Progression Of Follicular Lymphoma Research Articles

Cost Effectiveness Analysis of Rituximab Maintenance In Patients with Untreated High Tumour Burden Follicular Lymphoma After Response to Immunochemotherapy: A UK National Healthcare Services Perspective.

AbstractAbstract 3833 Introduction:The PRIMA Phase III study demonstrates that rituximab (R) maintenance therapy after induction immunochemotherapy in previously untreated follicular lymphoma (FL) significantly improves progression-free survival (PFS) with little additional toxicity (Salles et al., 2009). Utilising clinical evidence from PRIMA, this economic analysis evaluated whether R-maintenance therapy in FL patients after response to R-chemotherapy induction is a cost-effective option compared to observational (Obs) practices in the UK National Healthcare System.METHODS: A transition state (Markov) model was developed with FL patients having a complete/partial response to 1st line R-chemotherapy induction being assigned across 4 health states reflecting their disease status; progression-free in 1st line maintenance (PF1), progression-free in 2nd line, progressive disease (PD) or Death. The model was developed over a 25 year horizon to capture the lifetime of an average patient. This required extrapolation of PFS beyond the PRIMA trial follow-up period (median 38.37 months; PFS hazard ratio 0.55; 95% CI [0.44-0.68]; Clinical Study report addendum) using the best parametric fit (Gompertz). The monthly probability of dying in PF1 was based on the maximum of either the observed PFS deaths in PRIMA or background mortality. The disposition of 1st line R-maintenance patients after progression was based on ESMO guidelines (Dreyling et al., 2010) and PRIMA. Due to extensive censoring of overall survival in PRIMA (95% and 97% in the respective R and Obs arms), the probabilities of progressing or dying in second-line or third line were obtained from the EORTC 20981 trial (van Oers et al., 2010). Predicted time in each health state was weighted using FL utility scores (Pettengell et al. 2008) to account for quality of life and estimate the Quality Adjusted Life Years (QALYs).Costs associated with the average dose of R-maintenance, post-progression treatments and managing grade 3/4 adverse events observed in PRIMA were incorporated into the relevant health state. Drug administration, patient monitoring and pharmacy costs were informed by expert opinion and the NHS schedule of reference costs.RESULTS: The average overall survival in the 1st line R-maintenance cohort was projected to be 1.27 years longer on average than in the Obs cohort (10.31 vs 9.05); and associated with an additional 1.17 QALYs. This is largely due to patients treated with 1st line R-maintenance spending more time in progression-free in first line (1.17 years). Total costs were £14,129 higher in the 1st line R-maintenance than the Obs arm and were driven by the cost of the study drug and its administration. However, this was partially compensated by the lower costs of rituximab therapy in 2nd line (cost saving £198) and the lower costs of supportive care incurred at disease progression (cost saving £906).The incremental cost-effectiveness ratios (ICERs) for 1st line R maintenance was £14,712 Life Year Gained and £15,983 per QALY gained, well below an assumed willingness to pay threshold of £30,000. Although there is uncertainty associated with the progression of FL and relapse treatment costs, the ICER did not exceed £21,155 per QALY despite a wide variation in each parameter value used in both the probabilistic and deterministic sensitivity analysis.CONCLUSIONS: The cost-effectiveness of R-maintenance in FL patients after response to R-chemotherapy is well within the acceptable willingness to pay ceiling and remains valid under most plausible sensitivity scenarios. This provides adequate reassurance that the superior clinical benefits of 1st line R-maintenance are sufficient to justify the additional costs over observational practice. Disclosures:Papadakis:F. Hoffmann-La Roche Ltd: Employment. Boyer:F. Hoffmann-La Roche Ltd: Employment. Bashir:F. Hoffmann-La Roche Ltd: Employment. Ball:F. Hoffmann-La Roche Ltd: Employment. Aultman:F. Hoffmann-La Roche Ltd: Employment. Carr:F. Hoffmann-La Roche Ltd: Employment.

Abstract 4924: Gene-specific hyper and hypomethylation in follicular lymphoma progression

Abstract Epigenetic mechanisms involved in the origin and transformation of indolent lymphomas to aggressive forms are poorly understood. Based on the comparison of methylation states of follicular lymphoma (FL) pre and post- transformation, it has been suggested that DNA methylation changes occur during the early stages of lymphomagenesis. To obtain a more complete understanding of methylation aberrations responsible for progression and transformation of FL, we analyzed 20 untreated FL biopsies (10 low grade, 10 high grade), 5 diffuse large B cell lymphoma (DLBCL) biopsies, and 5 benign reactive lymph node (RLN) biopsies by using methyl-DNA immunoprecipitation coupled with analysis with Agilent 244K human promoter ChIP-on-chip microarrays. Our analysis revealed statistically significant differences in methylation status for 120 genes between FL samples and RLN samples and for 23 genes between low and high grade FL samples. To investigate the biological significance of these findings, we used DAVID functional annotation clustering algorithms and Gominer software. We found that the most methylation-enriched functional groups were comprised of genes with roles in cell cycle control, adhesion and motility, transcription regulation, homeobox, and developmental genes. In order to validate the DNA methylation differences of the significant gene regions, we utilized the MassARRAY EpiTYPER assay. Our data identified 13 genes (CYP26B1, GRM7, HS3ST2, DOCK5, GRB10, SST, TAC1, PALM2-AKAP, FLJ40125, ST6GAL2, WDR35, NCAM1, SATB2) with increased levels of DNA methylation in FL samples compared to controls; 3 of these genes (ST6GAL2, WDR35, NCAM1) exhibited increased methylation in high grades FLs and 1 gene (SATB2) in low grades FLs. We are currently investigating gene specific DNA hypomethylation in low versus high grade FL and FL versus DLBCL. Our data are the first to characterize the global changes in methylation that occur in FL progression. Our findings suggest that high grade FL are associated with hypermethylation of a specific subset of genes compared to low grade subtypes. Ongoing efforts include investigating the functional relevance of these methylation changes by treating, in vitro, FL cell lines with demethylating agents to evaluate changes in gene expression levels and correlating with phenotype alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4924.

The Effect of Microenvironmental CD40 Signals on TRAIL‐ and Drug‐induced Apoptosis in Follicular Lymphoma Cells

Follicular lymphoma (FL) cells are malignant counterparts of germinal centre (GC) B cells. Microenvironment of FL B cells has an important role in the progression of FL and might also have an impact on the treatment of FL. CD40 is an important mediator of microenvironmental survival signals in GCs. Here we studied responses of CD40 signalling on TRAIL-, dexamethasone- and doxorubicin-induced apoptosis in three human FL cell lines. In two of the FL cell lines, CD40 protected cells from apoptosis which was entirely dependent on the activation of NF-kappaB. In one of the FL cell lines, CD40 induced apoptosis itself. However, inhibition of NF-kappaB induced apoptosis in all three FL cell lines. Therefore, our results indicate that inhibitors of NF-kappaB or critical downstream anti-apoptotic targets of NF-kappaB instead of blocking CD40 antibodies in combination with TRAIL or other cytotoxic agents should be considered in the treatment of FL in order to prevent the protective effect of the microenvironment.

CD44 expression during tumor progression of follicular lymphoma

Follicular lymphoma often progresses to diffuse type lymphoma. To elucidate the mechanisms of the diffuse evolution of follicular lymphoma, we investigated the expression pattern of CD44 in 28 cases of follicular lymphomas (FLs) using an immunohistochemical method and semi-quantitative PCR-Southern blot analysis. The FLs were divided into four groups: i) intrafollicular (IF); ii) infiltrative (INF); iii) partially follicular (PF); and iv) minimally follicular (MF), according to the histological classification by Lukes and Collins. Immunohistochemical analysis of CD44 using antibodies against CD44 common (CD44C) epitopes showed that CD44 was expressed in the diffuse area in the INF (0/8 cases), PF (12/12 cases), and MF (2/2 cases) lymphomas, whereas CD44 was not expressed in the lymphoma cells within the area of follicular growth of IF (0/6 cases) and INF (0/8 cases). Semi-quantitative PCR-Southern blot analysis showed that CD19-selected B cells from the FLs were expressed as a product of 482 base pairs (bp) corresponding to a CD44 standard form (CD44s) (5/5 cases). Additionally, the lymphoma cells from the PF were expressed as products of 600 and 1100 bp and the cells from MF were expressed as products of 600, 900, and 1100 bp with the CD44 exon 10 or 11 probes. The results indicated that the expression of CD44s and CD44 variants containing exon 10 and 11 were up-regulated according to the diffuse evolution of the follicular lymphoma.

The effect of microenvironmental CD40 signals on TRAIL- and drug-induced apoptosis in follicular lymphoma cells

Follicular lymphoma (FL) cells are malignant counterparts of germinal centre (GC) B cells. Microenvironment of FL B cells has an important role in the progression of FL and might also have an impact on the treatment of FL. CD40 is an important mediator of microenvironmental survival signals in GCs. Here we studied responses of CD40 signalling on TRAIL-, dexamethasone- and doxorubicin-induced apoptosis in three human FL cell lines. In two of the FL cell lines, CD40 protected cells from apoptosis which was entirely dependent on the activation of NF-κB. In one of the FL cell lines, CD40 induced apoptosis itself. However, inhibition of NF-κB induced apoptosis in all three FL cell lines. Therefore, our results indicate that inhibitors of NF-κB or critical downstream anti-apoptotic targets of NF-κB instead of blocking CD40 antibodies in combination with TRAIL or other cytotoxic agents should be considered in the treatment of FL in order to prevent the protective effect of the microenvironment.

Histiocytic/dendritic cell sarcoma arising from follicular lymphoma involving the bone: a case report and review of literature

Histiocytic/dendritic cell sarcomas arising from follicular lymphoma are very rare and poorly understood lesions. We describe a case, which is unique in that it presented with a hipbone lesion simultaneously with axillary lymphadenopathy. Biopsy of the axillary lymph node showed a low-grade follicular lymphoma. The hipbone lesion was comprised two cell populations, one representing diffuse large B cell lymphoma and the other a histiocytic/dendritic sarcoma. The cells of all three lesions contained an IGH/BCL2 rearrangement, suggesting that both diffuse large B cell lymphoma and histiocytic/dendritic sarcoma differentiation developed from the same low grade precursor (follicular lymphoma). This case illustrates that sarcomatous transdifferentiation of follicular lymphoma can be an unpredictable local phenomenon and that it can occur extra nodally in the bone marrow. It may also occur concurrently with the progression of follicular lymphoma to a diffuse large B cell lymphoma.

Genetic Abnormalities Involved in the Development and Progression of Follicular Lymphoma.

Background: Follicular lymphoma (FL) is the most common indolent B-cell lymphoma and remains incurable by current therapeutic approaches. Clinical course is variable, and transformation into an aggressive lymphoma (t-FL) with marked worsening of prognosis occurs in 20–60% of patients. While Bcl2 gene translocation is a critical initiating event in the majority of FL cases, evidence indicates it is not sufficient for the development of a FL. Characterization of the genetic alterations subsequent to Bcl2 translocation will lend insight into the oncogenic pathways that contribute to FL pathogenesis and the molecular mechanisms underlying variability in clinical course.Methods: To define recurrent genomic copy number alterations (CNA) in FL, we performed high resolution array comparative genomic hybridization (aCGH) using the Affymetrix 500K SNP array platform. aCGH data were generated on a series of 112 FL cases with available gene expression profiling (GEP) and clinical information. Gene expression data were correlated with copy number data using the Gene Expression and Dosage Integrator (GEDI) algorithm developed at the NCI.Results: Selecting for abnormalities occurring in >10% of cases, the minimal common region (MCR) for 38 losses and 31 gains were defined. Novel common regions included gains on 15q11, 16p11, 5p14 and 19q13, and losses on 3q29, and 16p13. The MCR identified by aCGH were also compared with our existing cytogenetic data on 360 FL cases. MCR residing within the most frequent cytogenetic imbalances (>5%) were selected for analysis at the gene level to further refine these regions. These include gains on 1q21, 2p16, 7q11, 8q24, 12q13, 17q21, 18q21, 21q11, and X, and losses on 1p36, 6q, 10q, 13q34, and 17p13. Recurrent amplifications were detected for the 2p16, 15q11, and 17q21 MCR, while frequent uniparental disomy (UPD) was found to overlap the region of loss on 1p36. Recurrent UPD was also noted on 6p, 12q, 15q and 16p. For the majority of selected MCR, global expression of the genes residing in the MCR demonstrated an association with copy number status. Within these abnormalities, individual genes showing significant correlation with copy number were also identified.Conclusion: The combination of high resolution aCGH and GEP facilitated the identification of functionally relevant genes within the chromosomal abnormalities in FL. Delineation of these molecular targets will provide insight into the oncogenic pathways that contribute to FL disease pathogenesis and may provide novel therapeutic targets.

Prognostic significance of secondary cytogenetic alterations in follicular lymphomas

Follicular lymphoma (FL) is an indolent lymphoma with a long median survival. Transformation to a more aggressive histology (TLy) is a major cause of mortality. The critical events leading to TLy are unknown. We assessed the prognostic significance of secondary cytogenetic alterations on overall survival (OS) and transformation from 210 diagnostic FL biopsies. We analyzed serial and transformed karyotypes for recurrent alterations that predict transformation. Over 10 years, 31% of cases developed TLy. The only alteration in diagnostic karyotypes that correlated with an inferior OS was an additional X chromosome in males only (P = 0.005) suggesting that other mechanisms including epigenetic factors and over-expression of genes on the X chromosome may play a role in FL pathogenesis. In transformed karyotypes, 8q24 (MYC) translocations were common (14/37) and resulted in a median survival of 3 months posttransformation (P = 0.01). In serially obtained biopsies (28 pts), 43% of the later biopsies lacked the cytogenetic alterations found in the original FL karyotype, suggesting that karyotypic progression of FL is not strictly linear in all cases. Consequently, studying clonal evolution in FL using serial biopsies may not represent the full complexity of genetic alterations leading to transformation.

Langerhans’ cell histiocytosis mimicking relapse in a patient with follicular lymphoma

Dear Editor, A 27-year-old man was admitted for evaluation of peripheral lymph nodes with no other systemic symptoms. Initial laboratory tests, peripheral blood smears and bone marrow examinations were unremarkable. The thoracoabdominal computed tomography (CT) scan showed thoracic lymph nodes and a bulky retroperitoneal adenopathic mass. After the lymph node biopsy, the patient was diagnosed with follicular lymphoma grade 3 (World Health Organization) stage 4-A, IPI 1/5, FLIPI 2/5. He received chemotherapy with CHOP–rituximab and local radiotherapy to the abdominal mass. The patient achieved criteria of complete remission uncertain (gallium scintigraphy negative), and he initiated maintenance therapy with rituximab. Three and a half years after, the patient noticed a progressive pain in the left area of the scalp, and he asked for evaluation. On physical examination, he presented a swelling on the left parieto-temporal area of the head that was painful at palpation. A cranial radiography showed an isolated radiolucent lesion on the left parietal zone of the skull. A cranial CT scan described a lytic lesion of 12 mm in diameter in the same area, with edema of subcutaneous tissue. Thoraco-abdominal CT scan and bone marrow biopsy were normal. Bone scintigraphy and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) showed a locally increased uptake of 2×2 cm along the left parietal bone with extension to soft tissue. A surgical biopsy was decided, but a complete resection of the lesion was finally performed. The pathologic appearance of the lesion was a diffuse infiltration by cells with histiocytic appearance, vesicular nuclei, folded and grooved nuclear membranes, inconspicuous nucleoli, and abundant pale eosinophilic cytoplasm. This cluster of cells surrounded a large aggregation of eosinophils with central necrosis. Immunostaining showed cell positivity for CD1a and S-100 antibodies and negativity for CD79a, CD20, CD3, and CD5, and Ki67<5%, confirming the diagnosis of Langerhans’ cell histiocytosis (LCH). Six weeks later, FDG-PET was again performed, showing no abnormalities. No further treatment was administered. Two years later, the patient is in complete remission of either lymphoma or LCH. Histiocytic disorders represent a very heterogeneous and sometimes unclear group of diseases. The association with Hodgkin’s disease is well known, but association with other lymphoproliferative disorders is rare. To our knowledge, this is the second case of follicular lymphoma associated with LCH. In the first case, LCH was diagnosed simultaneously with the lymphoma. By contrast, in our case, LCH was diagnosed 3 years after the lymphoproliferative disease, when there was no evidence of follicular lymphoma progression. The origin of LCH is not clear, and there is Ann Hematol (2008) 87:675–676 DOI 10.1007/s00277-008-0459-y

Transformation of Follicular Lymphoma to Precursor B-Cell Lymphoblastic Lymphoma With c-mycGene Rearrangement as a Critical Event

Progression of follicular lymphoma (FL) to a higher-grade lymphoma occurs in 25% to 60% of cases and frequently indicates a poor prognosis. The transformation is accompanied by alteration in morphologic features and clinical behaviors. Herein, we report a case of FL occurring in a 59-year-old woman with the development of a precursor B-cell lymphoblastic lymphoma (PBC-LBL) during a 6-month period. Both lymphomas had identical bcl-2 and immunoglobulin heavy-chain gene breakpoints as assessed by polymerase chain reaction amplification and direct sequencing, indicating that the 2 tumors originated from a common precursor B-cell clone or the PBC-LBL arose from clonal evolution of its preceding FL and "dedifferentiated" into a lymphoblastic stage. Immunohistochemical studies revealed that both lymphomas displayed typical phenotypic characteristics. A c-myc gene translocation was observed in the PBC-LBL but not the FL by fluorescence in situ hybridization. The c-myc translocation is also reported in similar cases in the literature and likely is crucial in the pathogenesis of the PBC-LBL.

Gene Expression Model of Survival and Transformation in Follicular Lymphoma (FL): A Study by the LLMPP.

Background: FL is a common NHL that has a broad spectrum of clinical outcomes. Over time some pts will transform to an aggressive histology (Tly) associated with inferior survival. In 2004, the LLMPP constructed a model that was predictive of overall survival (OS) based on the gene expression profiles (GEP) of 191 specimens taken from pts with untreated FL. The genes associated with survival were derived from the non-neoplastic immune response (IR) cells. However the risk of developing Tly was not addressed in this study. Thus we re-analyzed the GEP with updated clinical data. Our goal was to validate our previous model with extended follow-up and to create a model that would predict the risk of developing TLy.Methods: 170 of 191 previously untreated FL pts had updated clinical information but only 142 had transformation outcome. Transformation was defined as biopsy proven DLBCL or clinically based on the presence of at least one of the following: hypercalcemia, a sudden rise in LDH >twice baseline, unusual extranodal growth or rapid discordant nodal growth. Raw CEL files from Affymetrix U133A arrays were pre-processed and normalized using Bioconductor's GCRMA package. Models were developed using SignS package (http://signs/bioinfo.cnio.es/), with 10 times cross-validation. All gene lists produced in these analyses were then re-tested for association with outcome using Bioconductor's Globaltest package. Over Representation Analysis of signature components was performed using Dchip.Results: The median OS of these patients was 8 yrs. A new 7-component survival model (85 genes) was developed that was significantly associated with survival (p= 2.9×10−13). In Globaltest, these gene lists were associated with survival at a level of (p=2.6×10−5). The previous model using IR-1 and IR-2 signatures was associated with survival at a level of p=2.6×10−4. Although there is little overlap between the 2 models, the new model confirms the importance of IR genes and extracellular matrix genes as being prognostically important. Interestingly, one component containing 10 genes on chromosome 6q was associated with a superior survival (p<1×107). 27% developed Tly over a median follow-up time of 11.2 yrs (69% biopsy proven). Our transformation model included 53 genes divided into 3 components (p=0.001). The Globaltest analysis for association of these genes with transformation was significant (p=0.018). 54 genes overlapped between the survival genes and transformation genes that were present in >1 cross validation run. These were significantly enriched in genes important in immune response like T cell and macrophage activation.Conclusion: Our survival model is stable and confirms the importance of key genes involved in the immune response and lymph node remodeling. It also introduces new genes that are potentially important for survival. Our transformation model may shed light on the mechanisms involved in the progression of FL to DLBCL but it is less stable and less reliable than our survival model at predicting outcome.

The Cost-Effectiveness of Rituximab, Cyclophosphamide, Vincristine and Prednisolone (RCVP) Compared with CVP for the Treatment of Follicular Non-Hodgkin's Lymphoma (NHL) in the UK.

Background: A large phase III randomised control trial (Marcus et al, Blood 2005, 105;4) has demonstrated that rituximab plus CVP compared with CVP alone improved time to treatment failure (p<0.0001) and a trend towards overall survival benefit (p=0.06). No analysis has yet been published relating to long-term clinical outcomes and costs of adding rituximab as a first-line treatment. Cost-effectiveness (CE) analysis aims to evaluate the additional costs and patient benefits (expressed as quality adjusted life years – QALYs) of an intervention (RCVP) compared with an alternative intervention (CVP). Some healthcare systems have CE thresholds to judge if the incremental cost-effectiveness ratio (ICER) is within acceptable limits; these thresholds vary across countries.Aims: To estimate the long-term clinical outcomes, direct health-service costs and incremental CE of RCVP compared with CVP for the first-line treatment of patients with follicular NHL.Methods: A CE analysis was developed using a state-transition Markov model, which simulated the lifetime progression of follicular NHL patients. Baseline patient characteristics were assumed equal to those in the Marcus et al study, with a mean age of 53. The CE of RCVP was evaluated using an ICER, based upon the UK health-service perspective. The model starts from the time of receiving first treatment until death. Patients could be in one of 3 discrete health states: progression-free survival, progression or death. The risk of disease progression for RCVP and CVP patients was derived directly from the results of the Marcus et al study, based upon a median trial follow-up of 42 months. The risk of death following disease progression was based upon outcomes reported by a UK registry of second-line follicular NHL patients (n=249). Patient quality of life was incorporated within the analysis using utility scores for each health state from a survey of follicular NHL patients (n=165) who completed the EQ-5D questionnaire. Cost and QALYs were discounted at 3.5%. Costs for patient monitoring and drug costs for second-line and later treatments were taken from published literature. Second-line and later treatments were assumed equivalent in both arms of the model.Results: Over the lifetime of a patient rituximab as a first-line treatment strategy generated higher total costs, but greater QALYs, compared with CVP alone. The average lifetime health-service costs per patient were £20,347 ($37,650) and £9,977 ($18,462) per patient for RCVP and CVP, respectively. Life expectancy was estimated as 9.4 yrs for RCVP and 7.2 yrs for CVP (undiscounted). Average QALYs for RCVP and CVP were 5.7 and 4.5 per patient, respectively (undiscounted). The ICER (discounted) of RCVP compared with CVP was £8,290 ($15,340) per QALY. Although there is uncertainty associated with the progression of follicular lymphoma and treatment costs, the ICER did not exceed £21,500 ($39,784) despite a wide variation in each parameter used in the analysis.Conclusions: Rituximab in combination with CVP is a cost-effective treatment option for the first-line treatment of follicular NHL. The clinical advantages of RCVP compared with CVP alone are predicted to generate an increase in life expectancy and QALYs. The resulting ICER for RCVP is well within the UK threshold of CE.

Molecular pathways in follicular lymphoma

Follicular lymphoma (FL) is one of the most common B-cell non-Hodgkin's lymphomas. The initiating genetic event found in approximately 90% of FL is the t(14;18), causing constitutive expression of the antiapoptotic BCL-2 protein. The exact secondary alterations leading to full FL development are still poorly defined. In this review, we address (i) the genetic pathways associated with tumorigenesis and progression of FL, (ii) the role of micro-environmental factors with emphasis on B-cell receptor ligands and (iii) lymphoma models in mice and what they teach us about lymphomagenesis in man.

Allelic loss during progression of follicular lymphoma

We performed loss of heterozygosity (LOH) analysis on matched lymph nodes before and after progression of follicular lymphoma (FL), and found novel LOH on chromosome arm 12p. This LOH has not been previously reported in association with FL transformation. Other sites of frequent LOH include chromosome arms 6q and 9p. LOH was observed in both transformed FL and relapse FL. These data suggest that altered tumor suppressor genes exist on 6q, 9p, and 12p that have an important role in the progression of FL. Genetic changes accumulated in relapsed FL in the absence of histological changes compared to initial diagnosis.

A novel t(2;6)(p12;q23) appearing during transformation of follicular lymphoma with t(18;22)(q21;q11) to diffuse large cell lymphoma

Follicular lymphoma is characterized genetically by t(14;18)(q32;q21), whereas t(18;22)(q21;q11), a rare variant form of t(14;18), has been preferentially observed in chronic lymphocytic leukemia (CLL). We describe here an unusual case of follicular lymphoma with a t(18;22)(q21;q11), that progressed to diffuse large cell lymphoma with a novel t(2;6)(p12;q23). Spectral karyotyping revealed that add(2)(p12) and add(6)(q23) were derived from a t(2;6)(p12;q23). Fluorescence in situ hybridization analysis confirmed rearrangements of the BCL2 gene at 18q21 and the BCL6 gene at 3q27. Our results indicate that a reciprocal translocation involving 6q23 could be implicated in the progression of follicular lymphoma and that t(18;22) may have a specific role in the pathogenesis of follicular lymphoma as well as CLL.

Cytogenetic characterization of diffuse large cell lymphoma using multi-color fluorescence in situ hybridization

We have employed multi-color fluorescence in situ hybridization (M-FISH) to characterize the cytogenetic changes in 20 diffuse large B-cell lymphomas (DLBCL), that contained complex and partially characterized karyotypes. The M-FISH analysis helped to delineate 94% of the unidentified abnormalities and assisted in redefining some unidentified/misidentified karyotypic changes. Recurrent breakpoints observed in ⩾20% cases included 14q32, 3p21, 3q27, 22q12, 1q25, and 18q21 (in decreasing order), and 1p22, 1q21, 4q31, 6q21, and 8q24 (in four cases each). Numerical gain of chromosomes 7, 9, 12, and X and loss of chromosomes 1, 4, 6, 17, and Y, were noted in ⩾20% of cases. The minimum deleted regions encompassed 6q21∼q25, 1p22∼p36, 1q32∼q44, 2p23∼p25, 4q31∼q35, 13p13∼q14, and 17p11∼p13. Two cases presented with a sole structural abnormality, and one contained a der(17)t(9;17)(p21;p13), which has not been reported earlier as a sole abnormality in DLBCL. Upon completely characterizing the karyotypes, we observed with interesting that in 55% of the cases, more than one BCL gene bearing regions was involved in translocations. In the remaining 45%, where only one or none of the BCL gene regions was involved in a rearrangement, we observed the loss of chromosomes 6 and/or 17 or partial deletions of 6q and/or 17p or gain of 7 and/or 12. Our findings suggest that, although BCL2 and BCL6 are most often implicated in DLBCL, the possibility of the disruptions of BCL3, BCL8, BCL9, and BCL10 as a “primary event” in DLBCL cannot be ruled out. Most often, a combination of events may be necessary for the genesis of DLBCL or progression of follicular lymphoma to DLBCL. Overall, M-FISH has enhanced our ability to provide a comprehensive karyotypic analysis, and has helped in defining the importance of BCL3, BCL8, BCL9, and BCL10 carrying breakpoints in DLBCL.

Relative distribution of tumour cells and reactive cells in follicular lymphoma.

Follicular lymphoma is the most common low-grade B-cell lymphoma. It is characterized by at least a partial follicular growth pattern in the majority of cases, by the morphological resemblance of the tumour cells to follicle centre centroblasts and centrocytes, and by the distinctive expression of Bcl-2 protein as a consequence of a translocation between chromosomes 14 and 18, resulting in the juxtaposition of Bcl-2 and the immunoglobulin heavy chain locus. It is not known whether the follicular growth pattern of follicular lymphoma is a consequence of properties of the tumour cells, or whether the tumour cells invade and gradually occupy a niche generated by a normal T-cell-dependent B-cell response. This study has identified cases of follicular lymphoma in which the tumour cells are apparent within a normal reactive germinal centre background. The reactive background has been investigated in these cases and also in cases showing a more characteristic appearance, in which entire malignant follicles appear to be Bcl-2-positive, as assessed by microdissection and analysis of clonality by the polymerase chain reaction (PCR). A reactive oligoclonal background was observed in all cases studied, characteristic of a normal follicle centre response. These data suggest that the progression of follicular lymphoma is dependent on the normal germinal centre microenvironment. Disruption of this dependence might be considered as a novel therapeutic strategy.

Analysis of secondary chromosomal alterations in 165 cases of follicular lymphoma with t(14;18).

Follicular lymphoma is characterized by the t(14;18) in up to 85% of cases. Almost all cases display evidence of secondary chromosomal alterations at initial diagnosis. The influence of recurrent secondary changes on disease progression has not been fully determined. The purpose of this study was to define the full spectrum of recurrent karyotypic events present at diagnosis in a large cohort of cases and to evaluate the sequence of cytogenetic evolution in relation to morphologic progression. A total of 165 cases of follicular lymphoma with t(14;18) were ascertained for which complete clinical information, histopathology, immunophenotype, and karyotype were available. One hundred sixty cases showed secondary alterations with an average of 7.9 additional changes per case. Recurrent alterations seen at the 10% or greater level included +X, +1q21-q44, +7, +12q, +18q, del(1)(p36), del(6q), del(10)(q22-q24), the development of polyploidy and sidelines, and the presence of extra marker chromosomes and chromosomal additions. Changes that correlated with morphologic progression included del(1)(p36), del(6q), del(10)(q22-q24), +7, the total number of abnormalities, the number of markers and additions, and the presence of polyploidy. The most frequent second event arising after the t(14;18) was duplication of the der(18)t(14;18). This study demonstrates that the number and type of secondary chromosomal alterations in follicular lymphoma is highly variable between cases, but that a relatively small number of changes are seen repeatedly in different combinations. A consistent pattern of cytogenetic evolution could not be identified. Potentially significant gene duplications or amplifications may be disguised within marker chromosomes and additions. Additional cytogenetic investigation is required to decipher the karyotypic complexity associated with the progression of follicular lymphoma.

Somatic Mutation of the 5′ Noncoding Region of the BCL-6 Gene Is Associated with Intraclonal Diversity and Clonal Selection in Histological Transformation of Follicular Lymphoma

Follicular lymphoma (FL) is a B cell non-Hodgkin's lymphoma (NHL) that frequently displays a t(14;18) translocation. Clonal evolution and histological transformation of FL is frequently associated with the accumulation of secondary genetic alterations. It has been demonstrated that the BCL-6 gene can be altered by chromosomal rearrangements and by mutations clustering in its 5' noncoding region in a significant fraction of FL and diffuse large cell lymphoma (DLCL). To elucidate the role of the BCL-6 gene alterations in the histological transformation and clonal progression of FL, we analyzed serial biopsy specimens from 12 patients with FL. Two cases of FL showed no histological alteration in the second biopsy, and 10 cases of FL showed morphological transformation to DLCL in the second biopsy. Southern blot analysis was used to detect rearrangement of the BCL-6 gene, polymerase chain reaction-single strand conformation polymorphism and sequence analysis were performed for identification of mutations in the 5' noncoding region of the BCL-6 gene, and immunohistochemical analysis was applied to reveal the BCL-6 protein expression. No BCL-6 gene rearrangement was detected in any of the samples, but a total of 58 mutations were found in the 5' noncoding region of the BCL-6 gene in seven cases. In five cases, both the FL and the clonally related FL or DLCL, and in two cases only the DLCL samples were mutated. The mutations were identical in multiple biopsy specimens of FL that did not show morphological transformation. In six patients where FL cells underwent morphological transformation, considerable intraclonal sequence heterogeneity was observed, indicating an ongoing type of somatic mutation. Based on the pattern of shared and nonshared mutations, the genealogical relationship of neoplastic clones could be established. In all of these cases, the histological transformation of FL was associated with the emergence of a subpopulation marked by new sites of mutations in the BCL-6 5' noncoding sequences. In three of these six cases, the histological transformation is also associated with the reduced expression of the BCL-6 protein. These findings demonstrate that mutation of the 5' noncoding region of the BCL-6 gene developed in the clonal evolution of FL, and at different time points in the lymphoma evolution different clonotypes dominate.

Localization by Chromosome Microdissection of a Recurrent Breakpoint Region on Chromosome 6 in Human B-Cell Lymphoma

Deletion of the long arm of chromosome 6 (6q) is one of the most common chromosomal alterations in human B-cell lymphomas. Conventional cytogenetic banding analysis and loss-of-heterozygosity (LOH) studies have detected several common regions of deletion ranging across the entire long arm (6q), with no defined recurrent breakpoint yet identified. We describe here a strategy combining chromosome microdissection and fluorescence in situ hybridization (Micro-FISH) to determine a minimal region of deletion along chromosome 6. Seven clinical cases and one cell line of follicular lymphoma containing a t(14;18) and one case of diffuse lymphoma, also with a t(14;18), were used for this study. All nine cases had previously defined abnormalities of chromosome 6 determined by cytogenetic analysis. The results of chromosome dissection were unexpected and in contrast to the suggestion of disparate breakpoints by conventional chromosome banding. Specifically, Micro-FISH analysis provided evidence for a common breakpoint at 6q11 in seven of nine cases. After Micro-FISH analysis, all of the presumed simple deletions of chromosome 6 were carefully reanalyzed and shown to actually represent either nonreciprocal translocations (three cases), interstitial deletions (five cases), or isochromosome (one case). The recurrent proximal breakpoint (6q11) was detected in seven of nine cases, with the minimal region of deletion encompassing 6q11 to 6q21. By analogy to other tumor systems, the identification of recurring breakpoints within 6q11 may suggest that a gene(s) important to the genesis or progression of follicular lymphoma can be localized to this band region.