Primary CNS T-cell Lymphomas: A Clinical, Morphologic, Immunophenotypic, and Molecular Analysis.

Primary central nervous system (CNS) lymphomas are relatively rare with the most common subtype being diffuse large B-cell lymphoma. Primary CNS T-cell lymphomas (PCNSTL) account for <5% of CNS lymphomas. We report the clinical, morphologic, immunophenotypic, and molecular characteristics of 18 PCNSTLs. Fifteen cases were classified as peripheral T-cell lymphoma, not otherwise specified, 2 of which were of γδ T-cell derivation and 1 was TCR silent; there was 1 anaplastic large cell lymphoma, ALK-positive and 2 anaplastic large cell lymphoma, ALK-negative. Median age was 58.5 years (range, 21 to 81 y), with an M:F ratio of 11:7. Imaging results showed that 15 patients had supratentorial lesions. Regardless of subtype, necrosis and perivascular cuffing of tumor cells were frequently observed (11/18 cases). CD3 was positive in all cases but 1; 10/17 were CD8-positive, and 5/17 were CD4-positive. Most cases studied had a cytotoxic phenotype with expression of TIA1 (13/15) and granzyme-B (9/13). Polymerase chain reaction analysis of T-cell receptor γ rearrangement confirmed a T-cell clone in 14 cases with adequate DNA quality. Next-generation sequencing showed somatic mutations in 36% of cases studied; 2 had >1 mutation, and none showed overlapping mutations. These included mutations in DNMT3A, KRAS, JAK3, STAT3, STAT5B, GNB1, and TET2 genes, genes implicated previously in other T-cell neoplasms. The outcome was heterogenous; 2 patients are alive without disease, 4 are alive with disease, and 6 died of disease. In conclusion, PCNSTLs are histologically and genomically heterogenous with frequent phenotypic aberrancy and a cytotoxic phenotype in most cases.

NOTCH1 intracellular domain immunohistochemistry as a diagnostic tool to distinguish T-lymphoblastic lymphoma from thymoma.

Distinction between lymphocyte-rich thymoma and T-lymphoblastic lymphoma/leukemia (T-LBL) can be problematic because of a predominance of precursor T cells in both, particularly if the epithelial component in a thymoma is undersampled. Because of very different clinical implications, accurate diagnosis is critical. The NOTCH1 signaling pathway is frequently activated in T-LBL and plays a central role in the pathogenesis of this disease. Antibodies to NOTCH1 intracellular domain (N1ICD), recognizing the active form of NOTCH1, have been developed. We hypothesized that detection of N1ICD would be useful in distinguishing T-LBL from thymoma and investigated a series of formalin-fixed, paraffin-embedded tissues for immunoreactivity with an N1ICD antibody using automated immunohistochemistry. Slides were scored using a 25% nuclear reactivity threshold for positivity. Hyperplastic tonsil showed positivity in few scattered interfollicular lymphoid cells, suprabasilar epithelial cells, and endothelial cells. Thymocytes from non-neoplastic thymus were largely negative for N1ICD. All thymomas tested (n=23) were negative for N1ICD, although epithelial cells and a small minority of thymocytes may be positive, requiring careful interpretation. All T-LBL cases (n=16) were scored positive for N1ICD: 8 (50%) of these showed diffuse and mostly strong immunoreactivity, whereas the remaining 8 (50%) had less extensive positivity, but with consistently >25% nuclear staining. In conclusion, normal thymocytes do not express significant levels of N1ICD. In keeping with this pattern, thymomas are negative for N1ICD, whereas a high percentage of T-LBL expresses N1ICD. Thus, N1ICD immunohistochemistry appears to be a useful method in distinguishing T-LBL from thymoma.

Frequency, interobserver reproducibility and clinical significance of equivocal peaks in PCR clonality testing using Euroclonality/BIOMED-2 primers.

AIMS: PCR studies for lymphoid clonality are now widely employed, especially using Euroclonality/BIOMED-2 primers. Criteria for interpretation as a clonal result, however, have proven controversial. This study examines the frequency and clinical significance of equivocal amplification patterns and measures the interobserver reproducibility of clonality interpretations. METHODS: At our institution, results of each primer set are first classified as clonal, non-clonal or abnormal (equivocal peak on polyclonal background). Final results for all primer sets are then collectively reported as positive (≥1 clonal result), negative (non-clonal results) or indeterminate (≥1 abnormal result) for a clonal population. Results of 274 consecutive clonality cases were reviewed, and the interobserver reproducibility of individual primer set reactions and final results was determined in a subset of 30 cases. RESULTS: 44/161 (27%) B-cell and 50/163 (31%) T-cell cases contained at least one abnormal peak. Of these, 29 (64%) and 31 (62%), respectively, showed clonal results in another primer set. Interobserver reproducibility was excellent for most primer sets and for final interpretations, but only fair to good for IGK V-J and TCRB D-J1+2 primer sets. A definitive diagnosis of lymphoma was rendered in 93%, 20% and 6% of B-cell cases and 90%, 42%, and 14% of T-cell cases positive, indeterminate or negative for a clonal population, respectively. CONCLUSIONS: Using a subjective approach, abnormal (equivocal) peaks are frequently observed in routine practice. However, most cases with abnormal peaks contain clonal rearrangements in other primer sets, facilitating overall interpretation of final results with excellent interobserver reproducibility.

Solitary extramedullary plasmacytoma of the penis.

Solitary extramedullary plasmacytomas are rare plasma cell malignancies, particularly outside the upper aerodigestive tract. A 90-year-old male presented with a penile mass suspicious for penile carcinoma. Pathology revealed the tumor to be an Epstein-Barr virus-associated plasmacytoma with no radiographic evidence of bone or other soft tissue involvement. There was no laboratory evidence of multiple myeloma.

Follicular lymphomas in children and young adults: a comparison of the pediatric variant with usual follicular lymphoma.

Follicular lymphoma (FL), a common lymphoma in adults, occurs rarely in pediatric and young adult patients. Most pediatric cases have been described as grade 3, but the criteria to distinguish the pediatric variant of FL (PFL) from usual FL (UFL) seen in adults are not well defined. We undertook a study of FL in patients under the age of 30. We identified 63 cases, which were analyzed by morphology, immunohistochemistry, and polymerase chain reaction analysis of IGH@ and IGK@ clonality. These data were correlated with clinical findings including stage, treatment, and outcome. Among the 63 cases, 34 cases were classified as PFL: 22 presenting in lymph nodes, 8 in the Waldeyer ring, and 4 in the testis. Clonal immunoglobulin gene rearrangement was detected in 97% of PFL cases, but fluorescence in situ hybridization analysis showed an absence of the BCL2/IGH@ translocation in all cases tested. Twenty-nine cases were classified as UFL, 28 of which presented in lymph nodes. The nodal PFLs were observed exclusively in male patients in both children and young adults with a median age of 15 years. They showed marked head/neck predilection, blastoid cytologic features with a high proliferation rate, lack of BCL2 protein and t(14;18), low clinical stage at presentation, and good prognosis. PFLs involving the Waldeyer ring were distinguished by MUM1 expression, 50% (3/6) of which carried IRF4 breaks. BCL2 expression was common (63%) in the absence of BCL2/IGH@ translocation. UFLs were more common in female patients, exclusively in young adults (median age, 24 y), with no cases reported in patients under the age of 18. Twenty-five of 29 cases were of grade 1-2, and 4 cases were classified as grade 3A. They exhibited a higher clinical stage at presentation. Eighty-three percent expressed BCL2. Our results indicate that histologic and immunophenotypic criteria can reliably separate PFL and UFL and that UFL is exceptionally rare in the pediatric age group. PFL associated with particular anatomic sites have distinctive features and should be evaluated separately in future clinical and biological studies.

Follicular lymphoma in situ: clinical implications and comparisons with partial involvement by follicular lymphoma.

Follicular lymphoma in situ (FLIS) was first described nearly a decade ago, but its clinical significance remains uncertain. We reevaluated our original series and more recently diagnosed cases to develop criteria for the distinction of FLIS from partial involvement by follicular lymphoma (PFL). A total of 34 cases of FLIS were identified, most often as an incidental finding in a reactive lymph node. Six of 34 patients had prior or concurrent FL, and 5 of 34 had FLIS composite with another lymphoma. Of patients with negative staging at diagnosis and available follow-up (21 patients), only one (5%) developed FL (follow-up: median, 41 months; range, 10-118 months). Follow-up was not available in 2 cases. Fluorescence in situ hybridization for BCL2 gene rearrangement was positive in all 17 cases tested. PFL patients were more likely to develop FL, diagnosed in 9 of 17 (53%) who were untreated. Six patients with PFL were treated with local radiation therapy (4) or rituximab (2) and remained with no evidence of disease. FLIS can be reliably distinguished from PFL and has a very low rate of progression to clinically significant FL. FLIS may represent the tissue counterpart of circulating t(14;18)-positive B cells.

Spontaneous perinephric hematoma due to acquired factor X deficiency in AL amyloidosis.

Spontaneous perinephric hematoma (SPH) is a rare entity whose diagnosis is challenging because of its varied clinical presentation and lack of any specific etiology. We report a 34-year-old African-American male who presented with left flank pain and was found to have a large left perinephric hematoma, in the setting of undiagnosed AL amylodosis. The case illustrates that while a SPH due to the vascular angiopathy of amyloid is rare, when amyloidosis is associated with abnormal coagulation studies or bleeding at multiple sites, it should be considered because of its protean systemic manifestations and potential response to chemotherapy.

Blastic plasmacytoid dendritic cell neoplasm in children: diagnostic features and clinical implications.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm is a rare malignancy that typically follows a highly aggressive clinical course in adults, whereas experience in children with this disease is very limited. DESIGN AND METHODS: This retrospective study analyzed the pathological and clinical findings of nine cases of blastic plasmacytoid dendritic cell neoplasm presenting in patients under the age of 18 years who were reviewed at our institution. We also identified 20 well-documented additional pediatric cases in the literature. RESULTS: In the combined analysis, the overall survival rate among the 25 patients with available follow-up, all having received chemotherapy, was 72% (follow-up ranging from 9 months to 13 years, with a median of 30 months). The event-free survival rate was 64%. Nine patients were alive 5 years after the original diagnosis, although only three of them had undergone hematopoietic stem cell transplantation--one in first complete remission and two in second remission. Of the seven patients who lacked cutaneous disease at presentation, 100% survived, including five who were alive more than 5 years after diagnosis, although only two had undergone stem cell transplantation. Among the 18 patients who presented with cutaneous disease and for whom follow-up data were available, only 11 survived (61%). Detailed immunophenotypic characterization and clinical features of all cases are presented. Unexpectedly, three of four cases of blastic plasmacytoid dendritic cell neoplasm tested showed focal positivity for S-100. S-100 was negative in 28 cases of acute myeloid leukemia evaluated for this marker. CONCLUSIONS: In contrast to adult cases, in which long-term survival depends on stem cell transplantation in first complete remission, blastic plasmacytoid dendritic cell neoplasms in children are clinically less aggressive. Treatment with high-risk acute lymphoblastic leukemia-type chemotherapy appears to be effective, and stem cell transplantation may be reserved for children who relapse and achieve a second remission. Outcomes were more favorable in cases that lacked cutaneous disease at presentation, although a comparison of cutaneous and non-cutaneous cases might be confounded by differences in treatment regimens. Focal expression of S-100 may be seen in concert with other markers of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells: physiologic roles and pathologic states.

Plasmacytoid dendritic cells (PDCs) have perplexed pathologists for decades, undergoing multiple adjustments in nomenclature as their lineage and functions have been characterized. Although PDCs account for less than 0.1% of peripheral blood mononuclear cells, they serve as a principal source of interferon-alpha and are also known as interferon-I producing cells (IPCs). Upon activation in vitro, they can differentiate into dendritic cells, and recent studies have substantiated a potential role in antigen presentation. Thus, PDCs may act as a link between innate and adaptive immunity. Normally found in small quantities in primary and secondary lymphoid organs, PDCs accumulate in a variety of inflammatory conditions, including Kikuchi-Fujimoto lymphadenopathy, hyaline-vascular Castleman disease, and autoimmune diseases, and in certain malignancies such as classical Hodgkin lymphoma and carcinomas. Demonstrating potential for neoplastic transformation reflective of varying stages of maturation, clonal proliferations range from PDC nodules most commonly associated with chronic myelomonocytic leukemia to the rare but highly aggressive malignancy now known as blastic plasmacytoid dendritic cell neoplasm (BPDCN). Formerly called blastic natural killer cell lymphoma or CD4/CD56 hematodermic neoplasm, BPDCN, unlike natural killer cell lymphomas, is not associated with Epstein-Barr virus infection and is generally not curable with treatment regimens for non-Hodgkin lymphomas. In fact, this entity is no longer considered to be a lymphoma and instead represents a unique precursor hematopoietic neoplasm. Acute leukemia therapy regimens may lead to sustained clinical remission of BPDCN, with bone marrow transplantation in first complete remission potentially curative in adult patients.

Galectin-3 induces death of Candida species expressing specific beta-1,2-linked mannans.

Lectins play a critical role in host protection against infection. The galectin family of lectins recognizes saccharide ligands on a variety of microbial pathogens, including viruses, bacteria, and parasites. Galectin-3, a galectin expressed by macrophages, dendritic cells, and epithelial cells, binds bacterial and parasitic pathogens including Leishmania major, Trypanosoma cruzi, and Neisseria gonorrhoeae. However, there have been no reports of galectins having direct effects on microbial viability. We found that galectin-3 bound only to Candida albicans species that bear beta-1,2-linked oligomannans on the cell surface, but did not bind Saccharomyces cerevisiae that lacks beta-1,2-linked oligomannans. Surprisingly, binding directly induced death of Candida species containing specific beta-1,2-linked oligomannosides. Thus, galectin-3 can act as a pattern recognition receptor that recognizes a unique pathogen-specific oligosaccharide sequence. This is the first description of antimicrobial activity for a member of the galectin family of mammalian lectins; unlike other lectins of the innate immune system that promote opsonization and phagocytosis, galectin-3 has direct fungicidal activity against opportunistic fungal pathogens.

Differential roles of SOCS family members in EpoR signal transduction.

To elucidate the roles of suppressor of cytokine signaling (SOCS) family members in erythropoietin (EPO) signaling, we explored SOCS gene regulation, mRNA stability, and protein function in two EPO-responsive hematopoietic cell lines. Using two independent approaches, one involving inhibition of specific signaling molecules and the other employing cell lines that express particular EpoR mutants and thereby activate only subsets of signaling cascades, we demonstrate that induction of SOCS1, SOCS2, SOCS3, and cytokine-inducible SH2-containing protein (CIS) in response to EPO stimulation appears to depend on Stat5 but not on mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). SOCS4 expression, in contrast, does not appear to be EPO inducible. Furthermore, we show differential stabilities of SOCS transcripts, with SOCS2 the longest-lived and SOCS1 and CIS the least stable, and provide evidence in support of EPO-independent expression of SOCS3 and SOCS4. In order to understand the effects of SOCS on EPO-mediated effects, we generated multiple stable cell lines that inducibly express particular SOCS proteins. Overexpression of SOCS1, SOCS3, or CIS negatively regulates EPO-mediated cell proliferation Stat5 phosphorylation, and activation of a Stat-dependent luciferase reporter. In contrast, SOCS2 is less effective, and SOCS4 is ineffective at counteracting EPO-mediated events. Thus, we have demonstrated differential regulation and function of various SOCS family members in EPO-dependent hematopoietic cells.

Erythroid expansion mediated by the Gfi-1B zinc finger protein: role in normal hematopoiesis.

In the search for genes expressed in hematopoietic stem cells, we identified that the expression of Gfi-1B (growth factor independence-1B) is highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, we analyzed its function in human hematopoiesis. Enforced expression of Gfi-1B in human CD34(+) hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B did not promote erythroid commitment, but enhanced proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biologic effects of Gfi-1B were mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription was dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression.

Erythropoietin receptor haploinsufficiency and in vivo interplay with granulocyte-macrophage colony-stimulating factor and interleukin 3.

Erythropoietin (EPO) and its receptor (EPOR) are critical for definitive erythropoiesis, as mice lacking either gene product die during embryogenesis with severe anemia. Here we demonstrate that mice expressing just one functional allele of the EpoR have lower hematocrits and die more frequently than do wild-type littermates on anemia induction. Furthermore, EpoR(+/-) erythroid colony-forming unit (CFU-E) progenitors are reduced both in frequency and in responsiveness to EPO stimulation. To evaluate the interaction between EPO and granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3), GM-CSF(-/-) or IL-3(-/-) mice were interbred with EpoR(+/)(-) mice. Deletion of either GM-CSF or IL-3 also leads to reduction in CFU-E numbers and hematocrits but does not significantly alter steady-state erythroid burst-forming unit numbers. These results suggest EpoR haploinsufficiency and promotion of in vivo erythropoiesis by GM-CSF and IL-3.

Regulation of Socs gene expression by the proto-oncoprotein GFI-1B: two routes for STAT5 target gene induction by erythropoietin.

SOCS proteins take part in a classical negative feedback loop to attenuate cytokine signaling. Although STAT family members positively modulate Socs gene expression, little else is known about Socs gene regulation. Here, we identify functional binding sites for GFI-1B, a proto-oncogenic transcriptional repressor, in the promoters of murine Socs1 and Socs3. Thus, mutating these sites relieved transcriptional repression, as determined by luciferase reporter assays of transiently transfected erythropoietin-responsive 32D-EpoR and HCD57 cells. Furthermore, cotransfection of Gfi-1B expression plasmid repressed reporter activity of wild-type (but not mutagenized) Socs1 and Socs3 promoters, strongly suggestive of direct GFI-1B binding to these promoters. In addition, overexpression of Gfi-1B resulted in reduced transcript levels of Socs1 and Socs3, but not Socs2 or Cis. Upon stimulation with erythropoietin, Socs transcripts were rapidly induced, whereas Gfi-1B mRNA was down-regulated. Interestingly, the latter effect appears to rely on STAT5 activity, but not on phosphoinositide 3-kinase or MAPK pathways. Thus, cytokine-mediated STAT5 activation allows relief of direct repression by GFI-1B of the Socs1 and Socs3 promoters, but apparently not of the Socs2 and Cis promoters. This constitutes a previously undescribed mode of controlling cytokine responsiveness, through the direct repression of a tumor suppressor (SOCS1) by a proto-oncoprotein (GFI-1B).