Follicles Of Follicular Lymphomas Research Articles

Immunohistochemical Characterization of H3K4Me3 in Reactive Lymph Nodes and Follicular Lymphoma

Abstract Introduction/Objective Genes involved in histone methylation are frequently mutated in non-Hodgkin’s lymphomas. For instance, frequent mutations in genes encoding histone methytransferases MML2 and EZH2 are present in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). The aim of this study was to characterize the immunohistochemical expression of H3K4Me3 in benign/reactive lymph nodes (LNs) with comparison to follicular lymphoma (FL). Methods/Case Report Immunohistochemical staining with an anti-H3K4Me3 antibody was performed on FFPE whole slide section from patients with benign/reactive LNs (n=21), low grade (grade 1-2) FL (n=21). H3K4Me3 reactivity was scored for staining intensity and percentage of lymphocytes showing reactivity. Results (if a Case Study enter NA) The majority of the reactive LN sections (15 out of 21 cases) showed a distinct distribution of H3K4Me3 staining, with the majority of cells in the mantle zone and the interfollicular zones showing moderate-strong staining, whereas reactive germinal centers (GCs) showed significantly decreased or close to negative staining. Neoplastic follicles in all the FL cases contained positive cells with significantly stronger staining compared to that in the germinal centers in benign lymph nodes. The interfollicular zones, while diminished in FL due to expanded neoplastic follicles, showed retained H3K4Me3 staining. The difference in staining intensity between follicles and mantle/interfollicular zones became indistinct in FL. Conclusion H3K4Me3 expression in benign/reactive LNs is characterized by positive expression in lymphocytes in interfollicular and mantle zones and significantly decreased in GCs. However, the expression pattern is different in FL, which showed significantly increased expression in the follicles compared to that in reactive GCs. In FL, the expression in GCs is similar to that in the interfollicular and mantle zone lymphocytes. It is reported that about 90% of FL have MLL2 mutation and MLL2 is the enzyme responsible for the methylation of H3K4. So far, it is unclear whether the mutation of MLL2 in FL affects the overall methylation activity of the enzyme. In our study, there is increased methylation of H3K4 in the follicles of FL, which raises the possibility that MLL2 mutation somehow increases H3K4 methylation.

Characterization of the Immunogenomic Landscape of Follicular Lymphoma

Background: Follicular Lymphoma (FL) is an indolent, but incurable form of non-Hodgkin lymphoma (NHL), with variable clinical behavior. The immune environment plays a major role in dictating the biology of FL. For example, gene expression profiling experiments have demonstrated that recurring “immune response” gene signatures in diagnostic FL specimens were more accurate in predicting clinical outcomes than gene expression patterns in FL cells themselves. Furthermore, immune cells, including T cells, are often found infiltrating or surrounding malignant follicles in FL, and our group has identified the presence of highly clonal T cell populations in most FL samples, suggesting that FL cells express antigens that drive the expansion of lymphoma-specific T cells. In addition, inactivating mutations in B2 microglobulin are relatively common in FL, indicating that down-regulation of antigen expression may occur as a result of host immune surveillance. Lastly, FL is responsive to immunotherapies, such as PD-1 antibody blockade. Importantly, the FL antigens that promote spontaneous immune recognition remain unknown. Emerging data has revealed a critical relationship between the genetic landscape and the immunogenicity of cancer. Somatic mutations in malignant cells encode for altered proteins (i.e. neoantigens) that are “sensed” as foreign by T cells, and the mutational “load” of a given tumor has direct implications for the host immune response.Methods: To address the hypothesis that somatic mutations generate neo-antigens which are the targets of T cell recognition in patients with FL, whole exome sequencing (WES) was performed on FACS-purified FL B cells (CD10+CD19+ cells) and matched T cells from 7 FL biopsy samples. HLA A, B and C haplotypes were predicted using PolySolver, OptiType, Phlat and Seq2hla software with >90% concordance. HLA binding affinity was predicted for all possible 8-11-mer peptides generated from each somatic mutation and corresponding wild-type peptides using NetMHCpan v2.8. These tiled peptides were analyzed for their predicted binding affinities (IC50 nM) to HLA class I molecules. An IC50 <150 nM was considered a predicted strong binder, ≥150 but ≤500 nM an intermediate to weak binder, and >500 nM a non-binder.Results: A median number of 71 somatic mutations were identified in each sample (range, 8-135). Non-synonymous single nucleotide variants (SNV) were cataloged, and in silico analysis was utilized to predict potential high-affinity epitopes capable of binding patient-specific HLA molecules. We achieved an average of 90% proportion of nucleotides > Phred quality score 30 of total bases, according to the base calling accuracy (range, 89.02%-92.06%). G>A/C>T transitions were the most common nucleotide substitutions observed. Consistent with published reports, recurrent somatic mutations were identified in EZH2, CREBBP, IGLL5, MUC21, PCLO, and PIM1. We also identified recurrent mutations in BCL2 and MLL2 (frameshift insertions) known to dominate the mutational landscape of FL. Mutations in HLA class II alleles were common, occurring in 3/7 samples. In silico analysis was next employed to identify putative neo-antigens. A total of 139 candidate neo-antigenic peptides were predicted across all patients. On average, 19 neo-antigens were predicted for each patient (range, 3-41). No public neo-antigens were identified, suggesting that the neo-antigens arising in FL are patient-specific. This observation is consistent with what has been described for other cancer subtypes. Functional validation of lead candidate neo-antigen peptides is ongoing.Conclusion: While overall numbers of somatic mutations are modest in FL, putative neo-antigens were identified in all patient samples analyzed. Our findings suggest that immune therapies incorporating neo-antigen-specific approaches, such as personalized vaccines, should be explored in FL patients. DisclosuresNo relevant conflicts of interest to declare.

The architecture of neoplastic follicles in follicular lymphoma; analysis of the relationship between the tumor and follicular helper T cells.

CD4+ T-follicular helper cells are essential for the survival, proliferation, and differentiation of germinal center B cells and have been implicated in the pathogenesis of follicular lymphoma (FL). To further define the role of these cells in FL, we used multiparameter confocal microscopy to compare the architecture of normal and neoplastic follicles and next generation sequencing to analyze the T-cell receptor repertoire in FL lymph nodes (LN). Multiparameter analysis of LN showed that the proportion of T-follic-ular helper cells (TFH) in normal and neoplastic follicles is the same and that the previously reported increase in TFH numbers in FL is thus due to an increase in the number and not content of follicles. As in normal germinal centers, TFH were shown to have a close spatial correlation with proliferating B cells in neoplastic follicles, where features of immunological synapse formation were observed. The number of TFH in FL correlate with the rate of B-cell proliferation and TFH co-localized to activation induced cytidine deaminase expressing proliferating B cells. T-cell receptor repertoire analysis of FL LN revealed that follicular areas are significantly more clonal when compared to the rest of the LN. These novel findings show that neoplastic follicles and germinal centers share important structural features and provide further evidence that TFH may play a role in driving B-cell proliferation and genomic evolution in TFH. Our results also suggest that targeting this interaction would be an attractive therapeutic option.

T-Cell Clustering in Neoplastic Follicles of Follicular Lymphoma

The nonneoplastic microenvironment is abundant in follicular lymphoma. Its composition has been reported to be associated with the course of the disease. Lack of animal models hampers studies of interaction between lymphoma and bystander cells. We aimed to identify indicators of cellular interaction exemplified by nonrandom distribution of cell types within neoplastic follicles. Physiological germinal centers and follicles in follicular lymphoma were stained to identify macrophages, all T, follicular T-helper, dendritic and B cells. Density of cell types and cell distribution (spatial point pattern) were analyzed by digital image analysis. The density of all T, follicular T-helper and dendritic cells was higher in the dark zone than in the light zone of physiological germinal centers. Densities of cell types in follicular lymphoma were intermediate between the light and the dark zone. All cell types analyzed showed a completely random spatial distribution pattern within the dark and the light zone, respectively. In follicular lymphoma B cells and macrophages displayed complete spatial randomness. In contrast, all T cells, follicular T-helper cells and dendritic cells showed clustering of each individual cell type within a radius of 6–10 μm in the lymphoma. We conclude that the distribution of nonneoplastic cells within follicles of follicular lymphoma is not random. T cells and dendritic cells form clusters within the follicles, suggestive of sites of interaction between microenvironment and lymphoma cells. These clusters might help to understand the interaction of lymphoma cells with the microenvironment and might provide a structure for therapeutic intervention.

Composite lymphomas: Experience from a tertiary cancer center in Kerala, South India.

Composite tumors are defined as tumors in which there are two different intermixed histologic types. Our objective was to study the clinical and pathologic features of five cases of composite lymphoma. Our study included five patients of composite lymphoma diagnosed over a period of 5 years. Clinical presentation, hematological parameters including peripheral smear, bone marrow aspirate, and histopathological examination of lymph node including immunohistochemistry (IHC) were studied. Treatment and follow-up details were also noted. All the five cases were in the adult age group ranging from 44 to 72 years. All the cases were composite follicular lymphoma (FL) and mixed cellularity classical Hodgkin lymphoma (CHL). Diagnosis in all cases was suspected on morphology by identification of distinct neoplastic follicles in FL and classic Reed-Sternberg cells in CHL and confirmed by IHC. Although rare, composite lymphomas should be kept in mind. Careful histopathological examination of lymph node with identification of distinct morphological features along with IHC helps to arrive at the definitive diagnosis.

Identification of TRA-1-60-Positive Cells As a Potent Refractory Population in Follicular Lymphomas

Despite receiving Rituximab-combined chemotherapy, approximately half of follicular lymphoma (FL) patients suffer tumor recurrence, a major clinical issue. Retrieval of lymphoma stem-like cells from these poorly understood, intractable lymphomas would thus greatly ameliorate the cancer therapeutics. Here we demonstrate that the TRA-1-60-expressing cells are a unique population in FLs, converge to the conventional stem cell marker Oct3/4 and ALDH1-positive population, and strongly resist current agents against B-cell lymphomas.TRA-1-60 expression was detected in minor populations of resting B-lymphocytes inside germinal centers in reactive lymphoid tissue, whereas scattered TRA-1-60-expressing cells were detected in the marginal area of neoplastic follicles of FLs, close to small blood vessels. Translocation t(14;18) was thus detected in DNAs from the TRA-positive cells in all five patients’ samples, which evinced the identical pattern of gene fusion as that of TRA-negative cells in the same tissue in 4 of 5 fresh frozen FL samples. Retrospective histological comparison between the recurrent and cognate primary tissues revealed that the number of TRA-1-60-positive lymphoma cells from R-CHOP treated FL subjects had increased four-fold relative to primary tissue, a finding corroborated by in vitro assay on Rituximab treated 2 cell lines, wherein TRA-positive cell numbers increased over ten to thirty-fold compared to the untreated sample. Moreover, TRA-1-60-positive cell population was markedly resistant to current therapeutic agents for B-cell lymphomas. Concordantly, small numbers of TRA-1-60-positive cells implanted subcutaneously into NOD/SCID mice evinced potent tumor-initiating capacity in vivo, where tumors were twelve-fold larger in volume (p=0.0021<0.005) and thirteen-fold heavier in weight (p=0.0015<0.005) compared to those xenografted from the TRA-negative cells.In conclusion, while further investigation for molecular insights will be necessary, it is now clear that TRA-1-60 should be a prominent focus among cellular markers in follicular lymphoma research, by which we ultimately hope to explain clinical intractability against combination-Rituximab and other therapies, and to better understand the stemness among a range of lymphoma cell types. DisclosuresMaeda:Mundipharma KK: Research Funding.

Abstract 3356: Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker

Abstract Gene expression profiling experiments have demonstrated that the immune microenvironment in follicular lymphoma (FL) plays an important role in controlling the behavior of the disease. FL patients with tumors enriched for T cell-related transcripts have a more favorable prognosis. As T cells have been shown to mediate anti-tumor immunity in other cancers, they may also be critical in controlling the progression of FL. T cells are often identified in and around malignant follicles in FL by immunohistochemistry, but it is not known whether their presence relates to their specificity for antigens expressed by FL cells. Hypothesizing that at least a fraction of T cells in FL are truly lymphoma antigen-specific, we have begun to characterize the T cell receptor (TCR) repertoire of FL tissues using a next generation sequencing platform, and have deeply sequenced T cell receptor (TCR) cDNAs of T cell subset populations present in pretreatment FL biopsy specimens. Regulatory T cell (Treg) TCRs in FL tissues revealed a highly oligoclonal expansion, compared to those in control lymph nodes. Furthermore, an inverse correlation was observed between the diversity of Treg and CD8+ TCRs in FL specimens. Interestingly, a tumor from an FL patient, who has received no anticancer treatment for more than 10 years, was found to have missense mutations in the peptide-binding domain of both HLA class I and class II molecules, which might have presented tumor-specific antigens and enhanced host immune responses. Although further verification is required, our data suggest that the T cell repertoire is skewed and restricted in FL and support the evolving understanding of the microenvironment in this disease. Citation Format: Justin Paul Kline, Xiao Liu, Girish Venkataraman, Jiaying Lin, Kazuma Kiyotani, Sonali M. Smith, Magdeline Montoya, Yusuke Nakamura. Diversity of CD8+ and rgulatory T cells is inversely correlated in follicular lymphoma: A potential predictive biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3356. doi:10.1158/1538-7445.AM2015-3356

Functional expression of CD137 (4-1BB) on T helper follicular cells

CD137 (4-1BB) is a surface protein initially discovered to mark activated T lymphocytes. However, its broader expression pattern also encompasses activated NK cells, B cells and myeloid cells, including mature dendritic cells. In this study, we have immunostained for CD137 on paraffin-embedded lymphoid tissues including tonsils, lymph nodes, ectopic tertiary lymphoid tissue in Hashimoto thyroiditis and cancer. Surprisingly, immunostaining mainly decorated intrafollicular lymphocytes in the tissues analyzed, with only scattered staining in interfollicular areas. Moreover, pathologic lymphoid follicles in follicular lymphoma and tertiary lymphoid tissue associated with non-small cell lung cancer showed a similar pattern of immunostaining. Multispectral fluorescence cytometry demonstrated that CD137 expression was restricted to CD4+ CXCR5+ follicular T helper lymphocytes (TFH cells) in tonsils and lymph nodes. Short-term culture of lymph node cell suspensions in the presence of either an agonistic anti-CD137 monoclonal antibody (mAb) or CD137-ligand stimulated the functional upregulation of TFH cells in 3 out of 6 cases, as indicated by CD40L surface expression and cytokine production. As a consequence, immunostimulatory monoclonal antibodies targeting CD137 (such as urelumab and PF-05082566) should be expected to primarily act on this lymphocyte subset, thus modifying ongoing humoral immune responses in patients with autoimmune disease and cancer.

Connexin 43 Communication Channels in Follicular Dendritic Cell Development and in Follicular Lymphomas

Follicular dendritic cells (FDC) show homo- and heterocellular metabolic coupling through connexin 43 (Cx43) gap junctions and support B cell selection and maturation in germinal centers. In follicular lymphomas B cells escape apoptosis while FDC develop abnormally. Here we tested Cx43 channels in reactive FDC development and follicular lymphomas. In culture, the treatment of FDC-B cell clusters (resembling to “ex vivo” germinal centers) with Gap27 peptide, mimicking the 2nd extracellular loop of Cx43 protein, significantly impaired FDC-B cell cluster formation and cell survival. In untreated cultures of intact clusters, cell proliferation showed a moderate reduction. In tissues, Cx43 protein levels run parallel with the density of FDC both in reactive germinal centers and in malformed follicles of follicular lymphomas and showed strong upregulation in newly generated and/or degrading bi-/multinuclear FDC of rudimentary processes. However, the inverse correlation between Cx43 expression and B cell proliferation seen in reactive germinal centers was not detected in follicular lymphomas. Furthermore, Cx43 levels were not associated with either lymphoma grade or bone marrow involvement. Our results suggest that Cx43 channels are critical in FDC and “ex vivo” germinal center development and in the persistence of FDC in follicular lymphomas but do not affect tumor progression.

Multiparameter Microscopy Analysis of the Follicular Lymphoma Microenvironment and Normal Germinal Centers: In Vivo evidence That Follicular Helper T Cells Form Synapses with Neoplastic B Cells and Are Associated with Proliferation and Expression of Activation Induced Cytidine Deaminase

The tumor microenvironment plays a central role in the pathogenesis of follicular lymphoma (FL) and has been shown to influence prognosis. The biological basis for this and the contribution of individual cell types however, remain unclear. In this study we compared the cellular content and structure of neoplastic follicles in FL with their normal counterparts in reactive lymph nodes (LNs). We specifically focused on follicular helper T cells (TFH) which, in normal germinal centers (GCs), form immune synapses with antigen responsive B cells triggering B cell proliferation and expression of activation induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class switch recombination. This is of relevance because off-target AID activity is thought to play a role in generating the mutations that characterize progressive FL.A limitation of previous studies of the FL microenvironment is the use of either single parameter immunohistochemistry which fails to accurately define the complex populations of cells involved, or flow cytometry on disaggregated cells which results in the loss of architectural information. In this study we used multiparameter confocal immunofluorescent (IF) microscopy to investigate in vivo the phenotype, distribution and interaction of CD4+ T cells in FL and to determine to what extent these are similar to normal GCs.Confocal IF microscopy was performed on multiple sections of formalin fixed paraffin embedded LN biopsy specimens from 20 patients with untreated FL, comparison was made with reactive LNs (n=5) and chronic lymphocytic leukemia (CLL) LN biopsies (n=5). Each section was stained with a combination of up to 4 simultaneously applied primary antibodies against CD3, CD4, CD20, PD1, ICOS, BCL6, AID, and Ki67, and fluorescently labelled secondary antibodies. Microscopy was performed using a Nikon TiE fluorescent microscope equipped with A1R Si Confocal imaging system; images were analyzed using NIS software.Results show that CD4+ T cells in FL are mainly located in the inter-follicular regions but they were also identified within the follicles in all cases. Combination staining with anti-CD4, PD1, and ICOS revealed that 23% (95%CI 18-27) of CD4+ T cells within follicles co-express PD1 and ICOS consistent with a TFH phenotype which is significantly higher than in inter-follicular areas where only 5% (95% CI 3-7) of CD4+ cells had this phenotype (p<0.001). PD1+ ICOS+ T cells were positive for the transcription factor BCL6, further confirming the TFH phenotype. There was no significant difference in the proportion of CD4+ cells that were TFH in FL follicles and reactive LN GCs. In CLL cases, 54% of CD4+ cells expressed PD1 but only 9% co-expressed PD1 and ICOS, significantly lower than either FL follicles or GCs (p<0.001). Automated analysis of 3D z-stacks demonstrated a very close spatial relationship between proliferating tumor cells and TFH in FL with a mean of 42% (95%CI 35-48) Ki67+ tumor cells in direct contact with TFH cells. No association was seen between the extent of co-localization and histological grade. A similar pattern of co-localization of TFH cells next to proliferating B cells was also identified in the light zones of reactive GCs. Of note, we also identified features of synapse formation between TFH cells and proliferating tumor cells; TFH cells demonstrated projections that encompass the tumor cell with distortion of the T cell nucleus and increased CD4 and PD1 expression at sites of cell contact (Figure 1). These findings were similarly present in reactive GCs. Finally, AID was expressed in proliferating GC B cells and in proliferating tumor cells in FL. AID expressing cells were found to be in close contact with PD1+ T cells in both GCs and FL.Our findings show many parallels between the follicles of FL and normal GCs. In particular the proportion of CD4+ T cells with a TFH phenotype and their localization in direct contact with proliferating AID+ B cells were very similar. Of note, features of immune synapses were observed in both GCs and FL. Taken together, the data suggest that TFH cells have an important role in the pathogenesis of FL just as they are vital in the normal GC reaction. Interruption of this interaction is a potential therapeutic target. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Differentiating germinal center‐derived lymphomas through their cellular microenvironment

Recent studies on normal and malignant B-cells have provided evidence that the germinal center (GC) of lymphoid follicles exerts a role in B-cell physiology and malignancy. GC-derived lymphomas include both B-cell and T-cell lymphomas. Remarkably, tumor cells of GC-derived lymphomas proliferate in close association with cellular environment that retains key features of normal GC cellular microenvironment. Neoplastic follicles in follicular lymphoma contain, in addition to follicular dendritic cells (FDC) other non-neoplastic cells including macrophages and GC T-cells. In addition to aggregates of FDCs, the background infiltrate of nodular lymphocyte predominant Hodgkin lymphoma includes small B-cells, T-cells, and histiocytes. Typically, most of the lymphocyte predominant (LP) cells are ringed by CD3+/CD4+ T-cells expressing CD57, PD1, BCL6, and MUM1/IRF4. By contrast, Reed-Sternberg cells of classic Hodgkin lymphoma (cHL) are surrounded by CD3+/CD4+ T-cells expressing CD40L. Unlike cHL and other peripheral T-cell lymphomas, the AITL microenvironment characteristically contain a prominent proliferation of high endothelial venules and FDC. Thus, these findings shed new light on the characterization of GC-derived lymphomas and may help in the differential diagnosis and acknowledge several novel pathogenetic mechanisms on these lymphomas. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

Significance of C4d deposition in the follicular lymphoma and MALT lymphoma and their relationship with follicular dendritic cells

We evaluated the deposition of C4d in follicular lymphomas (FL) and extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma). Deposition of C4d was detected in 118 lymphoma tissues from patients with lymphoma and in 20 reactive hyperplasia lymphadens (RHL) using immunohistochemistical methods. FL, MALT lymphoma, and RHL were studied using double staining for CD35/C4d and Bcl-2/C4d. We studied 26 FL tissues, 19 of which showed C4d deposition. C4d deposition was detected around the follicular dendritic cells (FDCs) in the neoplastic follicles. There was no significant difference between the positive ratio of C4d and the grades of FL. We studied 12 MALT lymphoma tissues, six of which displayed C4d deposition. In these tissues, C4d deposition was detected in the peripheral region of partially colonized follicles in the form of an irregular ring, but was not found in the central region. C4d deposition was negative in completely colonized follicles. There was no C4d deposition in diffuse large B-cell lymphomas, mantle cell lymphomas, B-small lymphocytic lymphomas, T-lymphoblastic lymphomas, peripheral T-cell lymphomas, and anaplastic large cell lymphomas. C4d around the FDCs in the neoplastic follicles was a specific indicator for FL. C4d deposition in partially colonized follicles of MALT lymphoma was completely different from that in neoplastic follicles of FL, forming a key point for differential diagnosis.

Presence of Preserved Reactive Germinal Centers in Follicular Lymphoma Is a Strong Histopathologic Indicator of Limited Disease Stage

Follicular lymphoma (FL) typically presents as a systemic disease (stages III/IV). We repeatedly observed in cases with conserved reactive follicle structures (so-called partial infiltration) an association with a limited clinical stage (I/II). In this study, we analyzed 53 lymph node biopsies of FL with conserved reactive follicle structures. In 44 cases (83%) of the patients with partial infiltration, a limited stage of disease (Ann Arbor stage I/II) was found, whereas only 9 of 53 cases (17%) suffered from a systemic disease. In those cases with at least one follicle totally spared by lymphoma, 95% of the patients (38 of 40 cases) presented with a limited stage (I/II) of disease, compared with only 20% (10 of 49 cases) in a control group with full-blown infiltration (P < 0.001). Analyzing systematically all 321 FL cases sent to the Reference Center Würzburg in the year 2001, reactive follicle remnants were detected in 34 of 321 (10.6%) cases with 26 of 34 (76%) tumors showing limited stage (I/II) disease, including all 18 cases with at least one totally spared follicle. Our results therefore show a clear association between the occurrence of preserved reactive follicles in FL and limited disease stage.

Centrocytoid Plasma Cells of the Germinal Center

Germinal centers within the lymph node follicles are T-cell-dependent, antigen-driven B-cell proliferations that develop from the rapid clonal expansion of a few founder cells. The end results of this B-cell expansion are memory B cells or plasma cells. Two morphologic forms of plasma cell can be recognized in the germinal center: classic plasma cells, characterized morphologically by peripherally clumped arrangement of nuclear chromatin, and cells with a nuclear morphology more resembling that of the centrocytes, which the authors have termed "centrocytoid plasma cells." In this study the authors examined the distribution and immunohistochemical characteristics of these two populations of germinal center plasma cells. The centrocytoid plasma cells were arranged in a band stretching from the junction of the dark and light zone to the periphery of the germinal centers, while the classic plasma cells were mainly present at the germinal center periphery. Both marked with CD38, CD138, and VS38c, recognized markers for plasma cells; however, EMA and CD31 were present only in the classic form of plasma cell. The proliferation marker Ki67 was present in less than 1% of the cells labeling with CD138. Others have demonstrated Ki67 in 50% of the cells labeled with Blimp-1, which is consistent with Blimp-1 appearing earlier than CD138 in ontogeny. CD10 is co-expressed with CD138 in about 10% of cells and CD45 with CD138 in about 5% of cells. Their topographic features, together with the progressive acquisition of plasma cell markers, suggest that the centrocytoid plasma cells may be the precursors of the classic plasma cells. Of note, both the forms of plasma cell were absent in follicles of follicular lymphoma, which supports the concept that in this disease, lymphocytes fail to differentiate and mature beyond the centrocyte stage.

Differential expression of cytokines, chemokines and their receptors in follicular lymphoma and reactive follicular hyperplasia: Assessment by complementary DNA microarray

Follicular lymphoma (FL) is pathologically categorised as a low-grade B-cell lymphoma and histopathologically shows follicular proliferation of neoplastic B cells. In the neoplastic follicles of FL, the presence of T cells, macrophages and follicular dendritic cells (FDCs) suggests that these cells may promote a favourable environment for the growth of FL cells. Because FL cells are generally associated with FDCs, FDCs may be considered an important source of cytokines and chemokines. FDCs form the framework for germinal centres and also provide networks for nodules of FL. To evaluate the gene expression in neoplastic follicles of FL and reactive follicles of reactive follicular hyperplasia (RFH), we performed gene expression profiling of FL (n=5) and RFH (n=5) using complementary DNA (cDNA) microarray of cytokines/chemokines and their receptors. FL and RFH exhibited a diffuse down-regulated profile compared with normal peripheral blood cells, which were used as controls, although some genes displayed up-regulated profiles. Hierarchical clustering analysis separated FL and RFH into two distinct groups based on their gene expression profiles. FL cases exhibited significantly higher expression of interleukin 3 receptor alpha (IL-3Ralpha) than RFH. Immunohistochemically, neoplastic follicles of FL frequently expressed IL-3Ralpha, especially in FDCs, but not in FL cells. However, IL-3Ralpha expression was rare or weak in the reactive follicles of RFH. These findings suggest the importance of the micro-environment for FL cell growth. Further studies of cDNA microarray should provide new insight into the molecular pathology of FL and may allow the design of improved therapies.

Clonality analysis of follicular lymphoma using laser capture microdissection method.

Whether a common and a single clone present, or not, among follicles of follicular lymphoma (FL) was examined in 12 cases with FL. Histologic grade was I in 6 cases, II in 3, and III in 3. DNA was selectively extracted from the neoplastic follicles of paraffin-embedded samples with use of laser capture microdissection method, and used for PCR-based analysis of rearrangement of immunoglobulin heavy chain variable region gene. Three different follicles in each case of FL were microdissected. Semi-nested PCR was performed using two sets of primers (Fr2A and Fr3A). In PCR with Fr2A primers, nine of 12 cases showed a common band among neoplastic follicles. The remaining three cases showed no PCR products. With Fr3A primers, eight of 12 cases showed a common band among follicles of the same case. The other four cases showed oligoclonal bands, among them presence of a common band was difficult to assess. Oligoclonal bands were more frequently observed in PCR with Fr3A than that with Fr2A and in grade I or II than in grade III cases. In total, 11 of 12 cases showed a common band in PCR with either Fr2A or Fr3A primers. In two cases, DNA extracted from whole section was amplified with both Fr2A and Fr3A or only Fr3A primers, showing smear or oligoclonal bands. These results showed the presence of a single clone of cells in neoplastic follicles of FL and the usefulness of PCR-based rearrangement analysis of immunoglobulin heavy chain gene combined with microdissection methods for differential diagnosis of FL from follicular hyperplasia.

Histiocytic and dendritic reticulum cells in follicular structures of follicular lymphoma and reactive hyperplasia. A quantitative electron microscopical analysis.

Histiocytic reticulum cells (HRC) and dendritic reticulum cells (DRC) are integral parts of germinal centres. These cell types are also present in follicles of follicular lymphomas, the neoplastic analogues of physiological germinal centres. In this study the distribution and ultrastructural appearances of HRC and DRC present in normal germinal centres and in neoplastic follicles were established by means of morphometric methods. The number of HRC was significantly lower in malignant follicles than in their reactive counterparts. Quantitative analysis of the cytoplasm and phagolysosomes suggest that HRC are smaller and that their activity is lower in malignant follicles. DRC were present in smaller numbers in these structures, as measured by nuclear counts and their relative volume within the follicles. The ultrastructural features indicate that DRC in follicular lymphoma are functionally less active than in reactive lymph nodes. The possibility that differences between the reticulum cells from reactive and neoplastic follicles may be related to the absence of an immune reaction in malignant follicular lymphoma is discussed. The frequency and appearance of HRC and DRC are suitable as additional parameters to differentiate reactive secondary germinal centres from their malignant analogues.

T-lymphocyte subsets in follicular lymphomas compared with those in non-neoplastic lymph nodes and tonsils

Using monoclonal antibodies on frozen sections, the authors define the anatomic localization of T-lymphocyte subsets in follicular lymphomas as well as in nonneoplastic lymph nodes and tonsils. The percentage of Leu-1+ T cells in the follicles of follicular lymphomas (20 per cent) was virtually identical to that seen in the follicles of nonneoplastic lymph nodes or tonsils (22 per cent). There were 50 per cent T cells in the interfollicular regions of follicular lymphomas and 75 per cent in the paracortical regions of the nonneoplastic specimens. In neoplastic follicles the number of Leu-3a+ cells was 67 per cent of the number of Leu-1+ cells, whereas, virtually the entire T-cell population in the nonneoplastic follicles expressed the Leu-3a antigen. These T cells of helper phenotype may facilitate the neoplastic process or an immune response against it or may be bystanders to the B-cell proliferation. Hum Pathol 13:618-625, 1982

Non‐Hodgkin's lymphomas: An immunohistochemical and histological study

Immunohistochemical and histoogical studies have been performed on paraffin sections of 19 cases of non-Hodgkin's lymphoma (NHL). All the cases were lymphocytic in type and, on the basis of the National Lymphoma Investigation classification, 11 were follicular (six small, three mixed small and large, and two large cell types) and eight were diffuse (four intermediate, three poorly and one well-differentiated types). Marshall's metalophil method revealed a population of dendritic histiocytes in and around the follicles of follicular lymphomas. The distribution of the dendritic cells within the neoplastic follicles resembled the distribution of similar cells in reactive follicles, lending support to the concept of an origin for lymphoma follicles from their reactive counterparts. In the diffuse lesions the dendritic cells were large and more pleomorphic than in the follicular lesions, but these features were not so pronounced as those previously observed in Hodgkin's disease. The PAP sequence was used to demonstrate Ig, and as judged by the types of light and heavy chains in the lymphoma cells, the cases were divided into three groups: Group 1 (eight cases) in which the lymphoma cells contained monotypic Ig; Group 2 (six cases) in which monotypic Ig was probably present; and Group 3 (four cases) where no evidence of monotypic Ig secretion was found. Monotypic Ig was most commonly found in follicular lymphomas, mu kappa secretion being the most frequently identified combination of heavy and light chains. The majority of cases (73 per cent.) were thus clearly derived from B lymphocytes. However, the fact that monoclonality was evident in only a proportion of cases suggested that lymphomas may be polyclonal initially and proportion of cases suggested that lymphomas may be polyclonal initially and that monoclonality is a later development. In addition to the lymphoma cells, normal mature plasma cells containing a high concentration of intracellular Ig were present in all but one of the lesions. The Ig was polytypic, cells containing kappa and lambda chains being present in roughly equal numbers and gamma chains pre-dominating. Extracellular Ig (gamma, mu, kappa, lambda) was also present in many lesions. Collections of small non-lymphomatous lymphocytes were also present in all cases. In eight lesions these appeared to have polytypic surface Ig (mu, kappa, lambda). Dendritic cells mingled with these lymphocytes. Collections of small lymphocytes non-reactive for Ig were also present. These had no association with dendritic histiocytes and might have been T cells. It is concluded that in most cases immunohistochemistry alone provides an insufficient basis for the diagnosis of lymphoma and that disturbance of cellular morphology and tissue architecture remain the most useful criteria on which the diagnosis of lymphoma rests.