Ayako Ichikawa

Expression of programmed cell death ligand 1 is associated with poor overall survival in patients with diffuse large B-cell lymphoma

Programmed cell death ligand 1 (PD-L1) is expressed on both select diffuse large B-cell lymphoma (DLBCL) tumor cells and on tumor-infiltrating nonmalignant cells. The programmed cell death 1 (PD-1)/PD-L1 pathway inhibits host antitumor responses; however, little is known about how this pathway functions in the tumor microenvironment. The aim of this study was to determine the clinicopathological impact of PD-L1(+) DLBCL. We performed PD-L1/PAX5 double immunostaining in 1253 DLBCL biopsy samples and established a new definition of PD-L1(+) DLBCL. We also defined the criteria for microenvironmental PD-L1(+) (mPD-L1(+)) DLBCL (ie, PD-L1(-) DLBCL in which PD-L1(+) nonmalignant cells are abundant in the tumor microenvironment). Of the 273 patients whose clinical information was available, quantitative analysis of PD-1(+) tumor-infiltrating lymphocytes (TILs) was performed. The prevalence rates of PD-L1(+) and mPD-L1(+) DLBCL were 11% and 15.3%, respectively. Both PD-L1(+) and mPD-L1(+) DLBCL were significantly associated with non-germinal center B-cell (GCB) type and Epstein-Barr virus positivity. The number of PD-1(+) TILs was significantly higher in GCB-type tumors and lower in mPD-L1(-) and PD-L1(+) DLBCL. Patients with PD-L1(+) DLBCL had inferior overall survival (OS) compared with that in patients with PD-L1(-) DLBCL (P = .0009). In contrast, there was no significant difference in OS between mPD-L1(+) and mPD-L1(-) DLBCL (P = .31). The expression of PD-L1 maintained prognostic value for OS in multivariate analysis (P = .0323). This is the first report describing the clinicopathological features and outcomes of PD-L1(+) DLBCL. Immunotherapy targeting the PD-1/PD-L1 pathway should be considered in this distinct DLBCL subgroup.

Clinicopathological analysis of a composite lymphoma containing both T- and B-cell lymphomas

Composite lymphomas (CLs) have been reported in 1–4.7% of all lymphomas, however, CLs containing both T‐ and B‐cell lymphomas (CTBLs) are very rare. Here, we examined the clinical and pathological features of 29 CTBLs. These CTBLs included 21 patients with angioimmunoblastic T‐cell lymphoma (AITL) and diffuse large B‐cell lymphoma (DLBCL), two with adult T‐cell leukemia/lymphoma and DLBCL, one with AITL and Follicular lymphoma, one with Lennert lymphoma and DLBCL, one with subcutaneous panniculitis‐like T‐cell lymphoma and DLBCL, one with peripheral T‐cell lymphoma (PTCL) and DLBCL, one with cutaneous T‐cell lymphoma and DLBCL, and one with PTCL and chronic lymphocytic leukemia. Eighteen of 27 patients (67%) were shown to be Epstein‐Barr virus (EBV)‐encoded RNA‐positive in their B‐cell lymphoma component. T‐cell and B‐cell clonality were confirmed by flow cytometry, Southern blot analysis, and/or polymerase chain reaction (PCR). Using Southern blot analysis, clonal immunoglobulin heavy chain (IgH) and T‐cell receptor (TCR) rearrangements were detected in 11 of 21 and 15 of 24 cases, respectively. Using PCR analysis, clonal IgH and TCR rearrangements were detected in 7 of 8 and 7 of 7 Southern blot‐negative cases, respectively. Our results suggested that PCR analysis was useful in diagnosing CTBL.

Comparison of CD20 expression in B-cell lymphoma between newly diagnosed, untreated cases and those after rituximab treatment

Few studies have statistically investigated reduced CD20 expression in B‐cell lymphoma after rituximab therapy and genomic mutation of CD20 associated with reduction. We examined CD20‐positive rate in follicular lymphoma (FL) and diffuse large B‐cell lymphoma (DLBCL) by flow cytometry (FCM) and immunohistochemical staining (IHS), comparing 138 cases after rituximab therapy with 360 initial, not yet treated cases. Sequence analysis of exons 3 to 8 of CD20 was performed on 22 cases with low CD20‐positive rate after rituximab treatment. The results showed a statistical correlation between CD20‐positive rate in FCM and IHS. By FCM, the CD20‐positive rate among post‐rituximab cases was significantly lower than among initial cases in DLBCL, non‐germinal center origin B‐cell type (average values [avg] 57.8 and 87.9, respectively) (P < 0.0001), FL2 (avg, 93.9; 103.2) (P = 0.0083), and FL3A (avg, 90.6; 100.7) (P = 0.033). Stratified analyses of post‐rituximab cases showed significantly lower CD20‐positive rate in cases that were resistant at the start of the treatment and cases with progressive disease during rituximab therapy before biopsy. Sequence analysis showed silent mutation of exon 4 (632 C/T) in seven cases, although this number was not statistically significant. These results suggest the influence of B‐lymphoma subtype and a therapeutic effect before biopsy on CD20 expression at relapse and contribute to a better therapeutic approach for relapse cases after rituximab therapy. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02307.x, 2012)

The Wnt signaling pathway and mitotic regulators in the initiation and evolution of mantle cell lymphoma: Gene expression analysis

For an accurate understanding of mantle cell lymphoma (MCL), molecular behavior could be staged into two major events: lymphomagenesis with the t(11;14) translocation (initiation), and evolution into a more aggressive form (transformation). Unfortunately, it is still unknown which genes contribute to each event. In this study, we performed cDNA microarray experiments designed based on the concept that morphologically heterogeneous MCL samples would provide insights into the role of aberrant gene expression for both events. A total of 15 MCLs were collected from the files, which include a total of 237 MCL patients confirmed by histology as CCND1-positive. We posited four stepwise morphological grades for MCL: MCL in situ, MCL with classical form (cMCL), MCL with aggressive form (aMCL), and MCL with intermediate morphology between classical and aggressive forms at the same site (iMCL). To identify genes involved in initiation, we compared the tumor cells of MCL in situ (n=4) with normal mantle zone B lymphocytes (n=4), which were selected by laser microdissection (LMD). To identify genes contributing to transformation, we selected the overlapping genes differentially expressed between both cMCL (n=4) vs. aMCL (n=5) and classical vs. aggressive areas in iMCL (n=2) obtained by LMD. A significant number of genes (n=23, p=0.016) belonging to the Wnt signaling pathway were differentially expressed in initiation. This specific activation was confirmed by immuno-histochemistry, as MCL in situ had nuclear localization of phosphorylated-β-catenin with high levels of cytoplasmic Wnt3 staining. For transformation, identified 60 overlapping genes included a number of members of the p53 interaction network (CDC2, BIRC5 and FOXM1), which is known to mediate cell cycle progression during the G2/M transition. Thus, we observe that the Wnt signaling pathway may play an important role in initial lymphomagenesis in addition to t(11;14) translocations, and that specific mitotic regulators facilitate transformation into more aggressive forms.

High frequency of t(14;18) in Hodgkin's lymphoma associated with follicular lymphoma.

t(14;18) is a common cytogenetic abnormality in B‐cell lymphoma, especially in follicular lymphoma (FL), but is rarely seen in Hodgkins lymphoma (HL). However, due to the small number of cases, the incidence of t(14;18) in HL associated with FL remains unclear. In this study, we applied chromogenic in situ hybridization and fluorescence in situ hybridization for t(14;18) on paraffin‐embedded tissue from four HL associated with FL cases and 11 HL cases without a history of FL for comparison. t(14;18) was present in all of the three successfully‐tested HL associated with FL cases and one case without a history of FL. The frequency of t(14;18) was significantly high in HL associated with FL (p = 0.013). All Hodgkins and Reed‐Sternberg (HRS) cells having t(14;18) showed immunoreactivity for BCL2 and were negatively stained for nuclear factor‐κB (NF‐κB), even in Epstein‐Barr virus (EBV)‐infected cases. However, HRS cells without t(14;18) showed BCL2 and NF‐κB immunoreactivity in 33% and 57% of cases, respectively. There was an inverse correlation between t(14;18) and NF‐κB. In conclusion, we assume the incidence of t(14;18) in HL associated with FL is higher than previously believed and BCL2 expression derived from t(14;18) may play a role in the pathogenesis of HL associated with FL.

Clinicopathological features of double-hit B-cell lymphomas with MYC and BCL2, BCL6 or CCND1 rearrangements.

Double‐hit (DH) lymphomas are B‐cell lymphomas characterized by chromosomal rearrangements, specifically of MYC and either BCL2, BCL6 or CCND1. We reviewed 22 cases of DH lymphomas. BCL2/MYC DH lymphomas constituted the majority of these DH lymphomas (17 cases; 77%), followed by BCL6/MYC (2 cases; 9%) lymphomas. Assessing morphological features using the 2008 World Health Organization classification system, 15 cases (68%) were determined to be B‐cell lymphoma, unclassifiable with features intermediate between diffuse large B‐cell lymphoma (DLBCL) and Burkitt lymphoma (BCLU) (10 cases; 45%), or as DLBCL (5 cases; 23%), and 2 cases (9%) were classified as morphologically untransformed follicular lymphoma. Burkitt lymphoma was rare (1 case; 5%) among DH lymphomas. Nineteen cases were treated with R‐CHOP or a high dose chemotherapy regimen. After a median follow‐up of 11 months, 7 patients had died, and the 1‐year survival rate was 62.5%. High dose chemotherapy did not improve the outcome. We suggest that screening of genetic variations to detect DH lymphomas is required in diagnosing all lymphomas, even those determined morphologically to be follicular lymphoma.

CD5-positive follicular lymphoma characterized by CD25, MUM1, low frequency of t(14;18) and poor prognosis

CD5‐positive follicular lymphoma (FL), although rare, has been described in a number of case reports. However, a statistically valid, clinicopathological comparison between CD5‐positive FL and CD5‐negative FL has never been performed because of its rarity. We statistically compared clinicopathological characteristics of 22 cases of CD5‐positive FL, diagnosed by immunohistochemistry, flow cytometry and morphological findings, with those of 62 cases of FL without CD5 expression (control cases). CD5‐positive FL patients showed a higher tendency of peripheral blood involvement (P = 0.076) and a higher frequency of CD25 expression (P = 0.0004) and MUM1 protein expression (P = 0.0008), and a lower frequency of t(14;18)(q32;q21) (P = 0.017). The overall survival (OS) curve of CD5‐positive FL was significantly worse than that of control cases (P = 0.0266), although progression‐free survival curves did not show a significant difference (P = 0.7899). Moreover, CD5 expression was shown to be an independent poor prognostic factor for OS in both univariate analysis [Hazard Ratio (HR), 3.63; P = 0.0464] and multivariate analysis (HR, 57.16; P = 0.0001). CD5‐positive FL showed different clinicopathological characteristics from FL lacking CD5 expression. These results suggest that CD5‐positive FL should be considered a different type of FL, and its clinicopathological management should be conducted differently.

A spindle cell variant of diffuse large B‐cell lymphoma is characterized by T‐cell/myofibrohistio‐rich stromal alterations: analysis of 10 cases and a review of the literature

Spindle‐shaped diffuse large B‐cell lymphoma (Sp‐DLBCL) has been recognized as a rare morphologic variant of DLBCL. However, the biological processes that contribute to the specific features of Sp‐DLBCL remain poorly understood. In this study, a combined immunophenotypic and genetic analysis was performed in 10 Sp‐DLBCL. First, we investigated several unique markers for anaplasia (CD30, ALK, CD68, and EBER‐ISH), mesenchyma (SMA, desmin, and vimentin), and B‐cell differentiation (CD10, BCL6, and MUM1). We also performed conventional cytogenetic and fluorescence in situ hybridization studies to look for common chromosomal break points (BCL2, BCL6, and MYC). We found that most Sp‐DLBCLs were germinal center B cell‐like and that none had any other specific phenotypes or any karyotypic abnormalities. Instead, T cells, CD68‐positive macrophages and SMA‐positive myofibroblasts were significantly increased in Sp‐DLBCL when compared with conventional GCB origin DLBCL cases (n = 10) (P = 0.012, P < 0.001, and P < 0.0001, respectively). To further characterize Sp‐DLBCL, we next compared the expression of fibroblast growth factor 2 (FGF2) and transforming growth factor‐β1 (TGFβ1) between the two types of DLBCL. Finally, we confirmed that the number of FGF2‐ and TGFβ1‐positive stromal cells was markedly increased in Sp‐DLBCL and that the difference between these and conventional GCB origin DLBCLs was significant (P < 0.0001 and P = 0.0017, respectively). Thus, T‐cell/myofibrohistio‐rich stromal alterations in Sp‐DLBCL, especially those mediated by TGFβ1 and FGF2, may play a role in the transition of lymphoma cells into those with spindle‐shaped features.

Detection of Tax-specific CTLs in lymph nodes of adult T-cell leukemia/lymphoma patients and its association with Foxp3 positivity of regulatory T-cell function

Human T-cell lymphotropic virus type (HTLV)-1 Tax is a viral protein that has been reported to be important in the proliferation of adult T-cell leukemia/lymphoma (ATLL) cells and to be a target of HTLV-1-specific cytotoxic T lymphocytes (CTLs). However, it is not clear how Tax-specific CTLs behave in lymph nodes of ATLL patients. The present study analyzed the immunostaining of Tax-specific CTLs. Furthermore, ATLL tumor cells are known to be positive for forkhead box P3 (Foxp3)and to have a regulatory T (Treg)-cell-like function. The association between T-reg function and number and activity of Tax-specific CTLs was also investigated. A total of 15 ATLL lymphoma cases with human leukocyte antigen (HLA)-A24, for which Tax has a high affinity, were selected from the files of the Department of Pathology, School of Medicine, Kurume University (Kurume, Japan) using a polymerase chain reaction (PCR) method. Immunostaining was performed for cluster of differentiation (CD) 20, CD3, CD4, CD8, T-cell intracellular antigen-1 and Foxp3 in paraffin sections, and for Tax, interferon γ and HLA-A24 in frozen sections. In addition, the staining of Tax-specific CTLs (HLA-A24-restricted) was analyzed by MHC Dextramer® assay in frozen sections. In addition, the messenger RNA expression of Tax and HTLV-1 basic leucine zipper factor were also evaluated by reverse transcription-PCR. Immunohistochemical staining of Tax protein in lymphoma tissue revealed the presence of positive lymphoma cells ranging from 5 to 80%, and immunohistochemical staining of HLA-A24 revealed the presence of positive lymphoma cells ranging from 1 to 95%. The expression of Tax and HLA-A24 was downregulated by viral function. Foxp3, a marker for Treg cells, was expressed in 0–90% of cells. Several cases exhibited Tax-specific CTL (HLA-A24-restricted)-positive cells, and there was an inverse correlation between Tax-specific CTLs and Foxp3. However, neither Tax nor HLA-A24 expression was associated with CTL or Foxp3. Our study indicated the possibility that ATLL cells, which expressed Tax, target of CTL, evade the CTL-mediated immune control by expression of Foxp3 as a Treg function.

Composite lymphoma of peripheral T-cell lymphoma and Hodgkin lymphoma, mixed cellularity type; pathological and molecular analysis

Composite lymphomas (CLs) are defined as two unrelated lymphomas occurring at the same time within the same tissue. The incidence of these tumors is low. Of all possible combinations between lymphomas, the least frequent are the ones combining peripheral T‐cell lymphoma (PTCL) and Hodgkin lymphoma (HL). We recently identified five cases of CL composed of PTCL and classical HL, mixed cellularity type. We investigated histological and clinical features of these cases. Immunostaining was performed on paraffin sections. PTCL cells were positive for CD8 and TIA‐1 in four of the five cases. Hodgkin and Reed‐Sternberg (HRS) cells were positive for CD30 and weakly positive for PAX5 in all cases, positive for CD15 in three of five cases, positive for CD20 in one of five cases, and negative for EBER. Monoclonal rearrangement of the T‐cell receptor (TCR) and immunoglobulin heavy chain (IGH) genes was confirmed by polymerase chain reaction (PCR) using whole paraffin sections. We concluded more precisely the monoclonality of the IGH rearrangement of HRS cells based on single‐cell PCR for IGH and DNA sequencing analysis after laser microdissection of single cells in one case. HL can occur in CD8‐positive and TIA‐1‐positive PTCL. Clinicians should recognize the possibility of these CL.