Biology Of Follicular Lymphoma Research Articles

Base-Pair Resolution of Somatic and Germline-Derived Genome Rearrangement Breakpoints in Follicular Lymphoma.

Abstract 439 Introduction:Follicular lymphoma (FL) is the most common indolent lymphoid malignancy in North America with approximately 20,000 new cases of this incurable cancer diagnosed each year. In approximately 85% of patients, FL is associated with the reciprocal translocation t(14;18)(q32;q21), which results in a fusion between IGH and BCL2 genes and consequent over-expression of the anti-apoptotic protein BCL2. This translocation likely represents an initiating event for FL, requiring additional mutational events for the onset of clinical disease. To investigate the relationship between genome rearrangements and FL we identified rearrangement locations in the genome followed by detailed, fine-structure analysis of the rearrangements to ascertain their effects on genes and other features of biological interest. Patients and Methods:We used a whole-genome bacterial artificial chromosome (BAC) fingerprint-based approach, termed Fingerprint Profiling (FPP, Krzywinski, M. et al. 2007), to detect genome rearrangements relative to the reference human genome in neoplastic B cells purified from 24 FL patient biopsies. Analysis of 2,640,707 BAC fingerprints revealed 721 candidate genomic rearrangements. To validate these observations and provide base-pair resolution of the rearrangement breakpoints we performed paired-end massively parallel sequencing, on the Illumina Genome Analyzer II platform, of the breakpoint-containing regions captured in the BAC clones. Sequence reads were assembled into contigs using our in-house de novo assembly algorithm ABySS (Assembly By Short Sequences, Simpson, J. et al. 2009) then aligned to the reference human genome. Following manual annotation of the breakpoint junctions PCR primers were designed to assay patient tumour and matched constitutional DNA and thus determine whether the observed genome rearrangements were somatic (acquired) or germline in origin. Results:727 BACs with apparent large-scale genome rearrangements, representing 354 distinct genome rearrangements across 20 patients, were sequenced in 95 pools, generating 72 Gbp of sequence. The 354 distinct events include 163 deletions, 71 inversions, 27 insertions, 83 translocations and 10 duplications, ranging in size from 3 kb to 67 Mb. PCR assays for 194 of the distinct events have been performed thus far identifying 80 distinct somatic and 114 germline-derived structural variations at base-pair resolution. Of the somatic events 5 are present in two or more of the 20 patients analyzed including a 720 kb inversion of 3q27.3 that results in expression of a BCL6-ST6GAL1 fusion transcript. Identification at base-pair resolution of breakpoint sequences enabled a detailed study of breakpoint and fusion mechanisms. We classified breakpoint junctions into 4 groups; those with microhomology (48%), those with sequence additions (28%), those with blunt fusions (20%) and those with flanking low copy repeats (4%). We were particularly interested in establishing the origin of the observed nucleotide sequence additions in 97 breakpoint junctions. The sequence additions ranged in size from a single nucleotide to 454 bp. In one case we have unambiguously mapped a 53 bp sequence, lying within one of the 3q27.3 inversion breakpoints, to chromosome 5q12.3. This finding is consistent with the recently proposed fork stalling and template switching (FoSTeS) DNA replication-based mechanism and thus represents a novel mechanism in FL lymphomagenesis. Conclusions:We have successfully employed high-throughput clone fingerprinting and sequencing to identify numerous novel somatic and germline genome rearrangements from FL primary tumour samples. Furthermore, base-pair resolution of rearrangement breakpoints provides mechanistic insights. With the complete inventory of somatic and germline events in hand we will be able to propose recurrent structurally altered genes in FL patients for validation in independent datasets and improve our understanding of FL biology. Pathway analyses to identify emerging themes from somatic mutations are also being performed. The PCR assays we have developed will also be of utility in identifying germline predisposition alleles in larger FL patient cohorts. Disclosures:No relevant conflicts of interest to declare.

Prognostic tools in follicular lymphomas

Despite significant improvements in treatment modalities over the 10 years, the clinical course of patients with follicular lymphoma (FL) remains heterogeneous. Thus, prognostic indexes are still required to direct treatment choices and for the design of clinical trials. Investigators have conducted a variety of studies aimed at integrated assessment of biological and clinical features in order to identify novel prognostic factors and scoring systems. Genetic studies focused on tumor cells and the tumor microenvironment represent a step forward in understanding the biology of FL and are likely to provide new prognostic tools for future clinical use. Several prognostic factors have been identified and are currently used in combination to establish prognostic scores and to support therapeutic decisions. The FL International Prognostic Index (FLIPI) is currently used for defining individual risk of death. More recently, FLIPI2 was developed by the same group that built FLIPI as a new model for prognostic definition of patients with FL. The model was defined using prospectively collected data from patients who also received the monoclonal therapeutic antibody rituximab and stratifies patients into three risk categories for disease progression. Since many biological factors are not yet clinically validated or easily assessable, clinical data still represent the major source of prognostic information. The progressive development of new and more effective therapies for the treatment of FL makes the study of prognosis a dynamic and evolving area of clinical research.

Immunoglobulin G Fc receptor polymorphisms do not correlate with response to chemotherapy or clinical course in patients with follicular lymphoma

Recently, immunoglobulin G Fc receptor (FcγR) polymorphisms have been found to correlate with the clinical response to rituximab or idiotype vaccine in patients with follicular lymphoma. Two critical questions are whether the FcγR polymorphisms correlate with the clinical outcomes after chemotherapy alone in patients with follicular lymphoma and whether they can be explained by linking to underlying biology of follicular lymphoma. This is an important issue because the clinical decisions about the use of antibody therapy may be based on the FcγR polymorphisms of these patients. Here, we analyzed the FcγRIIIa 158 V/F, FcγRIIa 131 H/R, and FcγRIIb 232 I/T polymorphisms in a group of 188 patients with follicular lymphoma who were treated with chemotherapy without rituximab initially. In the current study, FcγR polymorphisms neither correlated with response rate or time to progression after induction chemotherapy, nor with time to the initial therapy or overall survival after diagnosis. Our results confirm that the correlation between FcγR polymorphisms and clinical outcome is specific to immunotherapy such as rituximab and idiotype vaccination, and not due to any effect on the underlying clinical behavior of the disease or chemotherapy response.

Are Mast Cells a Biologic Predictor of Outcome in Follicular Lymphoma?

Background: The tumor microenvironment is important in the biology of follicular lymphoma (FL). In addition to macrophages, which have been shown to be associated with outcome in FL, mast cells (MCs) have the ability to influence the microenvironment through secretion of numerous biologically active molecules such as growth factors and cytokines/chemokines. Increased numbers of tumor-infiltrating MCs have been suggested as an unfavorable prognostic marker that also supercedes the predictive value of lymphoma-associated macrophages (LAM) in FL patients treated with immunochemotherapy. This study examines the effects of tumor-infiltrating MCs in FL patients at Cleveland Clinic.Design: Tissue microarrays were constructed from 94 newly diagnosed cases of FL from 09/01/1985 to 12/13/2002 that had been previously characterized for CD68+ LAMs and demonstrated that extrafollicular (EF) LAMs (>16.8/high power field) were a poor prognostic marker (Kelley T et al 2007). Immunohistochemistry for tryptase was performed on these microarrays to enumerate MCs per high powered (400x) field. In each core, all available lymphomatous tissue was evaluated (mean of 8.13 hpf/case, range 1–9). The cell counts were compared to the available clinical follow-up data, using overall survival as the measurable outcome.Results: The FL patients (44 women, 50 men) had a mean age at diagnosis of 59 years and a mean follow-up of 95.8 months. The patients were treated heterogeneously; the great majority did not receive immunochemotherapy as initial treatment. MC data was available in 91 patients. The mean MC count was 4.14 MCs/hpf (range 0–17.33). As shown previously, > 16.8 EF LAMs/hpf was a predictor of poor overall survival; hazard ratio of 2.28, 95% CI: 1.22–4.24, P = .009. In the entire cohort, MCs were not associated with overall survival. However, in the subgroup with low LAM (CD68 <16.8/hpf), increased MCs (>3.3/hpf) was associated with better overall survival, HR = 0.40, 95% CI: 0.16–0.99, P = .049.Conclusion: In our data set, increased LAMs in FL are associated with poor overall survival. Overall, MCs do not appear to predict outcome; however, in the subgroup of patients with low LAMs, increased MCs are associated with a favorable overall survival. This suggests a complex interaction between cellular constituents of the tumor microenvironment that affects patient outcome. The application of immunochemotherapy or other immunomodulatory therapies may alter these interactions and outcomes. Further work is needed to better understand these interactions and to validate biomarkers in therapy specific contexts.

In Follicular Lymphoma (FL), γδt-Lymphocytes (γδT) Are Present and Expandable from Peripheral Blood and Rare in Tumour Lymph Nodes, Mostly in Peri-Follicular Areas.

Background: The tumour microenvironment plays an important role in the biology of FL. Different cell populations have been explored, including T-regulatory lymphocytes, macrophages, and T-cell subpopulation. The involvement of γδT in lymph nodes from FL patients or from inflammatory diseases has been rarely documented. So far, their histological pattern and prognosis significance are unknown and must be defined in order to develop new therapeutic programs including in vivo or ex vivo expansion of γδT, as developed particularly in B-cell malignancies by us and different groups. In this study, we analyzed from FL patients 1/the number of circulating γδT and their ex vivo expansion, 2/ the presence and distribution of γδT in tumour lymph nodes, and different chemokines, in comparison to inflammatory lymph nodes (ILN), by immunochemistry.Patients and Methods: Circulating γδT cells were counted in peripheral blood from patients having FL by FACS analysis. Blood samples from 34 patients were collected and expanded in vitro by using γδT ligands, referred to as “phosphoantigens”, including IPH1101 (used in clinical trials) and interleukin 2. Tumour samples from 51 patients (35 at diagnosis and 16 at relapse) having FL were collected from a single institution. Immunochemistry was used to study numbers and distribution of CD8, γδT cells, and the expression of CCL19, 21 and SDF1 chemokines. CCR7/γδT cells were analyzed by double immunofluorescence. Results were compared to 28 samples from patients having ILN.Results: The mean of circulating γδT was 0.36% (0.03–2.5) representing a mean of 2.2% of the CD3 cells. The mean percentage of γδT cells obtained after in vitro culture was 85% (2.1–95) with a mean 220-fold expansion (2-1050). The median number of γδT cells (cells/mm2) in FL lymph node was 18 versus 47.5 in the ILN (mean: 30 versus 82,5), P<.00001. The median of CD8 cells was 1235 in FL as compared to 1503 in ILN (mean: 1290 versus 1524). CD8+ cells had different localization (i.e. intra-and /or extra-follicular localization), but γδT were strictly peri-follicular in both clinical situations. Immunohistochemistry of the high endothelial venules (HEVs) and lymphatic vessels (LV) of 14 FL and 14 ILN were performed. We observed a significant difference (P= 2.10−7) in the expression of only the CCL19 chemokine between FL and ILN, with a poor staining for CCL19 in FL lymph nodes. CCL21 and CXCL12 do not present a difference in their expression levels. The stroma was reactive for all these chemokines, while the SDF1/CXCL12 chemokine shows a topographic difference in the distribution of stromal cells around HEV.Conclusions: These observations suggest that γδT cells are present and expandable in PB from patients having FL including patients with advanced disease. In addition, γδT are not abundant in lymph nodes of patients with FL compared to ILN, but γδT conserve their CCR7+ phenotype. This deficiency could be explained by migratory problems provoked by a lack of CCL19 chemokine expression. As γδT have been demonstrated to kill tumour cells, including B-malignant cells, they could be considered as essential targets for immune therapy in different cancers including B-cell malignancies, but their activation and trafficking has to be considered.

Low Ratio of Inter-Follicular CD8/FOXP3-Positive Regulatory T-Cells Is Associated with a favourable Overall Survival in Follicular lymphoma (FL).

Background: By using gene expression profiling, a major role for T-lymphocytes and macrophages (M) have been observed, defining 2 prognostic subgroups. Then, conflicting results were published with both positive and negative impacts of different T-cells subpopulations, particularly for T-regulatory (Tregs) cells. In addition, CD8 and CD4 T-cells were mentioned as key cells in progression and/or transformation process and CD68+ M associated with a poor outcome. Functional studies have suggested that follicular dendritic cells have to be considered as important cells in comprehensive biology of FL, and that the function is probably associated with the proximity of tumour cells, different in intra-or inter-follicular location. We investigate the role CD8+ T cells, FOXP3+ Tregs and M, and calculate in large fields the different numbers of these cells, particularly focusing on their intra-versus inter-follicular localisation. To approach a more dynamical view of the immune response we correlate the ratio CD8+/Treg in these 2 compartments and correlated results with clinical parameters and outcome.Patients and Methods: Immunochemistry was used to study numbers and distribution of CD8, CD68, and Tregs cells in 89 patients with FL (65 at diagnosis and 24 at relapse). Since it can be expected that overall effect of Tregs depends on their number in relation to CD8+ effectors T cells, we determined the corresponding ratio for each patient and correlated the results with clinical parameters.Results: The median age of the patients was 58 years (range 23–91 years), and the male/ female ratio was 1.2. There were 67 % of the patients with grade1, 17 % grade 2 and 16 % grade 3a. Advanced stage (Ann Arbor IV) was observed in 24 %. The distribution of the population according to the FLIPI was the following: low risk 36 %, intermediate risk 45 % and high risk, 19 %. The interfollicular CD8/FOXP3+ cell ratio was significantly higher in patients with grade 3 tumours (2.04 versus 1.63) and with a high risk FLIPI index (2.99 versus 1.53) compared with those with grade 1–2 tumours or a low–intermediate FLIPI index. The same results were obtained with the intra-follicular CD8+/FOXP3 + cell ratio. The inter-follicular CD8/FOXP3 ratio was found to have prognostic value for overall survival with a 5-years OS of 82% versus 59% for a ratio of less or more than 1.68. Moreover, an inter-follicular FOXP3+ cell number of more than 86 cells/mm2 was correlated with a more favourable outcome (p = 0.03). When comparing the 2 groups at diagnosis and relapse, we observed a significant difference between the number of CD68 cells both in intra-follicular locations (39.23 at diagnosis versus 32 at relapse, p= 0.07) and inter-follicular locations (96 at diagnosis versus 62 at relapse, p=0.05). The CD8/FOXP3 ratio in inter-follicular locations was significantly different (1 .66 at diagnosis versus 2.2 at relapse, p=0.05).Conclusion: The presence of abundant FOXP3+ cells with a low CD8/FOXP3 ratio is probably the mark of an active immune response during tumour development, with lymphoma cells acting as targets or bystanders.

Combined FOXP3+ and PD1+ T Cell Density and Architectural Patterns Predict Overall Survival and Risk of Transformation in Uniformly Treated Patients with Follicular Lymphoma

Background: Follicular lymphoma (FL) is an indolent heterogeneous lymphoid neoplasm with a variable clinical course. Transformation into an aggressive lymphoma is a dominant clinical event frequently associated with inferior survival. No consistent biological markers are predictive of survival or risk of transformation (RT). Recent studies have highlighted the role of the microenvironment, including regulatory T cells (Treg) as important prognostic factors in FL. Using tissue microarrays (TMA), we previously identified FOXP3+ T cell distribution but not cell density within the tumor is an independent adverse prognostic factor for both overall survival (OS), progression-free (PFS) survival and RT in advanced-stage FL pts treated uniformly with an aggressive treatment regimen (ASH 2007). Programmed death-1(PD1) belongs to the immunoglobulin CD28 receptor family and is characteristically expressed by germinal center associated T cells. They have imunoregulatory activity and are FOXP3-negative. Using the same cohort we assessed the role of PD1+ cells alone and in combination with FOXP3+ cells in FL prognosis.Methods: Between 1987 and 1993, 126 pts were enrolled on a phase II study of BP-VACOP chemotherapy with involved region radiotherapy. All patients were treatment naive, < 61y and had advanced-stage FL. Paraffin blocks were available for 105 pts. TMAs consisted of duplicate 1.0mm cores of diagnostic biopsies and were immunostained with both FOXP3 and PD1 (NAT105) specific antibodies. Both cell content and immuno-architectural patterns were determined and correlated with OS and RT including univariate and multivariate analysis.Results: There were 101 evaluable cases. The median follow-up of the living pts was 14 years. Histologic grade included 78 grade 1, 17 grade 2 and 6 grade 3a FLs. The IPI was predictive of OS (RR = 2.5, 95% CI =1.5–4.4, p = 0.002), but not RT (p = 0.14). Cases revealed zero to 731 FOXP3+ cells per core (median of 242 cells per core) and zero to 654 PD1+ cells per core (median of 102 cells per core). There was a predominantly intrafollicular or perifollicular localization of positive cells (“follicular pattern”) in 38 and 43 pts for FOXP3 and PD1, respectively. There was no significant correlation between cell density (χ2=0.1) or pattern (χ2=0.8) between markers. Separately, cell density did not impact prognosis. Yet, when quartiles were combined and dichotomized, cases with high cell content of FOXP3+/PD1+ cells correlated with inferior OS and increased RT in univariate (p = 0.002 and p = 0.01) and multivariate Cox models (RR = 2.18, 95% CI =1.2–3.9, p = 0.01 & RR = 3.4, 95% CI =1.3–8.8, p = 0.015). In univariate analysis, a PD1 follicular pattern correlated with inferior OS (p=0.027) but did not impact RT. When combined with follicular FOXP3+ cases, a subset of 15 “double follicular” FOXP3 & PD1 cases correlated with inferior OS in univariate (p=0.0016) and multivariate Cox models (RR = 2.1, 95% CI =1–4.3, p = 0.04). This subset of cases also showed a trend for increased RT (p = 0.09).Conclusions: Combined cell density and distribution within FL of immuno-regulatory T cell subsets are important predictors of OS & RT in advanced-stage FL patients treated uniformly with an aggressive treatment regimen. Our results reinforce the importance of the microenvironment in FL biology and further suggest an active role for immuno-modulatory T cells in pro-tumoral immunity.

IL-4 protein expression and basal activation of Erk in vivo in follicular lymphoma

Follicular lymphoma (FL) is characterized by constitutive expression of Bcl-2 as a consequence of t(14;18). Evidence suggests factors in the lymph node microenvironment, related to intratumoral T cells, macrophages, and dendritic cells, play a role in the disease process. We generated proteomic cytokine profiles of FL (N = 50) and follicular hyperplasia (FH; N = 23). A total of 10 cytokines were assayed using ultrasensitive multiplex enzyme-linked immunosorbent assays: IL-1beta, IL-2, IL-4, IL-5, IL-8, IL-10, IL-13, IL-12p70, tumor necrosis factor-alpha, and interferon-gamma. Each cytokine showed overall lower protein concentrations in FL, with the exception of IL-4, which was nearly 5 times higher in FL than FH (P = .005). Using reverse-phase protein microarrays (RPMAs), we evaluated the activation state of several intracellular signaling proteins downstream of cytokine receptors. Basal Erk phosphorylation was approximately 4 times greater in FL than FH (P < .001), with similar findings for Mek; Stat-6 showed weak basal phosphorylation that was approximately twice as high in FL than in FH (P = .012). In conclusion, the FL microenvironment contains increased levels of IL-4, with prominent tumor basal phosphorylation of Erk. These findings suggest IL-4, Erk, and possibly Stat-6 may play a role in the biology of FL and may serve as targets for future therapies.

The presence of TP53 mutation at diagnosis of follicular lymphoma identifies a high-risk group of patients with shortened time to disease progression and poorer overall survival

AbstractThe International Prognostic Index and the Follicular Lymphoma International Prognostic Index are widely used for the risk assessment of follicular lymphoma (FL). Although molecular studies have provided insight into the biology of FL, no molecular marker has impacted on treatment stratification. Because TP53 mutations are associated with poor prognosis in hematologic malignancies, we investigated the prognostic value of TP53 mutation at diagnosis in FL. Heterozygous TP53 mutation was detected in 12 of 185 (6%) analyzed cases. Mutation was associated with older age (P = .02) and higher International Prognostic Index score (P = .04). On multivariate analysis, TP53 mutation correlated with shorter progression-free survival (P < .001) and overall survival (P = .009). TP53 mutation was associated with low expression of the immune-response 1 gene expression signature (P = .016) and with an unfavorable gene expression-based survival predictor score (P < .001), demonstrating for the first time that molecular features of the malignant cell may correlate with the nature of the immune response in FL.

The follicular lymphoma microenvironment: From tumor cell to host immunity

Follicular lymphoma (FL) is a neoplasm derived from follicular germinal center cells. Like the normal components of this lymphoid structure, FL cells interact with various immune cells, such as the follicular helper T cells, suppressor regulatory T cells, dendritic cells, and histiocytes, that define the tumor microenvironment. Gene expression studies have shown that the nature of the tumor microenvironment predicts survival in patients with FL and may influence the response to immunotherapy and risk of transformation. The immune system may either promote or constrain tumor cell development, depending on the relative distribution and activation status of various cell subpopulations. The prognostic value of germline genetic variants of some immune genes suggests that the host genetic background may also influence the biology of FL. Some efforts have been carried out to validate those findings and provide clinical tools that may be used at the time of diagnosis.

Refining the treatment of follicular lymphoma

Many effective treatments are currently available for patients with follicular lymphoma (FL). However, given the heterogeneity of this disease, identifying the most beneficial treatment for an individual patient remains a challenge, although clinical, genetic and biological features can all potentially be used to refine therapies in individual cases. The Follicular Lymphoma international prognostic index (FLIPI) algorithm is a valuable prognostic tool for risk categorisation. Despite its current limitations, further investigation will help to develop the role of FLIPI in treatment decision–making, and will increase its value in identifying the optimal therapy for individuals. Biological factors such as bulky disease, over-expression of Bcl-2, or histological grade can help to identify patients at high risk of relapse, and distinguish between the benefits of early intervention vs. a watch-and-wait policy in early-stage FL. The tumor microenvironment plays an important role in the development of FL, and identification of biological and genetic markers could help clinicians determine the prognosis of individual patients. Although much work remains to be done, a greater understanding of the biology of FL will lead to the development of novel therapeutic targets and therapies, bringing individualised treatment a step closer.

Gene Expression Profiling of Highly Purified Malignant and Non-Malignant Cells: Characterization of the Tumor-Microenvironment Cellular Synapse in De Novo Follicular Lymphoma (FL).

Genetic and functional studies have demonstrated that FL cells retain the major features of normal germinal center (GC)-derived B cells, in particular the dependency on an active crosstalk with their specialized microenvironment. In agreement, microarray analyses have recently revealed that FL patient outcome is primarily predicted by molecular characteristics of tumor-infiltrating immune cells instead of tumor cells. However, our knowledge of the crucial interactions between malignant and non-malignant cells in FL remains limited by the use of whole biopsy specimen to perform gene expression profiling (GEP). We thus conducted GEP on both CD19pos B cells and CD19negCD22neg non-B cells purified from lymph nodes of 17 patients with de novo FL & 4 normal donors (CD20pos >94.5%, median=98.2%) and 9 de novo FL patients & 5 normal donors (CD20pos<6.7%, median=0.5%), respectively. Biotinylated cRNA were amplified according to the small sample labelling protocol and hybridized onto HGU133 Plus 2.0 arrays (Affymetrix). Raw data were normalized using GC-RMA methodology (ArrayAssist, Stratagene) and finally, based on a CV>80, 10870 probesets were selected for further analyses. Unsupervised hierarchical clustering (Eisen's software) allowed the correct classification of the 35 samples into the 4 groups: FL B-cell, Normal B-cells, FL non-B cells, and Normal non-B cells. Supervised analyzes were done using asymptotic non-parametric Mann-Whitney U-test (fold change ≥2, P<0.01) and confirmed by permutation analysis (500 permutations, false discovery rate <5%) using SAM software. We first established the list of the 841 probesets that were differentially expressed between FL and normal B-cells containing, 355 probesets overexpressed in malignant B cells including genes involved in GC B-cell biology (BCL6, MTA3, ID2, CD80, SDC4) and oncogenes as well (BCL2, AURK2) and conversely, 486 probesets downregulated in malignant B cells involving several interferon-stimulated genes for example. We then looked for the FL-specific microenvironment signature and pointed out the 1206 probesets that were differentially expressed between FL and normal non-B cells. Interestingly, all these genes were upregulated in the lymphoma context. Among them, we identified a striking follicular helper T-cell (TFH) signature (CXCR5, ICOS, CXCL13, CD200, PDCD1, SH2D1A) and an activated T-cell signature (IFNG, FASLG, GZMA, ZAP70, CD247). Notably, the TFH and activated T-cell signatures were not merely a surrogate for the number of T cells since many standard T-cell genes (i.e. CD2, CD4, CD7, LEF1, CD8A) were not induced in the FL microenvironment. Finally, in order to draw an overview of the FL-specific synapse between B and non-B cell compartments, we isolated a group of 2323 probesets that were differentially expressed between both compartments in FL and not in normal context. Using Ingenuity Pathway Analysis software we then identified among them FL-specific functional networks, including an IL-4- & an IL-15-centered pathway. Altogether, these data shed new light on our understanding of FL biology and could be a source of new therapeutics targeting the interplay between B cells and their microenvironment.

The Architecural Pattern of FOXP3+ T Cells Predicts Risk of Transformation in Patients with Follicular Lymphoma (FL).

Background: FL is an indolent heterogeneous lymphoid neoplasm with a variable clinical course. Transformation into an aggressive lymphoma (Tly) is a dominant clinical event frequently associated with inferior survival. No consistent biological markers have been identified that predict for Tly. Recent studies have highlighted the role of the microenvironment, including lymphoma-associated-macrophages (LAM) and Treg cells as important prognostic factors in FL. The nuclear expression of FOXP3 is considered a specific marker of regulatory T cells (Treg). However, most series have included pts variably treated with a wide spectrum of protocols. Using tissue microarrays (TMA) we recently demonstrated that both LAM content and follicular/perifollicular pattern of FOXP3+ T cells are independent adverse prognostic factors for both overall (OS) and progression-free (PFS) survival in advanced-stage FL patients treated uniformly with an aggressive regimen. However, the impact of either LAM or Tregs on Tly is largely unknown. Using the same cohort we assessed the role of these cell types for predicting Tly.Methods: Between 1987 and 1993, 126 pts were enrolled on a phase II study of BP-VACOP chemotherapy with involved region radiotherapy. All patients were treatment naïve, < 61y and had advanced-stage FL. Paraffin blocks were available in 105 patients. The TMAs consisted of duplicate 1.0mm cores of diagnostic biopsies and were immunostained with both CD68 and a FOXP3-specific antibody. Both CD68+ macrophage content and FOXP3+ T cell content and immuno-architectural patterns were determined and correlated with RT including univariate and multivariate analyses.Results: There were 102 evaluable cases. The median follow-up of the living pts was 14.3 years and the estimated 10-year OS and Tly rates were 63% and 18%, respectively. The IPI was predictive of OS (RR = 2.4, 95% CI =1.4–4.2, p = 0.002) but not Tly (RR = 2.2, 95% CI =.9–5.1, p = 0.07). Histologic grade included 76 grade 1, 20 grade 2 and 6 grade 3a FLs. Cases revealed 1 to 28 CD68+ cells per high power field (median 7 cells/hpf) and zero to 731 FOXP3+ cells per core (median of 242 cells/core). In 38 pts there was a predominantly intrafollicular or perifollicular localization of positive cells (“follicular Treg pattern”) and in 64 pts the pattern was diffuse. Neither LAM content nor number of FOXP3+ cells had any impact on Tly. However, the immuno-architectural pattern of FOXP3+ cells was predictive of Tly, with the “follicular” pattern showing increased risk of Tly in univariate analysis (p=0.005). A Cox multivariate model including IPI and LAM showed only the FOXP3+ Treg pattern as an independent predictor of Tly (RR = 3.9, 95% CI =.1.5–9.9, p = 0.004).Conclusions: Treg cell distribution within the tumor is an important predictor of Tly in advanced-stage FL patients treated uniformly with an aggressive treatment regimen. Our results reinforce the importance of a FOXP3 follicular pattern as a good surrogate for functionally active Treg cells within the tumor microenvironment and further highlight the important role of the tumor microenvironment in FL biology. [Display omitted]

Comprehensive Analysis of Copy Number and Allele Status Identifies Multiple Chromosome Defects Underlying Follicular Lymphoma Pathogenesis

Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic or -extrinsic factors. FL carries the hallmark chromosomal translocation t(14;18), deregulating the expression of Bcl-2, but this is not sufficient to explain either FL biology or clinical behavior. We have employed high-density genomic profiling technology using the Affymetrix 50K-XbaI oligonucleotide single nucleotide polymorphism-chip platform to interrogate the genomes of 58 fluorescence-activated cell-sorted (FACS) FL specimens for chromosomal copy number changes and 46 specimens for loss of heterozygosity (LOH). We report (a) previously unknown high-frequency copy-neutral LOH (uniparental disomy) in FL on chromosomes 1p (approximately 50%) and 6p (approximately 30%); (b) that del6q is complex, as reported, with at least two regions of minimal common loss at 6q13-15 and 6q23-24, and that in addition, approximately 8% of FL specimens contain a homozygous deletion at 6q23.3-24.1 that spans the negative NFkappaB regulator A20 and the p53 apoptosis effector PERP; (c) that combined analysis of chromosome 17p for LOH, copy number, and p53 mutations shows that most p53 mutations in FL do not involve del17p. Finally, we map high-frequency LOH with and without copy loss on chromosomes 9p, 10q, and 16p and genomic gains on 2p15-16 and 8q24.22-24.3. This comprehensive description of the pathologic anatomy of the FL genome uncovers novel genetic lesions and should aid with identification of genes relevant to FL biology and clinical behavior.

CD8+ T-Cell Content in Diagnostic Lymph Nodes Measured by Flow Cytometry Is a Predictor of Survival in Follicular Lymphoma

Follicular lymphoma is a heterogeneous disease with variable prognosis and clinical course. We hypothesized that the presence of nonmalignant T cells in the microenvironment of the tumor may affect the outcome. Using flow cytometry, we evaluated the T-cell subsets in the lymph node microenvironment of follicular lymphoma. All patients in South Stockholm County with indolent follicular lymphoma and with flow cytometry done on a diagnostic lymph node between 1994 and 2004 were included (N = 139). Diagnosis and grade (1, 2, and 3a) were confirmed by re-review. Flow cytometry results were reanalyzed. Lymphocyte subsets, the Follicular Lymphoma International Prognostic Index, grade, and clinical characteristics were evaluated in univariable and multivariable Cox analysis with respect to overall survival (OS) and disease-specific survival (DSS). Higher CD8+ T-cell levels correlated with longer OS and DSS, independently of the Follicular Lymphoma International Prognostic Index (OS, P = 0.017; DSS, P = 0.020) and independently of all other prognostic factors (OS, P = 0.001; DSS, P = 0.004). Median OS was not reached for patients in the upper quarter of CD8+ T-cell levels (>8.6%), 10.4 years for patients in the middle half (4.2-8.6%), and 6.0 years for patients in the lower quarter (<4.2%). Furthermore, patients who had not required treatment within 6 months from diagnosis had more CD8+ T cells (P = 0.011). Higher levels of CD8+ T cells predict a better prognosis, and these data support an important role for nonmalignant immune cells in the biology of follicular lymphoma. Evaluating the CD8+ T cells by flow cytometry at diagnosis may provide prognostic information.

Genomic Profiling of Follicular Lymphoma Identifies Novel High-Frequency Copy-Neutral Loss of Heterozygosity on Chromosomes 1p and 6p and Identifies Candidate Genes Involved in FL Biology.

Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic (genetic) or -extrinsic (infiltrating immune cells, infiltrating macrophage content) factors. FL carries the hallmark chromosomal translocation t(14;18), deregulating expression of Bcl-2, but this is not sufficient to explain either FL biology or clinical behavior. We have employed genomic profiling technology using the Affymetrix 50K-Xba1 oligonucleotide single nucleotide polymorphism (SNP) chip platform to interrogate the genomes of 58 FL specimens for loss of heterozygosity (LOH) and chromosomal copy number changes. Where available, these high-density genomic data were correlated with published FL gene expression data. Using this integrated approach, we provide data that suggest that i) Bcl11A is a likely target gene for the amplification on chromosome 2p15–16 in FL, ii) that the amplification of der(18)t(14;18) does not include Bcl-2, iii) high-frequency copy-neutral LOH exists at 1p36 (50%) and 6p (33%), and that 6p LOH possibly deregulates IRF4 and, iv) deletion 6q is non-overlapping, complex and possibly involves four or more gene loci of importance. In addition, high-frequency LOH (with and without chromosomal copy loss) exists on chromosomes 9p, 10q and 16p. These data should facilitate analysis of specific genes in FL and should allow for development of a SNP -and genomics-based assay for FL risk stratification and outcome prognostication.

Prognosis of follicular lymphomas

Follicular lymphoma (FL) is as an indolent neoplasia with median survival measured in decades. Nevertheless, some patients have poor progression-free survival and overall survival. Several treatment approaches are proposed for patients with FL, however criteria to rationalize treatment decisions are lacking. Studies have been performed to build up prognostic indices that are useful for defining risk-adapted treatment recommendations. Available indices are based on parameters that have an independent role in predicting patient survival and that are variably correlated with the features of the disease, with the characteristics of the patient and with the effects of treatment. Two new prognostic indices have recently been proposed for FL: the Italian Lymphoma Intergroup (ILI) index and the Follicular Lymphoma International prognostic Index (FLIPI). Both indices are based on large series of patients and exhibit differences in their ability to discriminate between patients with different probabilities of survival. In recent years, with the advent of gene expression profile studies, our knowledge of the biology of FL is changing as novel data become available about the lymphoma cell and about the role of the microenvironment; these studies have already provided novel prognostic tools for identifying patients with more aggressive disease. Further data and large international cooperative studies are needed to translate into clinical practice the novel acquisitions of biology and therapeutics.

Analysis of multiple biomarkers shows that lymphoma-associated macrophage (LAM) content is an independent predictor of survival in follicular lymphoma (FL)

AbstractWe studied the role of multiple biomarkers in determining outcome in follicular lymphoma (FL), concentrating in particular on the role of benign macrophages. The study group consisted of uniformly staged and treated patients with FL enrolled in a phase 2 trial between 1987 and 1993. All patients were younger than 61 years of age, had advanced-stage FL, and were treated with a multiagent chemotherapy regimen, BP-VACOP (bleomycin, cisplatin, etoposide, doxorubicin, cyclophosphamide, vincristine, and prednisone), followed by involved region radiation. The median follow-up of living patients was 12.5 years, and the median survival was 16.3 years. The International Prognostic Index (IPI) was predictive of overall survival (OS) (P = .003). Biopsy specimens from all cases were stained with an anti-CD68 antibody. Of the 99 evaluable patients with FL, 87 had less than 15 CD68+ macrophages/high-power field (hpf) (median, 7; range, 1-14) and 12 had more than 15 CD68+ macrophages/hpf (median, 20; range, 16-25) with a median OS of 16.3 vs 5.0 years, respectively (P < .001). A multivariate Cox model that included the IPI score, the histologic grade, and the lymphoma-associated macrophage (LAM) score, showed IPI and LAM to be independent predictors of OS (P = .009 and P = .004, respectively). The LAM content of FL predicts survival, and these data support a prominent role for nonneoplastic immune cells in the biology of FL. (Blood. 2005;106:2169-2174)

Multicolour fluorescence in situ hybridization analysis of t(14;18)‐positive follicular lymphoma and correlation with gene expression data and clinical outcome

In order fully to identify secondary chromosomal alterations, such as duplications, additions and marker chromosomes that remained unresolved by G banding, 60 cases of t(14;18)-positive follicular lymphoma (FL) were analysed by multicolour karyotyping techniques [multicolour fluorescence in situ hybridization (MFISH)/multicolour banding for chromosome 1 (MBAND1)]. A total of 165 additional structural chromosomal aberrations were delineated. An increased frequency of chromosomal gains involving X, 1q, 2, 3q27-q29, 5, 6p11-p21, 7, 8, 11, 12, 14q32, 17q, 18 and 21 and deletions of 1p36, 3q28-q29, 6q, 10q22-q24 and 17p11-p13 was revealed by the MFISH/MBAND1 analysis. Balanced translocations other than t(14;18) were uncommon, whereas unbalanced translocations were numerous. Deletion of 1p36 and duplication of 1p33-p35, 1p12-p21 and 1q21-q41 were regularly involved in chromosome 1 alterations, seen in 53% of the cases. A strong correlation was demonstrated between gains of individual chromosomal bands and increased gene expression, including 1q22/MNDA, 6p21/CDKN1A, 12q13-q14/SAS, 17q23/ZNF161, 18q21/BCL2 and Xq13/IL2RG. Unfavourable overall survival was associated with del(1)(p36) and dup(18q). These data support the notion that translocation events are primarily responsible for FL disease initiation, whereas the unbalanced chromosomal gains and losses that mirror the gene expression patterns characterize clonal evolution and disease progression, and thus provide further insights into the biology of FL.

Anti-Idiotype Antibodies Can Induce Long-Term Complete Remissions in Non-Hodgkin’s Lymphoma Without Eradicating the Malignant Clone

The immunoglobulin on the surface of B-cell lymphomas can be a tumor-specific target for monoclonal antibody therapy. Between 1981 and 1993, 45 individuals with low grade B-cell lymphoma were treated with 52 courses of custom-made anti-idiotype antibodies. The antibodies were used either alone or in combination with -interferon, chlorambucil, or interleukin-2 (IL-2). The majority of these patients responded to treatment, with a 66% overall and 18% complete response rate. Six patients (13%) experienced prolonged complete remissions, five of which are ongoing from 4 to 10 years after therapy and are the subject of this report. We asked whether residual lymphoma could be found in these patients with prolonged remissions. We performed enzyme-linked immunosorbent assay (ELISA) assays for idiotype protein or anti-idiotype antibodies in serum. Blood and bone marrow samples were examined by flow cytometry for idiotype positive cells, and by polymerase chain reaction (PCR) for clonal gene rearrangements of immunoglobulin CDR3 sequences or t(14;18) translocations. Using these sensitive and specific tests it was possible to detect very low levels of residual lymphoma in five of these patients who had been in clinical remission for 3 to 8 years before this evaluation. These five have continued without recurrence for up to 3 years since. Thus, we have found a pattern of residual inactive disease in patients treated with anti-idiotype antibodies. The biology of follicular lymphoma evidently includes the potential for tumor dormancy after therapies with varied mechanisms of action, resulting in clinical inactivity for many years. Thus, long-term control of the disease is possible at a clinical level despite persistence of the malignant clone. © 1998 by The American Society of Hematology.