characteristic of HCL. We wished to determine if this pattern was valuable in the diagnosis of HCL in leukopenic patients with low levels of neoplastic cells in the peripheral blood. The abnormal immunophenotype above was observed in 25 peripheral blood specimens from patients with unexplained cytopenias or suspected lymphoproliferative processes. Ten of the 25 blood samples exhibited this abnormal phenotype in less than 5% of circulating leukocytes (ranging from <1% to 4%). All 10 patients had other manifestations of HCL, including cytopenias (mean white blood cell count, 1.8 x 10(3)/mm(3); hemoglobin, 11.0 gm/dl; platelets, 74 x 10(3)/mm(3)), splenomegaly, and typical bone marrow morphologic changes. Eight of the 10 patients achieved an excellent response to one course of 2-CDA, with significant improvement of cytopenias (mean white blood cell count: 5.3 x 10(3)/mm(3); hemoglobin: 14.4 g/dl; platelets: 181 x 10(3)/mm(3)) and regression of splenomegaly. One patient had a partial response to alpha interferon and a subsequent complete response to 2-CDA, and one died during treatment. In conclusion, flow cytometric immunophenotyping of peripheral blood is capable of detecting low levels of circulating malignant cells in HCL, even in leukopenic patients. As such, it can be a very useful, non-invasive tool in the diagnosis of this disorder.

Congenital autoimmune neutropenia in two premature neonates.

Autoimmune neutropenia (AIN) has been reported in infants and children, but not in neonates. AIN is caused by antibodies produced by the patient against their own neutrophils; therefore, it differs from the more common alloimmune neonatal neutropenia and the neonatal neutropenia because of a maternal autoimmune disease in which antineutrophil antibodies of maternal origin cross the placenta. We observed 2 cases of congenital AIN in premature neonates. These are the youngest reported cases, and indicate that AIN can have a prenatal onset. Examination of the bone marrow biopsies revealed an increase in B lymphocytes and myeloperoxidase-positive cells with a maturation arrest at the myelocyte stage. Recombinant human granulocyte colony-stimulating factor effectively treated the neutropenia, as it does in infantile AIN. Ten months after the diagnosis, 1 of the patients still requires recombinant human granulocyte colony-stimulating administration.

A practical approach to evaluating and treating neutropenia in the neonatal intensive care unit.

Neutropenia is a relatively common problem in the NICU, recognized in as many as 8% of patients at some time during their hospital stay. In most instances, neutropenia among NICU patients is of short duration and has little influence on outcome. In other cases it is prolonged and severe, and constitutes a serious antimicrobial defense deficiency. When a neonatologist discovers a low blood neutrophil count, choices must be made regarding further evaluation and treatment. The authors hope that the information provided in this article is useful in making these choices.

Evaluation and treatment of thrombocytopenia in the neonatal intensive care unit.

Thrombocytopenia is a very frequent problem among sick neonates, affecting up to 35% of all infants admitted to the NICU. Although multiple clinical conditions have been causally associated with neonatal thrombocytopenia, the cause of the thrombocytopenia is unclear in up to 60% of affected neonates. This article provides neonatologists with a practical approach to the thrombocytopenic neonate, with an emphasis on conditions that could be life-threatening or could have significant implications for further pregnancies. An overview of the current therapeutic modalities is also presented, including a discussion of the possible use of recombinant thrombopoietic cytokines to treat certain groups of thrombocytopenic neonates.

Benign hematogone-rich lymphoid proliferations can be distinguished from B-lineage acute lymphoblastic leukemia by integration of morphology, immunophenotype, adhesion molecule expression, and architectural features.

Distinction of normal B-lymphoid proliferations including precursors known as hematogones from acute lymphoblastic leukemia (ALL) is critical for disease management. We present a multiparameter assessment of 27 bone marrow samples containing at least 25% hematogones (range, 25%-72%) by morphologic review. We used flow cytometry to evaluate B-cell differentiation antigen and adhesion molecule expression and immunohistochemistry on clot sections to evaluate architectural distribution. Flow cytometry revealed that intermediately differentiated cells (CD19+, CD10+) predominated, followed in frequency by CD20+, surface immunoglobulin-positive cells, with CD34+, terminal deoxynucleotidyl transferase (TdT)-positive cells as the smallest subset. Adhesion molecules (CD44, CD54) were expressed more heterogeneously compared with expression in acute lymphoblastic leukemia. Immunohistochemistry revealed that CD34+, TdT-positive cells were dispersed without significant clustering, while CD20+ cells exceeded CD34/TdT-positive cells in 24 of 25 cases. This multidisciplinary study demonstrates that hematogone-rich lymphoid proliferations exhibit a spectrum of B-lymphoid differentiation antigen expression with predominance of intermediate and mature B-lineage cells, heterogeneity of adhesion molecule expression, and nonclustered bone marrow architectural distribution.

Microsatellite instability is not a defining genetic feature of acute myeloid leukemogenesis in adults: results of a retrospective study of 132 patients and review of the literature.

The frequency of acute leukemia in children with constitutional DNA repair defects implicates defective DNA repair in leukemogenesis. Whether sporadic cases of AML also arise from an inherited genetic predisposition remains to be determined. Prior studies have reported microsatellite instability (MSI) in AML, particularly secondary and relapsed AML. These studies included small numbers of cases in which key features such as cytogenetic abnormalities were not reported. To determine whether defective DNA mismatch repair, reflected by MSI, is a defining feature of adult myeloid leukemogenesis, we retrospectively studied 132 AML cases including 28 de novo, 62 secondary, 22 relapsed/refractory, 15 cases of paired diagnosis/relapse. 110 patients were elderly (55+ years). The cases included a range of cytogenetic abnormalities. MSI was assessed at three loci (BAT 25, BAT 26, BAT 40) in DNA isolated from sorted leukemic blasts and paired T cell controls. Fluoresceinated PCR products were analyzed using an automated capillary electrophoresis system. Of the 132 AML cases, no single case demonstrated MSI. Our studies indicate that MSI, and defective DNA mismatch repair, is not a defining feature of the majority of adult patients with AML. Furthermore, our data does not support the hypothesis that MSI could be acquired during the progression of AML from diagnosis to relapse, as a consequence of therapeutic exposure.

A bone marrow biopsy technique suitable for use in neonates.

Thrombocytopenia and neutropenia are common among neonates in intensive care units. Bone marrow aspirations are sometimes performed as part of their evaluation. However, marrow biopsies have not been reported from living neonates. Since architecture and cellularity cannot generally be accurately assessed from marrow aspirates, we devised a biopsy technique which we successfully applied to five cytopenic neonates (three with severe persistent thrombocytopenia and two with idiopathic neutropenia). This technique used a 19 gauge, half-inch Osgood needle to obtain bone marrow clots from the tibias of small preterm neonates which enabled the assessment of marrow cellularity and architecture. On the basis of our initial experience we have ceased using the traditional bone marrow aspiration technique in neonates and now use this technique exclusively.

The major vault protein (MVP), a new multidrug resistance associated protein, is frequently expressed in multiple myeloma.

The major vault protein (MVP), a ribonucleoprotein complex which mediates the transport of xenobiotic toxins, has been implicated in multidrug resistance (MDR) not mediated by p-glycoprotein (P-gp) or multidrug resistance related protein (MRP). We evaluated, via immunohistochemistry, the presence of MVP in plasma cells of myeloma patients. Among 73 patients registered with the Southwest Oncology Group (SWOG), 52 patients (74%) were positive for MVP. The presence of MVP and P-gp were significantly associated (p < 0.01). A univariate analysis of response versus MVP positivity showed borderline statistical significance (p = 0.043) with no association with OS or PFS. In particular, MVP positivity at first biopsy was associated with non-responsiveness to therapy (7/7 patients, 100%). MRP was not present in any of 23 samples tested. An increased proliferative rate (Ki-67 > 5%) was significantly associated with shorter OS (log rank p-value = 0.0002). The collective work indicates that MVP protein is common and abundant in myeloma with potential relevance to therapeutic response.

The presence of CD34+ cell clusters predicts impending relapse in children with acute lymphoblastic leukemia receiving maintenance chemotherapy.

Early detection of relapse in children with acute lymphoblastic leukemia (ALL), as well as distinction of leukemic blasts from hematogones, can be difficult by morphologic examination alone. Using CD34 and terminal deoxynucleotidyl transferase (TdT) immunoperoxidase stains, we studied specimens from 25 children with ALL in morphologic remission to determine if we could identify children at risk of relapse. We studied morphologic remission bone marrow specimens from 9 patients who experienced relapse during the subsequent 6 months and 16 children who remained in complete remission, including 10 specimens with increased numbers of hematogones. Despite morphologic remission, clusters of more than 5 CD34+ and/or TdT-positive cells were identified before overt relapse in 6 of 9 cases of relapse, but were noted in only 1 of 10 specimens from children in continuous complete remission and none of 10 specimens with increased numbers of hematogones. Clusters of CD34+ or TdT-positive cells can identify individual patients at risk for imminent relapse. Hematogones may be differentiated from lymphoblasts by this method.

Rapid automated combined in situ hybridization and immunohistochemistry for sensitive detection of cytomegalovirus in paraffin-embedded tissue biopsies.

The authors questioned whether an automated kinetic mode assay of combined cytomegalovirus (CMV) late viral message and immediate and early antigens might result in a more sensitive and timely CMV diagnosis relevant to speedy treatment in the transplant setting. Toward this end, two cohorts were studied using automated in situ hybridization (ISH) for CMV as well as immunohistochemistry (IHC). The first cohort of patients consisted of 19 cases that were histologically positive (CMV-associated cytopathic change). A second cohort consisted of 10 cases that were histologically negative, yet culture positive. From the first cohort of histologically positive cases, 100% were positive by both ISH and IHC run on separate slides. In the second cohort, CMV was detected overall in 70% of cases (50% by ISH alone and 30% by IHC alone). These results indicate that a combined assay of ISH and IHC can detect more cases than routine hematoxylin and eosin staining or either assay alone. In two illustrative cases, used to demonstrate the feasibility of combining ISH and IHC, the authors used a combined two-color assay (ISH and IHC) performed sequentially on the same slide. The combined assays resulted in colocalized single cell message and protein in some cells and demonstrated more positive cells overall (some positive by IHC alone, some by ISH alone, and some by both) than either assay alone. The combined dual color assay can be completed within 4 to 5 hours giving the prospect of a same day result, which is faster than shell vial technique with immunofluorescence (24 to 48 hours) or culture (7 to 14 days). This study demonstrates that combining CMV message and protein assays results in a more sensitive assay and, when carried out in the kinetic mode, allows a speedy result relevant to early anti-CMV therapy.