Table of contents :

Epidemiology Pathogenesis Symptoms & signs Laboratory examinations Differential diagnosis Therapy  Prognosis Web resources

Epidemiology : average onset : 59 years; the second most frequent lymphoma subtype (35% of all adult NHLs in Western populations^ref); only diffuse large B cell lymphoma (DLBCL) is more common^ref. Clinically most patients are elderly and present with advanced stage disease.
Pathogenesis :

• t(14;18)(q32;q21) (85-90%), which juxtaposes the joining segment (J_H) of the Ig H chain locus on chromosome 14 and the bcl-2 proto-oncogene on chromosome 18^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. Data suggest that FL originates in the bone marrow^ref where the chromosomal translocation occursin a pre-B cell as an error in D-J or V-D joining^{ref1, ref2, ref3, ref4, ref5}. The frequency of the t(14;18) in FL suggests that activation of the bcl-2 proto-oncogene is a critical event in follicular lymphomagenesis. However, several lines of evidence suggest the t(14;18) translocation alone is not sufficient to produce the malignant phenotype. Transgeneic mice expressing a dysregulated bcl-2 demonstrate polyclonal follicular hyperplasia and extended B cell survival^ref. A clonal, malignant phenotype requires either a stochastic second event^ref or coexpression of a second proto-oncogene^{ref1, ref2}. Furthermore, FL is invariably a tumor of mature B cells with clonal H and L chain rearrangements. Provocatively, a recent report suggested that the t(14;18) translocation can be detected in some normal tonsil tissue by sensitive PCR assays^ref, suggesting its presence does not inevitably lead to lymphomagenesis. The finding of nonrandomly distributed mutations in the FL V gene in comparison with the GL V gene demonstrates that malignant clonal expansion occurred after the initiation of somatic mutation. In FL one of the IGH genes is involved in the t(14;18) and thus precludes involvement of this allele in a functional rearrangement. Similar to normal germinal center B cells, FL cells typically undergo a functional rearrangement of the remaining IGH locus, which shows frequent SHMs and evidence of ongoing mutation leading to intraclonal heterogeneity, suggesting persistent activity of the SHM machinery. Transgenic mouse models suggest that the t(14;18) alone is insufficient to produce lymphoma. Presumably the constitutive expression of Bcl-2 accompanied by the inhibition of programmed cell death allows early FL cells to remain alive and therefore at risk for subsequent molecular alterations needed to produce a fully malignant phenotype. Bcl-2 rearrangements in FL occurs at the :
• major breakpoint region (MBR) (70%) located in the 3' UTR of the last exon^{ref1, ref2, ref3}
• minor cluster region (mcr) (10%) located 20-30 kb downstream of the bcl-2 gene^ref
• other sites (20%) such as :
• variant cluster region at the 5' end of the bcl-2 gene
• intermediate cluster region (ICR) that lies between the MBR and the MCR
Normal DNA structure is described as bimodal or B-DNA. This corresponds to the normal dsDNA helix resulting from complementary base pairing. A non-B-DNA structure refers to areas of single stranded DNA where breakage would be more likely to occur. Previous dogma regarding the t(14;18) implicated the recombinase complex (RAG) and suggested that BCL2 translocations were the result of errors during primary VDJ recombination resulting from recombination recognition sequences (RRS) located on chromosome 18. In fact, this turns out not to be true. Although the RAG enzyme is involved, it is not the result of RRS on chromosome 18 but occurs because the MBR within the 3' UTR of the BCL2 gene forms a non-B-DNA structure that is recognized and cleaved by the RAG complex^ref. Proximity within the nucleus of chromosomes 14 and 18 may also play a role in promoting translocations. Breakpoints within the BCL2 region are clustered, but nonetheless span a large area of DNA. Thus, PCR strategies are associated with false-negative results, although when positive in a given case, they are useful for assessing MRD. Standard cytogenetic studies and locus-specific FISH techniques are much more sensitive^ref. In approximately 15% of patients, no t(14;18) translocation can be detected by either cytogenetic, Southern blot analysis, or polymerase chain reaction (PCR) techniques^ref (germline configuration). Tight clustering of the breakpoints at MBR and mcr, as well as the availability of consensus sequences of the J_H segments of the IgH make this a particularly favorable target for PCR amplification^{ref1, ref2, ref3, ref4}. In fact, it is possible to use 2 universal primers (one for each breakpoint) along with a primer derived from J_H region to amplify the majority of the translocations at MBR and mcr^{ref1, ref2, ref3}, with < 5% of translocations failing to be amplified.
Most patients with FL, even those in early stages, have circulating cells that carry the bcl-2 rearrangement providing a unique opportunity to identify and classify them according to their bcl-2 rearrangement without necessarily having to directly study their tumor tissue. When comparing tumor samples with blood or marrow, the only discrepancy found was in 4 of the 18 germline lymph node biopsy samples in which MBR (1) or mcr (3) rearrangements were found in PB, the BM, or both. In the remaining 64 cases (94.2% of all cases), there was agreement between the results of lymph node and PB/BM analyses. Of particular note, all patients germline for MBR or mcr in PB or BM also showed a germline pattern in lymph node tissue. Therefore, the rate of agreement between blood/marrow and tumor tissue was 94%. No patient with a bcl-2 rearrangement assessed in lymph-node tissue failed to be detected when combining PB and BM determinations. Of note is the fact that in 5.8% of the cases, a bcl-2 rearrangement was detected in PB or BM by PCR but was not observed in tumor tissue. Possible explanations for this discrepancy are :
• the tumor sample studied was only partially infiltrated by lymphoma
• some technical problems could have occurred in extracting DNA from tumor tissue
• the blood/marrow results could be spurious and could perhaps represent contamination
• the bcl-2 rearrangement detected was real but did not correspond to the tumor tissue.
This latter alternative cannot be completely ruled out in these 4 cases, because bcl-2 rearrangements have been described in PB of healthy individuals^{ref1, ref2, ref3}. However, when only 1 µg of DNA is loaded, as in this study, the bcl-2 rearrangement in blood is only detected in 6% of normal individuals^ref. When whole PB white cells are tested and not B lymphocytes, as in this study, this proportion is expected to be much lower. Relatively good concordance between the detection of lymphoma cells in blood and BM at diagnosis has been previously observed by different groups^{ref1, ref2, ref3}. However, in 20% of the patients tested, the bcl-2 rearrangement was detected only in the PB or in BM but not in both. 66% of these cases were positive in blood, but negative in BM. Because BM infiltration in FL is characteristically patchy and frequently not observed in marrow aspirates, this finding is not viewed as unusual. In fact, Gribben et al^ref previously observed that the results of PCR from BM biopsy specimens varied according to the site where the biopsy was performed^ref. Quantitative PCR of bone marrow BCL2/IgH⁺ cells at diagnosis predicts treatment response and long-term outcome in FL^ref
• t(2;18)(p11-12;q21)
• t(18;22)(q21;q11)
• +7

The typical changes that herald transformation to diffuse large B cell lymphoma (DLBCL) include a loss of the FDC meshwork, reduction in reactive T cells and an increase in centroblasts in proportion to centrocytes

• immune response 1 (IR1) contained many T cell signature genes and correlated with a survival advantage. The immune-response 1 signature included several T-cell–restricted genes but was not merely a measure of the number of tumor-infiltrating T cells, since a signature of pan–T-cell genes was not associated with survival. The immune-response 1 signature also included genes that were more highly expressed in monocytes than in T cells, suggesting that it reflected a mixture of immune cells
• immune response 2 (IR2) appeared to be associated with macrophages or perhaps FDCs and predicted for inferior survival. The immune-response 2 signature did not include T-cell–restricted genes but rather genes that are highly expressed in monocytes, dendritic cells, or both^{ref1, ref2, ref3, ref4, ref5, ref6}

• histology : Involved organs : lymph nodes, spleen, bone marrow, peripheral blood

Neoplastic cell types :
• centrocytes (alkaline phosphatase positive)
• centroblasts (alkaline phosphatase positive)
Infiltration pattern :
• follicular (75-80%)
• diffuse (20-25%)
WHO classification :
• grading :
• grade 1 (previously referred to as small cleaved B cell centrofollicular lymphoma) : 0–5 centroblasts/HPF (predominance of small centrocytes)
• grade 2 (previously referred to as mixed, small and large cell lymphoma) : 6–15 centroblasts/HPF. Prognosis : indolent but untreatable. CD10⁺19⁺20⁺22⁺79a⁺BCL2^{+(overexpressed in 100% due to t(14;18)}Bcl-6⁺
• grade 3 (previously referred by WF as follicular large cell lymphoma) : > 15 centroblasts/HPF. Prognosis : aggressive but responds to aggressive therapy
• grade 3a : > 15 centroblasts, but centrocytes are still present within follicles
• grade 3b : centroblasts form solid sheets with no residual centrocytes; further tentatively subclassified into 3 groups^ref :
• group I involved a t(14;18) and no 3q27 aberrations
• group II was without a t(14;18) and without 3q27 aberrations, but had other cytogenetic aberrations
• group III was without a t(14;18) but with aberrations involving 3q27
None of the FL grade 3B cases harbored both a t(14;18) and 3q27 aberration. These results, in particular the finding of a mutual exclusiveness of bcl-2 and bcl-6 rearrangement, indicate at least 3 different pathways of oncogenesis in FL grade 3B. FL grade 3B with bcl-2 rearrangement probably is part of the same entity as the other follicular lymphomas (1, 2, 3A), whereas the cases with 3q27 abnormalities or other unrelated translocations are more closely related to the majority of diffuse large B cell lymphoma (DLBCL) of germinal center cell origin.
Whether or not this distinction has clinical significance is unclear^ref. A small amount of data suggests that grade 3a FL is part of the spectrum of indolent grade 1 and 2 FL, while 3b FL may be more akin to de novo DLBCL^ref. Lastly, the presence of diffuse areas within a FL appears to confer more aggressive clinical behavior^ref
In addition, unusual cytological variants can be encountered that are not included in the WHO classification, including cases with large centrocytes and others with small centroblasts; in some cases the latter may resemble the cytomorphology of Burkitt lymphoma and may on occasion be associated with MYC oncogene translocations.
• variants :
• cutaneous follicle center lymphoma occurs uncommonly and characteristically involves the scalp. These cases are characterized by a lower frequency of t(14;18) and expression of Bcl-2 protein, yet the cells express CD10 and Bcl-6 in most cases. A recent study using polymerase chain reaction (PCR) and FISH suggests that primary cutaneous FL may not be related to primary nodal FL, as virtually all cases lack the t(14;18)^ref.
• diffuse follicle center lymphoma may be rarely encountered, but on closer inspection, most cases demonstrate vaguely follicular architecture that is underappreciated without the routine use of immunostains for FDCs. The latter represent the antigen-presenting cells of the lymphoid follicle and possess long cytoplasmic processes that provide the scaffolding for the secondary follicle. When a diagnosis of true diffuse follicle center lymphoma is considered, the pathologist is encouraged to demonstrate co-expression of CD10 and Bcl-6 as well as presumptive evidence of the t(14;18).
• approximately 10% of FL cases reveal the presence of a zone of cells resembling marginal zone B cells, immediately surrounding neoplastic follicles. Importantly, a residual benign mantle zone is not seen, helping to distinguish FL from a marginal zone lymphoma. The area of marginal zone differentiation within a FL takes on a distinct morphology with cells having moderate amounts of pale cytoplasm; moreover, the immunophenotype is also different, with downregulation of CD10 and Bcl-6 expression by cells within the marginal zone compartment. This aspect of FL pathology serves to highlight the importance of microenvironment influencing both morphology and immunophenotype.
• "in-situ" FL (rare) : scattered malignant follicles are identified within lymph nodes revealing mostly benign features. The malignant follicles are typically more monomorphic, but importantly strongly express Bcl-2 protein helping to identify them as neoplastic. Laser-capture microdissection together with PCR and sequencing suggests that these rare cases may represent early forms of FL, wherein malignant follicular B cells seed benign follicles^ref. These and other data provide insights into the trafficking of malignant B cells in FL.
Unresolved issues in FL pathology remain and to some extent contribute to problems with reproducibility. Diffuse areas in FL are often not identified, in part resulting from underutilization of FDC stains^{ref1, ref2}. Similarly, follicular areas in suspected de novo DLBCL are underappreciated. Variability in the cytology of cells within follicles contributes to inconsistent reporting of FL grade. FDCs are commonly misidentified as centroblasts, resulting in higher grade and, in general, there is poor inter-observer reproducibility for the counting of centroblasts. All of these factors contribute to inconsistencies in the diagnosis and grading of FL biopsies, and to some extent raise questions regarding the validity of grading and clinical decision making based on grade.
The cellular composition of FLs is also similar to that of reactive GCs, consisting of a mixture of centrocyte-like and centroblast-like cells, reactive T cells, follicular dendritic cells, and a few macrophages^{ref1, ref2}
Histological types :
• provisional cytologic grades:
• small cleaved cell lymphoma
• follicular, mixed small cleaved and large cell lymphoma : a mixture of small cleaved, large cleaved, and large uncleaved cells; large cells < 50% of the tumor. It has a low grade of malignancy. del(11)(q14;q23).
• follicular, predominantly large cell lymphoma (mildly aggressive) : a rare type with large cells that are either cleaved or noncleaved; it has a poorer prognosis than other types
• provisional subtype: follicular, predominantly small cleaved cell lymphoma : the most common type of follicular lymphoma; it has a low grade of malignancy and is characterized by the formation of malignant small cleaved follicular center cells.
• pleomorphic lymphoma / small non-cleaved cell lymphoma
• immunophenotype : CD5^-(uncommon)10 / CALLA^+/-11c^-19⁺20⁺23^-30 / Ki-1 Ag^+/-ref43^{- (uncommon)}79a⁺ IgM^mi/hi Bcl-2⁺ Bcl-6⁺. The proliferative rate as assessed using Ki-67 is characteristically low in FL and is a useful criterion for distinguishing FL from RFH
• cytogenetics : the t(14;18)(q32;q21) is the cytogenetic hallmark of FL, present in approximately 85% of grade 1 and 2 FL. The translocation results in the juxtaposition of the BCL2 oncogene into the IGH heavy chain locus on chromosome 14, leading to its constitutive expression. This molecular event is critical to follicular lymphomagenesis and is an early event, but may on its own be insufficient to produce FL. A marked heterogeneity of clonal evolution events characterizes FL at diagnosis and these alterations likely underlie the varied clinical behavior. The mean number of alterations is extremely variable, averaging 4.8, 6.5 and 19 for grades 1, 2 and 3 FL, respectively^ref. Correlation of the cytogenetic alterations with gene expression data using purified FL cells suggests that although translocation mechanisms are critical to disease initiation, most of the clonal evolution events that underlie disease progression and disordered gene expression result from copy number alterations and not balanced translocations^ref. Regions involved in these events include gains such as +X, +7, +12 or gain of 12q13-14, +18, and losses including del6q, del13q, 1p36-, 17p13- and del10q22-24^ref. Recent studies of FL suggest that distinct pathways of clonal evolution may exist and point to specific temporal sequences that follow the acquisition of the t(14;18)^ref. How these pathways correlate with gene expression has yet to be studied. The presence of the t(14;18), although characteristic of FL, is not specific, being found in 15–20% of diffuse large B cell lymphoma (DLBCL) and an almost equal percentage of mucosa-associated lymphoid tissue (MALT) lymphomas. Importantly, in MALT lymphomas the BCL2 oncogene is not involved but rather the translocations involve the MALT1 gene located 5 MB centromeric of the BCL2 gene. FLs lacking the t(14;18) exist and reveal distinct patterns of chromosomal alterations^ref. Translocations involving the BCL6 oncogene are overrepresented in this group and appear to travel with absent Bcl-2 protein expression. Moreover, Bcl-2 IHC studies suggest 2 subgroups with distinct cytogenetic, phenotypic and possibly clinical features :
• one with Bcl-2 protein overexpression unrelated to an IGH/BCL2 rearrangement
• a second without Bcl-2 expression.
Grade 3 FL is not a homogeneous entity, as recent cytogenetic data suggest at least 2 subgroups that appear to have a morphological correlate^ref.
• grade 3a FL with some residual centrocytes appears to represent the aggressive end of the clinical/morphological spectrum of indolent FL, closely related to grades 1 and 2 FL, and is characterized by the t(14;18).
• grade 3b FL on the other hand is characterized by sheets of centroblasts without admixed centrocytes, is CD10⁺ in only 50% of cases, is much less often associated with the t(14;18), often harbors the t(3;14) or variant involving the BCL6 oncogene and may be more closely related to de novo diffuse large B-cell lymphoma (DLBCL). Cytogenetic studies suggest that within the category of grade 3b FL, the t(14;18) and t(3;14) are mutually exclusive^ref. However, the clinical relevance of these distinctions remains controversial.

• early stage disease^ref : for the relatively small group of patients with stage I or II FL, radiotherapy (XRT) continues to be the standard of care based on studies showing long survivals and many patients without recurrence.
• involved-field irradiation (IF-XRT) : the long-term outcome at the Princess Margaret Hospital was recently updated showing cumulative relapse rates of 54% and 56% at 15 and 25 years, respectively, with only a 2% risk of relapse beyond 15 years (Petersen PM, Gospodarowitz M, Tsang R, et al. Long-term outcome in stage I and II follicular lymphoma following treatment with involved field radiation therapy alone. Proc ASCO. 2004;23:561). A recent report from Stanford University, however, suggests that deferring therapy until there are symptoms or other indications for treatment may yield survival that is comparable to that achieved with immediate XRT^ref. In an effort to improve on the results of IF-XRT, the MD Anderson Cancer Center group added multiagent chemotherapy and report excellent disease control^ref. A randomized trial comparing IF-XRT to combined modality therapy is ongoing. Use of functional imaging with positron emission tomography (PET) may improve the results of IF-XRT by more accurately identifying patients with localized disease
• radioimmunotherapy (RIT) is yet another approach that has the potential to improve outcomes in early stage FL by providing both local and systemic therapy with less toxicity but has not yet been studied in this setting.
• advanced stage disease :
• watch and wait : 3 randomized clinical trials have shown that the initiation of conventional therapy soon after diagnosis does not alter outcome in FL. The British National Lymphoma Investigation (BNLI) recently reported the results of a Phase III trial that randomized asymptomatic adults to either daily chlorambucil or observation^ref. With a median follow-up of 16 years, there has been no difference in overall or cause-specific survival (34% vs. 35% at 10 years). The median time to first systemic treatment for patients in the observation group was 2.6 years. For patients over the age of 70, the chances of not receiving chemotherapy or dying of lymphoma were 40% at 10 years. Complete remission rates were higher in the immediate treatment group, compared to patients who were observed and later treated (63% vs 27%), a difference that may be important if the ultimate goal is to administer post-remission therapy (e.g., anti-idiotypic vaccine) that is likely to be most effective in the presence of minimal residual disease. Carefully designed clinical trials are required to establish whether or not treatment with rituximab, chemoimmunotherapy, or radioimmunotherapy (RIT) will benefit the asymptomatic patient with advanced stage disease.
• chemotherapy : studies comparing single-agent chemotherapy with multiagent therapy in patients with advanced stage FL have not shown meaningful differences in clinical endpoints, including survival
• a recently reported trial by the Cancer and Leukemia Group B (CALGB) compared single-agent daily oral cyclophosphamide with CHOP (cyclophosphamide, hydroxydaunomycin, oncovin, prednisone)–Bleo^ref and showed no benefit to the more aggressive therapy. An unplanned analysis of the small subgroup with follicular, mixed lymphoma, however, demonstrated an improved failure-free survival and overall survival (OS) with combination chemotherapy. The achievement of durable remissions in patients with follicular mixed lymphoma (grade 2) is consistent with reports from the National Cancer Institute (NCI), also in small numbers of patients. Others, however, have failed to document a difference in clinical behavior between grades 1 and 2 FL or a benefit to more intensive therapy for grade 2 disease. Expert hematopathologists rarely agree when trying to distinguish between grades 1 and 2, further complicating matters. It is likely that molecular profiling, rather than morphology or clinical parameters, will soon be used to identify subgroups of patients most likely to benefit from specific treatments. The recently reported FL IPI was derived using clinical data from patients diagnosed between 1985 and 1992 and may no longer be the best clinically based predictor of outcome in the face of new therapies^ref.
• fludarabine, identified in the 1980s as an active agent in FL, has been incorporated into combination regimens with high response rates including molecular remissions. Although toxicity was satisfactory in a Phase I/II trial combining fludarabine + cyclophosphamide, the toxicity associated with this combination was unacceptable in an Eastern Cooperative Oncology Group (ECOG) Phase III comparison with CVP (cyclophosphamide, vincristine, prednisone), forcing the premature closure of the fludarabine-containing arm (Hochster HS, Weller E, Ryan T, et al. Results of E1496: A phase III trial of CVP with or without maintenance rituximab in advanced indolent lymphoma (NHL). Proc ASCO. 2004;23:556). Other regimens containing fludarabine have not been associated with the toxicity observed in the ECOG trial, but have not been shown to prolong the duration of remission when compared to other multiagent regimens. Prior therapy with purine analogues has also been associated with poor mobilization of peripheral blood progenitor cells in patients destined for transplant, further complicating decision-making regarding its use as initial therapy in patients who may require autologous hematopoietic stem cell transplant in the future.

 

FCM (fludarabine, cyclophosphamide, mitoxantrone)
FC (fludarabine + cyclophosphamide)
FCM (fludarabine + cyclophosphamide + mitoxantroneref
FND-Rref
• IFN-a has been studied extensively in FL patients both in combination with chemotherapy and as maintenance therapy with varying results. In most studies, IFN-a was associated with a prolongation of remission, but not OS. In a recently reported large Phase III trial conducted by Southwestern Oncology Group (SWOG), patients with indolent lymphomas were randomized to IFN-a or observation following induction with an intensive anthracycline-containing regimen and in some cases radiotherapy^ref. Neither PFS nor OS was affected by postremission therapy. Although results of randomized trials in FL and toxicities associated with IFN-a have led many clinicians to abandon its use altogether, a large Phase III study is currently ongoing in Germany testing standard versus intensive dose maintenance^ref. In addition, investigators at the MD Anderson Cancer Center are studying IFN-a in regimens including rituximab and fludarabine
• rituximab :
• first-line agent in untreated individuals. In the chemotherapy naïve, ORR are significantly higher (67% vs 46%, P = 0.0097; CR = 20%^ref) than in previously treated individuals^ref.
• in relapsed or refractory indolent lymphomas have shown overall response rates (ORR) of nearly 50% with median response durations of approximately 1 year, but only 6% of CR. Anyway ORR rise to 78% if prior ABMT/PBSCT (vs. 43% if not), 58% if FL (12% if other than FL), 53% if < 5 cm (vs. 42% if 3-7 cm and 38% if > 7 cm); 53% if chemosensitive, 46% if chemoresistant^ref. After the first infusion 20% experience Bcl-2 clearance, 50% before 4th infusion, and 60% at 3 months. Further studies confirm ORR = 27-59%, CR = 0-24%, and TTP =6-16 months^{ref1, ref2, ref3, ref4, ref5}. Extended therapy : 8 cycles give OR = 69% (CR = 17%)^ref or 76% (CR = 16%) (Kimby E, 2000)
• maintenance rituximab, repeated doses given at designated intervals after completion of 4 weekly treatments, has been studied in Phase III trials. ORR correlates with serum rituximab levels^ref.
• in a trial conducted by the Swiss Group for Clinical Cancer Research (SAAK), the median EFS was significantly longer in the maintenance arm than the "no treatment" arm (23 vs 12 months, P = 0.024), with this difference being more pronounced in the previously untreated subset (36 vs 19 months, P = 0.009)^ref.
• in a separate randomized trial, previously treated patients with responsive or stable disease following four weekly doses of rituximab were randomized to either receive repeated doses on schedule or at the time of disease progression^ref. In this trial, PFS was significantly better in patients receiving maintenance, although the overall duration of benefit from rituximab was the same in both arms. The total number of doses of rituximab administered was also greater in the maintenance arm.
In neither study were there differences in OS. A similar trial is ongoing in ECOG in previously untreated asymptomatic patients with low disease burden. Patients on the scheduled arm will receive only a single dose every 12 weeks, until progression, while those on the "retreatment" arm will be treated for progressive disease with four weekly doses until they no longer respond to rituximab. Pharmacokinetics will be studied in this trial in an attempt to correlate clinical outcome with serum levels. Differences in maintenance schedules may underlie differences in outcome between trials and these may relate to pharmacokinetics. Studies such as this one will help to determine the optimal approach to administration of R. Schema for E4402/CTSU E4402, "Randomized Phase III Trial Comparing Two Different Rituximab Dosing Regimens for Patients with Low Tumor Burden Indolent Non-Hodgkin’s Lymphoma."

When using serum rituximab > 25 mg/ml as the target value in maintenance (at 5 month, then after 3-5 months and finally after 3 months)^ref, in ..
• low-grade lymphomas ORR = 63% (CR = 36%)
• high-grade lymphomas ORR = 43% (CR = 0%)
When using serum rituximab > 100 mg/ml at month 3 as the target value in maintenance^ref
Other schemas include :
• at month 3, 5, 7 and 9 for up to 4 courses^ref
• at month 6 (4 courses), 12 (4 courses), 18 (4 courses), and 24 (4 courses) for up to 4 courses^ref
• at month 3, 6, 9, and 12^ref
Rituximab after R-CHOP : PFS = 38 vs. 15 month in untreated group (van Oers 169a, 2004)
Maintenance or re-induction ? PFS = 31.7 vs. 7 months^ref
• retreatment for patients who relapse following chemotherapy and then respond to rituximab yields responses in < 50%. Curiously, second remissions may last longer than the first^ref.
• after 1st retreatment : RR = 100%, TTP = 12.4 months, DR = 9.8 months
• after 2nd retreatment : RR = 40%, TTP = 17.8 months, DR = 16.3 months
• for patients refractory to rituximab :
• treatment with IL-2 to enhance ADCC is being studied clinically
• IFN-a_2a + rituximab : upregulation of CD20 and ADCC, increased hematological toxicity (IFN 3 MIU, not 6 MIU) => ORR = 45-99% (CR = 33-54%), DFS = 19-22.3 months^{ref1, ref2, ref3}
• G-CSF (already used in R-CHOP14 for aggressive lymphomas) + rituximab : upregulation of ADCC via neutrophils (upregulation of CD64 / FcgRI and down-regulation of CD16 / FcgRIII) : ORR = 42% (CR = 18%), DR = 18 months^ref
• although the majority of rituximab failures will still respond to radioimmunotherapy (RIT), the impact of previous rituximab therapy on results of subsequent treatment with chemoimmunotherapy requires study. "Preemptive" use of rituximab in asymptomatic patients could potentially lead to resistance early in the patient’s clinical course. The rate of transformation among patients treated with rituximab or RIT will need to be monitored. Long-term follow-up of patients will be a crucial part of all clinical trials containing novel therapies.
• chemoimmunotherapy : high response rates in multiply relapsed patients have led to the incorporation of rituximab into frontline treatment programs. In virtually every randomized trial reported to date, the addition of rituximab to chemotherapy increases time to treatment failure/progression :
• rituximab + CHOP (cyclophosphamide, hydroxydaunomycin, Oncovin, prednisone) (R-CHOP) works better than CHOP : in a mixture of untreated (n = 30) and previously treated (n = 8) patients with indolent lymphomas, response rates were high (ORR 100%, complete response [CR] 58%) and remissions durable (Czuczman M, Grillo-Lopez AJ, LoBuglio AI, et al. Patients with low-grade NHL treated with rituximab and CHOP experience prolonged clinical and molecular remission [abstract]. Blood. 2003;102:411a). The median TTP was 82 months, and half of the patients continued in remission at the time of the last update
• R-CVP (cyclophosphamide, vincristine, prednisone) works better than CVP (Marcus R, Imrie K, Belch A, et al. An international, multi centre, randomized, open-label, phase III trial comparing rituximab added to CVP chemotherapy to CVP chemotherapy alone in untreated stage III/IV follicular non-Hodgkins lymphoma [abstract]. Blood. 2003;102:28a) : phase III trials in previously untreated patients comparing R-CHOP to CHOP9
• R-FCM works better than FCM (fludarabine, cyclophosphamide, mitoxantrone) in both MCL and FL (Dreyling MH, Forstpointer R, Repp R, et al. Combined immuno-chemotherapy (R-FCM) results in superior remission and survival rates in recurrent follicular and mantle cell lymphoma—final results of a prospective randomized trial of the German Low Grade Lymphoma Study Group (GLSG) [abstract]. Blood. 2003;102:103a)^ref
• R-MCP (melphalan, cyclophosphamide, prednisone) is better than MCP^ref
Overall, chemoimmunotherapy appears to be superior to chemotherapy alone, for both chemotherapy-naïve patients and those who have been previously treated. The large US Intergroup trial comparing R-CHOP to CHOP followed by I-¹³¹-tositumomab will hopefully confirm the durability of remissions induced with chemoimmunotherapy. Schema for Phase III randomized study of CHOP with either rituximab or iodine I¹³¹ tositumomab (monoclonal antibody anti-B1) in patients with newly diagnosed FL. (* Rituximab 375 mg/m²)

In patients induced with chemotherapy, rituximab has been used as consolidation or maintenance with apparent benefit (Hochster HS, Weller E, Ryan T, et al. Results of E1496: A phase III trial of CVP with or without maintenance rituximab in advanced indolent lymphoma (NHL). Proc ASCO. 2004;23:556). In a Phase III trial, maintenance R following CVP improved PFS compared to observation (58% vs 34% at 4.5 years). An appropriately designed clinical trial is needed to compare the use of rituximab as part of induction therapy (e.g., in R-CHOP) to rituximab used only as post-remission therapy. New antibody reagents such as anti-CD22 (epratuzumab) and anti-CD80 (galiximab) are being studied individually and in combination with rituximab, and are likely to become the latest members of the armamentarium.
• radioimmunotherapy (RIT) : radioimmunoconjugates have the capacity to deliver high doses of irradiation to disease that is widespread, while having only limited effects on normal tissues. The radiolabeled anti-CD20 antibodies deliver ionizing irradiation to target cells and their neighbors but also retain their capacity for mediating complement-mediated cytotoxicity, ADCC, and apoptosis^{ref1, ref2}. Both radiopharmaceuticals have proven relatively easy to administer, safe and highly effective. They provide an alternative treatment modality that has the potential for combination with chemotherapy and other immunotherapeutic approaches^ref.
• Y-⁹⁰- ibritumomab tiuxetan, a high-energy ß-emitter, has been extensively studied in previously treated patients. In the pivotal trial, the overall response rate for relapsed or refractory follicular or transformed CD20⁺ B cell NHL was 80% (CR 30%) with a median duration of response of 14 months^ref. Y-90-ibritumomab tiuxetan is effective in patients with bulk disease and multiple prior therapies, although response rates are higher in patients treated earlier in their disease course. Response rates in patients refractory to rituximab are high (74% ORR), but the median duration of response is relatively short (6.4 months; range 0.5–25+ months). Y-90-ibritumomab tiuxetan is associated with predictable short-lived myelosuppression occurring 7–9 weeks post-therapy. Long-term follow-up of treated patients shows no excess risk of secondary leukemia or myelodysplasia. It is easily dosed on a body weight basis (0.4 mCi/kg) with a dose reduction (0.3 mCi/kg) for those with mild thrombocytopenia (< 100,000/µL). Subsequent treatment with chemotherapy is not precluded by prior RIT^ref. Studies are underway to establish the efficacy of programs in which Y-90-ibritumomab tiuxetan is used as first-line therapy or to consolidate first responses achieved with chemotherapy, rituximab, or the combination.
• I-¹³¹-tositumomab is both a a- and ß-emitter which is individually dosed according to dosimetry to deliver 75 cGy total body irradiation^{ref1, ref2}. Like Y-90-ibritumomab tiuxetan, it has proven effective in heavily pretreated relapsed and refractory patients. In the multicenter pivotal trial, heavily pretreated patients with refractory low-grade or transformed NHL had an ORR of 65% (20% CR) with a median duration of response of 6.5 months^ref. This compares favorably to a response rate of only 28% to the patients’ previous chemotherapy regimens and illustrates the impact of alternative treatment strategies in chemoresistant patients. Predictable myelosuppression is also associated with I-131-tositumomab administration. Myelodysplasia and acute leukemia have occurred following RIT, but only in patients who were previously treated with chemotherapy, and thereby already at risk. Among previously untreated patients, the ORR is 95% with a complete response rate of 75%. The 5-year progression-free survival rate has been reported to be 62.3% (Kaminski MS, Tuck M, Regan D, et al. High response rates and durable remissions in patients with previously untreated, advanced-stage, follicular lymphoma treated with tositumomab and iodine I-131 tositumomab (Bexxar) [abstract]. Blood. 2002;100:356a). In a Phase II trial, the percentage of CR/CR unconfirmed increased from 39% following CHOP chemotherapy to 60% following I-¹³¹-tositumomab consolidation.20 At 2 years, approximately 80% of treated patients remain disease free. The relative ease of treatment and potential durability of these treatments would argue for their introduction early in the therapy of patients with FL. The US Intergroup is now comparing 6 cycles of CHOP chemotherapy followed by I¹³¹-ibritumomab with R-CHOP in previously untreated patients with indolent lymphoma. It remains to be seen whether or not OS can be prolonged through the use of RIT as part of initial therapy. Thus far, it appears that subsequent therapy with chemotherapy and stem cell transplant is possible in patients who fail treatment with RIT. Use of abbreviated fludarabine before I¹³¹ tositumomab can reduce bone marrow involvement, when needed, to allow the use of RIT and can suppress HAMA responses. This sequential treatment regimen is highly effective as front-line therapy for follicular lymphoma, particularly for low- or intermediate-risk FLIPI patients^ref. A single one-week course of ¹³¹I-tositumomab therapy as initial treatment can induce prolonged clinical and molecular remissions in previously untreated patients with advanced FL. 95% of the patients had any response, and 75% had a complete response. The use of PCR to detect rearrangement of the BCL2 gene showed molecular responses in 80% of assessable patients who had a clinical complete response. After a median follow-up of 5.1 years, the actuarial 5-year progression-free survival for all patients was 59%, with a median progression-free survival of 6.1 years. The annualized rate of relapse progressively decreased over time: 25%, 13%, and 12% during the first, second, and third years, respectively, and 4.4% per year after three years. Of 57 patients who had a complete response, 40 remained in remission for 4.3 to 7.7 years. Hematologic toxicity was moderate, with no patient requiring transfusions or hematopoietic growth factors. No cases of myelodysplastic syndrome have been observed^ref. The tositumomab and I¹³¹ tositumomab therapeutic regimen produces high response rates in patients with relapsed or refractory low-grade, follicular, and transformed low-grade NHL, with a sizable subgroup of patients achieving long-term durable responses^ref. Re-treatment with I¹³¹ tositumomab in patients with low-grade, follicular, or transformed low-grade B-cell lymphoma who relapsed following a response to I¹³¹ tositumomab can produce second responses that can be durable^ref. Nurses play a vital role in the success of a Bexxar therapy program by providing patient and caregiver education, patient monitoring, and coordinating treatment schedules^ref
Radioimmunoconjugates targeting cell surface antigens such as CD22 (Y-90-epratuzumab) and HLA-DR10ß (Lym-1) have also shown promise. Whether cocktails of 2 or more radioimmunoconjugates can be used simultaneously or in sequence will require careful investigation. New strategies to improve the targeting of radioimmunoconjugates are also being studied.
• anti-idiotypic vaccine : in studies conducted at Stanford, immunized patients who mounted an anti-Id response experienced longer remissions than those who failed to generate an immune response to vaccination. In a subsequent trial at the NCI, patients who achieved complete remissions with chemotherapy but had molecular evidence of minimal residual disease had disappearance of residual malignant cells from the peripheral blood after vaccination. 3 Phase III trials to evaluate the efficacy of anti-idiotype vaccination in FL are in progress. Cytoreduction is first achieved with either chemotherapy or rituximab, followed by a series of vaccinations. While Phase III trials to validate the concept of vaccination in FL go forward, alternative methodologies, including DNA vaccines and dendritic cell based vaccinations are also being studied.
• novel agents : a long list of targeted therapies including bortezomib and Bcl-2 antisense are under clinical investigation in FL. They are most likely to be used in the future as part of multiagent and possibly multimodality therapy that incorporates other strategies.
• HSCT : after initial treatment and first recurrence, the natural history of FL is a continuum of repeated chemosensitive relapses of progressively shorter duration and ultimately death from progressive disease. Conventional chemotherapy has limited or no impact on survival, and the long-term benefit of newer treatments such as monoclonal antibodies, chemo-immunotherapy, or radioimmunotherapy is as yet unknown. HSCT advantages include the durability of responses and potential impact on survival, while the major disadvantages are immediate and long-term toxicities^{ref1, ref2}.
• autologous HSCT : indications and results : although ASCT in follicular NHL has been studied most extensively in the treatment of patients with recurrent disease, it also has been used as consolidation of first remission and in the treatment of patients with transformed lymphoma.
• ASCT for recurrent disease : chemotherapy at conventional dose for the treatment of patients with recurrent follicular NHL is likely to produce consecutive remissions of shorter duration each time. Several Phase II studies suggest, however, that salvage treatment followed by consolidation with ASCT can result in prolonged DFS^{ref1, ref2}. Freedman and colleagues reported the largest single institution experience. A total of 153 patients were treated with ASCT using autologous bone marrow purged in vitro with anti-B cell mAb. At a median follow-up of 5 years (range 2–13 years), the estimated 8-year DFS and OS were estimated as 42% and 66%, respectively^ref. All trials suggested an improved median duration of PFS compared to historic controls treated with conventional chemotherapy and prolonged PFS in a fraction of patients. On the other hand, with prolonged follow-up, recurrence rates of over 50% were generally observed and questions remain as to whether the durable responses were due to a treatment effect versus patient selection. Autologous transplantation performed in relapsed disease or partial remission^ref :

author (no. pts.)	conditioning	in vitro purging	stem  cell  source	median  follow  up (mo.)	PFS	OS	early treatment-related mortality (TRM), usually defined as occurring within 100 days after ASCT.	comments
Colombat (42)	Cy/TBI or  BEAM	purging in  40% BM pts	BM 88%,  PB 12%	43	58%  @ 43 mo	83%  @ 43 mo
Freedman (153)	Cy/TBI	Anti B MoAB + C'	BM	96	42%  @ 8 yr	66%  @ 8 yr	<1%	18 secondary  malignancies  (12 MDS)
Bierman (100)	Cy/TBI or  BEAM	none	BM 13%,  PB 87%	31	44%  @ 48 mo	65%  @ 48 mo	8%	2 secondary MDS
Apostolidis (99)	Cy/TBI	Anti B MoAB + C'	BM	66	63%  @ 66 mo	69%  @ 66 mo	4%	12 secondary  hematologic  malignancies
Voso (41)	Cy/TBI 80%  BEAM 20%	CD34+ selection  in some pts	PB	44	43%  @ 44 mo	72%  @ 44 mo	4%	5% secondary  malignancies
Bastion (48)	Cy/TBI	none	PB	21	53%  @ 21 mo	86%  @ 21 mo	8%	1 secondary MDS
Weaver (49)	Bu/Cy or	none  BEAC	PB	42	35%  @ 42 mo	55%  @ 42 mo		BuCy equivalent to  BEAC
Berglund (22)	BEAC +/–TBI	MoAB in 9 pts.	BM	74	72%  @ 74 mo	81%  @ 74 mo		50% transformed  NHL
Brice (83)	TBI 71%,  BEAM 29%	CD34 22%	PB 73%,  BM 27%	44	42%  @ 60 mo	58%  @ 60 mo		29% transformed  NHL
Verdonck (18)	Cy/TBI	none	BM	36	22%  @ 24 mo	33%  @ 36 mo
Cervantes  (34)	Cy/TBI 91%,	Anti B MoAB + C’  BEAM 9%	BM 70%,  PB 27%,  both 3%	40	18%  @ 24 mo	37%  @ 60 mo	4%
Cao (49)	Cy/TBI/VP 78% Cy/BCNU/VP 22%	Anti B MoAB	BM 39%  PB 61%	66	44%  @ 48 mo	60%  @ 48 mo
Van Besien (597)	TBI 31%  Non-TBI 69%	none	BM 15%  PB 85%	41	31%	55%	4%	TRM increases to 8% @ 5 yr
Van Besien (131)	TBI 34%  Non-TBI 66%	MoAB 11%  CD34+  selection 21%  in vitro chemotherapy 68%	BM 76%  PB 24%	49	39%	62%	8%	TRM increases to 14% @ 5 yr
Schouten (65)	Cy/TBI	Anti B MoAB 50%	BM	69	52%	72%		randomized study
Molina (58)	Cy/VP and either  BCNU or TBI	none	PB	62	42%  @ 60 mo	67%  @ 60 mo	2%	2 secondary MDS
Seyfarth (22)	Cy/TBI 38%  BEAM or  Bu/Cy 62%	CD34+ selection  in 95%	PB	48	38%  @ 48 mo	73%  @ 48 mo
Stein (36)	Cy/VP/TBI 75%  CBV 25%	none	BM 93%  PB 7%	NS	NS	53%	15%	1 secondary MDS

The European Bone Marrow Transplantation Registry (EBMTR)–sponsored CUP trial (conventional Chemotherapy, Unpurged autograft, Purged autograft) conducted between 1993 and 1997 is the only prospective randomized trial to address the role of ASCT in prolonging PFS and overall survival in patients with FL^ref. Due to slow accrual, the trial was closed after enrollment of 140 of a planned 250 patients. 65% of patients were in first relapse, the remainder in subsequent relapses. Patients were given three initial cycles of chemotherapy (usually a CHOP-like regimen), and 89 patients who attained at least a partial response were randomized to one of 3 treatment arms: further conventional chemotherapy, ASCT using purged autografts or ASCT using unpurged autografts. Too few patients were entered to assess the effect of ex-vivo purging. PFS and OS at 2 years after transplant for both patient groups randomized to ASCT were 55% and 71% compared to 26% and 46% for those receiving conventional chemotherapy, respectively. Hazard ratios for survival and PFS were 0.3 and 0.4, respectively, when comparing conventional chemotherapy with ASCT. These data are highly significant statistically and demonstrate that ASCT provides an important survival benefit in patients with chemosensitive recurrences of FL and should currently be considered a treatment of choice in this situation.
• ASCT as consolidation of first remission : ASCT has also been used for low-grade NHL patients in first complete or partial remission in order to prolong or render such remissions permanent. Most of them found very prolonged PFS in a substantial proportion of patients and favorable outcomes compared with historical controls. This led several groups to develop multicenter, randomized studies comparing ASCT consolidation versus IFN maintenance or observation. The German Low Grade Lymphoma Study Group randomized patients younger than 60 years of age with chemosensitive indolent NHL (mostly follicular NHL) in first partial or complete remission to ASCT versus maintenance interferon therapy^ref. 240 patients with FL were evaluable for the comparison of ASCT versus IFN maintenance. At a median follow-up period of 4.2 years, 31 relapses (27.2%) were observed in the ASCT study arm and 76 relapses (60.3%) in patients receiving IFN maintenance. In addition, 5 deaths occurred in remission (4 patients in the ASCT study arm and 1 in the IFN group). Accordingly, the PFS was significantly different in the 2 study arms. In patients receiving ASCT, the PFS was 79.1% after 2 years (95% confidence interval 71.4% to 86.9%) and 64.7% after 5 years (95% confidence interval 54.6% to 74.8%) in comparison to only 52.7% (95% confidence interval 43.8% to 61.7%) after 2 years and 33.3% (95% confidence interval 24.3% to 42.3%) after 5 years in the IFN study arm respectively (P < 0.0001). Autologous transplantation performed in first complete remission^ref :

author  (No. Pts.)	conditioning	in vitro purging	stem  cell  source	median  follow  up (mo.)	PFS	OS	early  TRM, treatment-related mortality usually defined as occurring within 100 days after autologous stem cell transplantation (ASCT).	comments
Freedman (77)	Cy/TBI	Anti B MoABb + C'	BM	45mo	63%  @ 3 yr	89%  @ 3 yr	3%	5 secondary  hematologic  malignancies.
Horning  (37)	Cy/TBI/VP	Anti B MoAB + C'	BM	78mo	60%  @ 10 yr	86%  @ 10 yr	5%	2 secondary  hematologic  malignancies
Corradini  (40)	Mitoxantrone/  Melphalan	none	PB	90mo	57%  @ 12 yr	60%  @ 12 yr	1%	11 pts had  transformed FL.  3 secondary  hematologic  malignancies
Voso  (70)	Cy/TBI	CD34+ selection  (10%); None (90%)	PB	44mo	78%  @ 44 mo	86% @  @ 44 mo	5%
Lenz  (140)	TBI/Cy	none	PB	53mo	65%  @ 60 mo	NS	1%	Randomized trial;  6.6% secondary  MDS in transplant  group.
Colombat  (27)	Cy/TBI	CD34+ selection  or Anti B MoAB + C'	PB	72mo	55%  @ 72 mo	64%  @ 72 mo		Randomized trial.  MDS increased in  transplant group.
Seyfarth  (33)	Cy/TBI 79%  BEAM 21%	none	PB	48 mo	76%  @ 48 mo	92%  @ 48 mo
Bociek  (43)	not stated	none	PB	36 mo	36%  @ 60 mo	63%  @ 60 mo
González- Barca (15)	Cy/TBI	immunomagnetic  purging	BM 9  PB 6	56 mo	83%  @ 56 mo	NS	13%	6/8 BM PCR  negative for BCL-2
Linassier  (42)	TBI 48%  non-TBI 52%	Immunomagnetic  purging 36%	BM 88%  PB 12%	43mo	66%  @ 43 mo	83%  @ 43 mo		No prognostic  factors identified

The French Groupe Ouest Est Leucémies Aiguës Myéloblastiques (GOELAM) group also reported in preliminary fashion that ASCT decreased recurrence rates but did not have any effect on long-term survival (Deconinck E, Foussard C, Bertrand P, et al. Value of autologous stem cell transplantation in first line therapy of follicular lymphoma with high tumor burden: final results of the randomized GOELAMS 064 Trial [abstract]. Blood. 2003;646). This may be due to the fact that patients undergoing ASCT appeared at increased risk for the development of myelodysplastic syndrome (MDS). In the absence of a demonstrated survival benefit, we do not routinely recommend ASCT in first remission for follicular NHL patients.
• technical considerations : several technical aspects of ASCT such as method of stem cell collection, ex vivo purging, conditioning regimen, and aspects of supportive care may have a considerable influence on the outcome of transplant and ideally should be investigated in randomized studies. For a variety of reasons these aspects of care generally have not been addressed in this fashion. Definitive answers to questions of the optimal preparative regimen or optimal stem cell sources therefore are not possible. Some recommendations can be provided by comparison of individual study results, case-control studies and perhaps best from multivariable analysis of large observational data sets.
• conditioning : transplant conditioning regimens for ASCT in follicular NHL can be classified into 3 variants: total-body irradiation (TBI)–containing; high-dose BCNU-based (BEAC, BEAM, CBV); and busulfan-based (BuCy, BuEtoposide). Largely for historic reasons, TBI–containing or BCNU-based regimens are used most commonly in FL. An IBMTR analysis indicated that use of TBI might be associated with an increased TRM^ref. On the other hand, TBI in the preparative regimen was also associated with decreased rates of disease recurrence. This observation, while far from definitive, suggests that TBI is a particularly effective but rather toxic treatment for FL and provides a rationale for the further development of radiolabeled monoclonal antibodies as part of conditioning regimens for transplant. A Phase II study of I-¹³¹-tositumomab combined with high-dose chemotherapy suggests an improved overall survival compared with historical controls receiving TBI^ref.
• purging : most patients with follicular NHL present in advanced disease stage and often with morphologic evidence of marrow involvement. Even in patients with histologically normal marrow, occult marrow involvement usually can be demonstrated by PCR. Further, occult lymphoma cells contaminating the stem cell infusate likely contribute to relapse after autologous transplantation. Ex vivo technologies to decrease tumor contamination include ‘positive’ selection (i.e., the in vitro enrichment of the graft for CD34 cells, a marker presumably lacking on lymphoma cells) as well as ‘negative’ selection (i.e., the removal of tumor cells by exposure to lymphoma-specific antibodies). The group from the Dana Farber Cancer Institute found that successful in vitro purging was associated with dramatically improved freedom from relapse in follicular NHL patients undergoing ASCT^ref. Furthermore, Ladetto et al showed that successful in vivo purging (i.e., the generation of PCR^- harvests after intensive induction chemotherapy) was associated with improved PFS^ref. Fouillard et al, by using various purging methods, also found a correlation between graft purging and outcome^ref. Only one group failed to find a correlation between PCR status of the infusate and recurrence^ref. The obvious corollary of these findings is that occult lymphoma cells contribute to recurrence in tumor-contaminated grafts. However, the absence of a control group makes it impossible to rule out the alternative explanation, namely that the ability to achieve a tumor-free graft is a surrogate for chemotherapy sensitivity. The CUP trial attempted to address this issue in a prospective fashion, but low patient numbers limited the study’s power. Indirect evidence of the role of a tumor-free graft comes from registry analysis. In an IBMTR study of follicular NHL, stem cell purging was identified as an independent predictor for PFS and OS^ref. Also a case-control study by Bierman et al showed that syngeneic transplants had a lower recurrence rate than purged autologous transplant, which in turn had a lower recurrence rate than unpurged autologous transplant^ref. We interpret these data as indicative of the importance of providing a tumor-free graft. Current technology limits the efficacy of graft purging and further research in this area is indicated. Recently, rituximab has been used as an "in vivo purging agent"^ref. Pilot studies indicate that rituximab administration eliminates PCR-detectable cells in a significant proportion of hematopoietic cell harvests but may be associated with late neutropenia and serious infection^ref. Whether transplant outcome will be improved by collecting PBSC after exposure to rituximab remains to be demonstrated.
• late toxicities of ASCT : although early morbidity is considerable, advances in supportive care have significantly reduced the immediate TRM associated with ASCT, even in elderly patients. On the other hand, late treatment-related side effects, such as MDS or secondary acute myeloid leukemia (sAML) occur with a 5- to 15-fold increased incidence^{ref1, ref2}. The etiology of posttransplant MDS is complex and relates to a combination of host-related factors, prior treatment, and conditioning. The outcome for patients who develop treatment related MDS/AML usually is very poor.
• allogeneic HSCT
• myeloablative transplantation : the exact role of allogeneic stem cell transplant (alloSCT) in follicular NHL remains somewhat difficult to define. Allotransplant initially was used in patients thought not to be candidates for ASCT due to extent of disease or marrow involvement. Several retrospective studies suggest that alloSCT is associated with a very low relapse rate and might be a curative treatment for FL. On the other hand, the benefit of alloSCT was offset by high TRM. The IBMTR reported data on 904 FL patients who underwent either ASCT or alloSCT between 1990 and 1999^ref. A total of 176 (19%) received alloSCT, 131 (14%) received purged ASCT, and 597 (67%) received unpurged ASCT. The 5-year TRM rates were 30%, 14%, and 8% and recurrence rates were 21%, 43%, and 58% after alloSCT, purged ASCT, and unpurged ASCT, respectively. Furthermore, the 5-year probabilities of survival were 51%, 62%, and 55% after alloSCT, purged ASCT, and unpurged ASCT. This communication confirms the high TRM yet low recurrence rate with alloSCT and overall similar long-term survival compared to patients undergoing ASCT. These data do not allow practitioners to make decisions for the use of alloSCT versus ASCT, and treatment recommendations continue to be guided by physician judgment. Over the past decade, advances in supportive care and better patient selection have resulted in improved outcomes for alloSCT. Also, the risk of secondary MDS after alloSCT is negligible. We currently recommend alloSCT for recurrent NHL patients under age 50 years who have an HLA-identical sibling. Use of a TBI-based conditioning may be preferred due to the decreased recurrence rate associated with that modality. Selected studies using myeloablative conditioning and allogeneic transplantation for FL^ref :

author (no. pt.)	conditioning	stem cell  source	median  follow-up  (mo)	PFS	OS	TRM	relapse	comments
Van Besien (113)	TBI 84%  Non-TBI 16%	BM	25	49  @ 36mo	49  @ 36mo	40%  @ 36mo	16%	transplants between 1984–1995. One third with small lymphocytic lymphoma
Peniket  (231)	varied regimens	NS	60	43%  @ 48mo	51%  @ 48mo	38%  @ 48mo	25%	case-matched study comparing  allo with auto. Allo has higher  TRM but low relapses
Van Besien  (176)	TBI 68%  Non-TBI 32%	BM 77%  PB 23%	36	45%  @ 60mo	51%  @ 60mo	24%  @ 12mo		transplants between 1990–1999
Mandigers  (15)	Cy/TBI	BM	36	67%		33%	13%	T cell depleted transplant
Stein  (15)	Cy/TBI 93%	BM 93%  PB 7%	60	NS	15%	53%	33%
Verdonck  (15)	Cy/TBI		25	70%	70%	27%	0	T cell depletion
Toze  (16)	NS	BM 100%	29	56%		25%	0	4 unrelated donors and 12  sibling donors
Forrest  (24)	Bu/Cy±MEL  92%	BM 16  PB 8	28	78%  @ 28 mo	78%  @ 28 mo	21%	0	no relapses noted

• reduced-intensity conditioning regimens, considered less toxic, are being used increasingly often. The underlying hypothesis of such a strategy is that fewer patients will develop TRM and graft-versus-leukemia (GVL) effects will be effective in curing NHL. Interesting preliminary results indicate that this premise may be the case^ref, and the potency of GVL effects is underscored by the sometimes spectacular responses to donor lymphocyte infusion^ref. Still, the considerable risks and toxicity associated with chronic GVHD cannot be underestimated^ref. Paradoxically some of the best reported results involve in-vivo or in-vitro T cell–depleted transplants^ref. Also, there is a certain arbitrariness to the definition of "reduced intensity" and a plethora of conditioning variants that renders interpretation of data difficult. The value of reduced-intensity alloSCT hopefully will be answered by a recently activated trial conducted by the Blood and Marrow Transplant Clinical Trials Network comparing ASCT versus alloSCT in follicular NHL patients. Studies using reduced intensity conditioning and allogeneic transplant for follicular lymphoma :

author (no. pts.)	conditioning	stem cell source	median  follow-up  (mo)	PFS	OS	TRM occurring in the first year after transplant	comments
Seattle (9)	flud/TBI 200	PB	> 12	7	8	0
Khouri (20)	flud/CY	PB	21	80%	80%	20%	64% cumulative incidence chronic GVHD
Faulkner (28)	BEAM Campath	PB	16	69%	74%	16%	no extensive chronic GVHD
Robinson (28)	flud/alkylator (92%)  BEAM Campath 8%	PB	NS	29%	39%	39%	higher than expected TRM  and relapse

• future directions : the limitations of ASCT transplantation include a high incidence of disease recurrence and risk of secondary MDS. While it can be considered the standard of care for patients with recurrent FL, it constitutes a very mediocre standard. Future improvements likely will focus on incorporation of targeted monoclonal antibodies into the preparative regimen and posttransplant period, novel stem cell purging strategies, and administration of additional treatments in the posttransplant period. Studies in alloSCT will focus on minimizing treatment-related complications that likely will include newer GVHD prevention approaches, use of new graft-versus-lymphoma maneuvers, and incorporation of more advanced techniques of donor lymphocyte infusion.

FCL transforms to a more aggressive diffuse large B-cell lymphoma (DLBL / DLBCL) in 25-65% of patients within 10 yrs of diagnosis

• age^ref
• stage^ref
• tumor burden
• bone marrow (BM) involvement
• B symptoms
• performance status (PS)
• serum LDH level
• anemia
• erythrocyte sedimentation rate (ESR)
• b₂ microglobulin^ref

• Romaguera score, 1991 : peripheral lymph node size (LN), degree of marrow involvement, and sex, in that order, to be important for FFS, while the number of extranodal sites (#ENS), LN, sex, and degree of marrow involvement were important for cause- specific survival. A tumor burden (TB) model was devised incorporating #ENS, LN, and degree of marrow involvement. 3 groups were identified with statistically significant differences in cause-specific survival and FFS. Those with low TB (one ENS exclusive of extensive marrow and nodal disease < 5 cm) had a 10-year cause-specific survival of 73% compared with 24% for patients with high TB (>= 2 ENS and nodal disease >= to 5 cm) (P < .001) and 40% for those with intermediate TB (either >= 2 ENS, or extensive marrow only, or nodal disease > 5 cm) (P = .050). Patients with low TB had a 10- year FFS rate of 32%, while the intermediate and high TB groups had 10% and 9% FFS, respectively (P = .003). Because sex was a very strong prognostic variable, we created a risk model for survival and FFS based on TB and sex. Females with low TB had the best prognosis (92% survival and 50% FFS at 10 years) and males with high TB had the worst outlook (median survival and FFS, 43 and 12 months, respectively). Other TB-sex combinations defined two groups with statistically significant differences in survival but comparable FFS^ref
• Scotland and Newcastle Lymphoma Group Therapy Working Party, 1991^ref and 1993^ref
• Groupe d'Etude des Lymphomes de l'Adulte, 1999^ref
• Intergruppo Italiano Linfomi score, 2000^ref :
• age (< 60 vs > 60) : RR = 2.6
• sex (F vs M) : RR= 1.8
• extranodal sites (0-1 vs > 2) : RR = 2.1
• serum LDH (normal vs elevated) : RR =  2.0
• B symptoms (absent vs present) : RR = 2.2
• ESR (< 30 vs > 30) : RR = 2.4
• the International Prognostic Index (IPI) initially designed for aggressive NHLs could also be used in indolent NHLs^{ref1, ref2, ref3, ref4, ref5, ref6}. However, some important prognostic factors may have been missed since the IPI was not designed to investigate prognostic factors in FL. Moreover, when using the IPI, very few patients (around 10-15%) with FL are classified in the poor-risk category. Because of this, the IPI is not appropriate to identify patients in whom intensive therapy has to be tested.
• Follicular Lymphoma International Prognostic Index (FLIPI), 2004^ref

Parameter	n	%	5-year survival, % (SE)	10-year survival, % (SE)	Log rank, P
sex	4167				.0025
Female		49	72.5 (1.0)	51.0 (1.4)
Male		51	68.7 (1.0)	46.2 (1.4)
age	4167				< 10-4
Younger than 60 y		63	78.1 (0.8)	58.4 (1.2)
60 y or older		37	57.7 (1.3)	32.3 (1.6)
cell type	3511				.1065
Small cell		50	71.8 (1.1)	48.6 (1.5)
Mixed		41	71.1 (1.2)	50.4 (1.7)
Large cell		9	66.1 (2.7)	40.0 (3.9)
Ann Arbor stage	4162				< 10-4
I-II		22	83.2 (1.2)	64.3 (2.0)
III-IV		78	67.0 (0.8)	44.1 (1.1)
B symptoms	3965				< 10-4
Absence		81	73.8 (0.8)	50.7 (1.2)
Presence		19	55.8 (1.8)	36.8 (2.3)
performance status (ECOG)	3602				< 10-4
0-1		88	72.4 (0.8)	50.0 (1.2)
> 1		12	58.6 (2.4)	37.8 (3.0)
Number of nodal sites	3322				< 10-4
0-4		65	77.0 (0.9)	54.6 (1.4)
5 or more		35	63.7 (1.4)	42.1 (1.9)
Number of extra nodal sites other than bone marrow	3741				< 10-4
0		62	76.1 (0.9)	55.4 (1.3)
1 or more		38	63.7 (1.3)	40.8 (1.7)
Bone marrow involvement	4016				< 10-4
Absence		52	75.6 (0.9)	56.2 (1.3)
Presence		48	65.7 (1.1)	40.4 (1.6)
Spleen involvement	3816				< 10-4
Absence		78	74.8 (0.8)	53.0 (1.2)
Presence		22	57.6 (1.7)	36.5 (2.1)
Serum b2 microglobulin	716				< 10-4
<= ULN		59	86.0 (1.7)	65.1 (3.9)
> ULN		41	65.0 (2.8)	42.5 (3.8)
ESR	2256				< 10-4
<= 40 mm/h		89	73.3 (1.0)	52.0 (1.4)
> 40 mm/h		11	43.6 (3.2)	28.5 (3.6)
Serum LDH	2565				< 10-4
<= ULN		79	76.6 (0.9)	53.9 (1.6)
> ULN		21	57.6 (2.1)	41.4 (2.7)
Thrombocyte count	3655				< 10-4
>= 150 x 109/L		88	72.2 (0.8)	50.5 (1.2)
< 150 x 109/L		12	59.9 (2.3)	36.1 (3.1)
Hemoglobin level	3813				< 10-4
>= 120 g/L		82	74.9 (0.8)	51.7 (1.2)
< 120 g/L		18	50.7 (1.9)	35.0 (2.3)
PB lymphocyte count	3122				< 10-4
>= 1 x 109/L*		80	73.3 (0.9)	51.5 (1.3)
< 1 x 109/L		20	62.5 (2.0)	39.2 (2.6)
Serum albumin level	2116				< 10-4
>= 35 g/L		90	72.3 (1.0)	50.3 (1.4)
< 35 g/L		10	48.3 (3.5)	25.8 (4.0)

• nonrandom chromosomal changes^{ref1, ref2, ref3}
• genetic instability^ref
• somatic mutations of the translocated BCL2 gene^ref
• rearrangements of the c-MYC gene^{ref1, ref2}, changes in the expression profiles of c-MYC and genes regulated by c-MYC^ref
• mutations of the P53 tumor suppressor gene have been related to aggressive behavior in FL^{ref1, ref2, ref3}
• inactivation of p16^INK4A and p15^INK4B by deletions, mutations, and hypermethylation has also been demonstrated^{ref1, ref2}
• mutations of the BCL6gene^ref (BCL6 translocation is not necessary for diffuse large B-cell lymphoma (DLBL / DLBCL) transformation but that BCL6 translocation in FL may constitute a subgroup with a higher risk to transform into aggressive lymphoma^ref). A previous study of BCL6 translocation in posttransformation DLBCL did not find a different prevalence compared with random FL cases^ref. However, the studied cases were analyzed at the posttransformation stage, and because BCL6 translocation can be lost during transformation, this study might have underestimated BCL6 translocation prevalence in FL cases that subsequently transform. The partner genes involved in the translocation with BCL6 are transcriptionally activated by a variety of stimuli that could affect the clinical behavior^{ref1, ref2}. What role does BCL6 translocation play in FL? Bastard et al^ref reported no difference in prognosis (overall survival) between BCL6 rearrangement⁺ FL cases and the negative cases^ref; however, the number of cases in their study was very small. They very recently reported that FL with BCL6 rearrangement and without t(14;18) constitutes a subgroup with distinct pathologic, molecular, and clinical characteristics^ref

	Akasaka 2003ref
	transformation	not known to transform	US 1994ref	France 1994ref	US 1995ref
BCL6 translocation (nongermline-altered PCR amplification by LDI-PCR or rearrangement by Southern blot)	16/41 (2 cases, which had a point mutation leading to the generation of a restriction site, were excluded from positive cases)	9/64 (2 cases, which had a point mutation leading to the generation of a restriction site, were excluded from positive cases)	2/31	11/80	6/42
ratio (%)	39.0	14.1	6.4	13.8	14.3
P value	.0048 (P value of transformation cases versus cases not known to transform, US 1994, France 1994, and US 1995, respectively)		.0020	.0025	.0134
methodology	LDI-PCR	LDI-PCR	Southern blot (Sac 4.0 (Sac 4.0 or F372 was used to detect BCL6 rearrangement as a probe for Southern blot ))	Southern blot (F372 (Sac 4.0 or F372 was used to detect BCL6 rearrangement as a probe for Southern blot))	Southern blot (F372 (Sac 4.0 or F372 was used to detect BCL6 rearrangement as a probe for Southern blot))

		case no.
partner	chromosomal locus	group known to transform	group not known to transform
involved gene		17 (14; 3 cases had 2 independent BCL6 partners)	8
IGH	14q32	2	2
CIITA	16p13	4	2
RhoH/TTF	4p13	2	0
MBNL	3q25	1	1
EIF4A2	3q27.3	1	0
U50HG	6q15	1	0
GRHPR	9p11.2	1	0
LRMP	12q12.1	1	0
LOC92656 (similar to NAPA)	19q13.33	1	0
SIATI	3q26.33	0	2
Currently uncharacterized		3	1
deletion of BCL6	not applicable	2	1
mutation of BCL6	not applicable	2	2
total	not applicable	21	11

• cytogenetic alterations such as loss or gain of whole chromosomes, deletions, and amplifications^{ref1, ref2, ref3, ref4}. However, the data on these chromosomal abnormalities are relatively scarce due to the technical difficulties of obtaining cytogenetic data from paired pretransformation and posttransformation specimens. These difficulties have been partially overcome by the use of comparative genomic hybridization (CGH), which has allowed the description of a high incidence of genomic imbalances in FCL^{ref1, ref2, ref3}. There are 3 studies comparing sequential FCL biopsies using conventional CGH, in which DNA is hybridized to normal metaphase chromosomes^{ref1, ref2, ref3}. A broad spectrum of alterations were observed accompanying the transformation process and affecting specific chromosomal regions. However, the analyses were limited by the low resolution of this form of CGH, which only detects DNA copy number aberrations > 10-20 megabases (Mb)^{ref1, ref2, ref3, ref4,ref5}. Moreover, with few exceptions^ref, the "target" genes within these amplification and deletion sites remain unknown. Summary of published reports on the genomic characterization of FCL transformation to DLBCL using CGH to chromosomes : the number of cases with the corresponding genomic abnormality is shown

	n	genomic abnormality
		+2p16	+X	+12q	+18q21	+7	+17q	-4q	+3q	-17q	-13q
Hough et alref	23	4	18	12	1	7	2	4	2	0	2
Goff et alref	6	2	1	1	1	1	0	1	0	1	0
Nagy et alref	5	5	1	0	0	2	0	0	0	0	2
Martinez-Climent et alref	10	2	2	2	2	1	2	2	1	1	3

Genome-wide microarray-based CGH (array CGH comprising approximately 2400 BAC and P1 clones for quantitative measurement of DNA copy number changes across the human genome with an average resolution of 1.4 Mb^{ref1, ref2}) and gene expression analysis^ref was performed on a panel of paired biopsy specimens of FCL at diagnosis and their clonally related posttransformation diffuse large B-cell lymphoma (DLBCL), as well as to 12 unrelated cell lines known or presumed to be derived from FCL cases, to determine the genomic deletions and amplifications more precisely and to correlate these genomic alterations with changes in the patterns of gene expression. FCL transformation is associated with the acquisition of a variable spectrum of genomic imbalances, some of which have not been previously reported in association with FCL transformation. In some cases these genomic changes are associated with changes in expression of genes within the targeted region upon transformation. This genomic heterogeneity confirms previous studies of patients with FCL using CGH to chromosomes^{ref1, ref2, ref3}. Moreover, array CGH allowed the structural analysis and delineation of the areas of imbalance in clinical specimens and in cell lines with higher accuracy and precision than CGH to chromosomes, distinguishing gene amplification loci within areas of gain and delineating the borders of the overrepresented loci. Array CGH also was capable of detecting small regions of genomic loss that are undetectable by CGH to chromosomes, including the homozygous loss of the P16 gene locus in the OZ cell line^ref. An additional advantage of array CGH was the use of small amounts of genomic DNA (0.2 µg), which makes it suitable for the analysis of clinical material. Areas of amplification and deletion identified in human tumors are strong candidates to contain genes important for cancer development and progression; these DNA alterations may influence gene expression. Indeed, it is a common assumption that chromosomal gains and losses exert "dosage effects" on the expression of at least some of the genes within these regions. However, confirmatory evidences for this hypothesis are scarce, especially in the process of FCL transformation to DLBCL^ref. Limited numbers of analyzed specimens, especially those harboring recurrent chromosomal aberrations, multiplicity of potentially affected genes in each aberrant chromosomal region and lack of knowledge of all the genes in each chromosomal locus make such analysis difficult and contribute to the lack of knowledge of the pathophysiological consequences of the observed chromosomal alterations. Therefore, only cataloging of the chromosomal changes and the accompanying gene expression alterations will result in data that will address these questions. We investigated the genes that may potentially become deregulated in the areas of gain/amplification and loss by reanalyzing the published^ref gene expression profiling. Our study identified a number of genes with deregulated expression that may be targeted by the genomic imbalances. However, many of the genes on the expression array that were located in these chromosomal areas did not show marked alteration in their expression within the measurement precision, and in addition, the majority of the genes that showed increased or decreased expression upon transformation were not located within the genomic regions deleted or amplified upon transformation. The limited number of cases with recurrent chromosomal aberrations prevented statistical analysis of the correlation between gene expression versus genomic changes. However, accumulation of similar data will enable such analysis in the future and will allow identification of which genes if any are deregulated by chromosomal aberrations. The analysis identified relevant genes that may be novel targets of chromosomal aberrations in FCL. For example, one of the most frequent imbalances was the gain of chromosome 18q, including in all cases chromosomal band 18q21 and the BCL2 gene locus in 18q21.3^{ref1, ref2, ref3}. As BCL2 gene expression was not altered upon transformation, other targets such as the gene PMAIP1 in band 18q21.3 may be activated by this genomic gain. Also frequent was the gain/amplification of 2p16, locus of REL and BCL11A genes. Both REL and BCL11A genes have been found coamplified in both B-NHL and Hodgkin disease^{ref1, ref2, ref3, ref4, ref5}. Data, however, did not identify marked changes in the expression of the REL gene upon transformation. Other changes acquired exclusively upon transformation included the gain of a segment in chromosome 12 from 12pter to 12q12. Trisomy of chromosome 12 is frequent in numerous B-cell malignancies, being the common region of amplification delineated to 12q12-q15^{ref1, ref2, ref3}. Recently, copy number gains at 12q12-q14 were reported exclusively in DLBCL cases transformed from previous FCL^ref. The repeated alterations of chromosome 12q in lymphoid malignancies suggest that this is a candidate area for further molecular analysis to localize target genes involved in the pathogenesis of B-cell malignancies and especially in FCL transformation. Our study identified overexpression of many genes along 12q, including CDK2 in 12q13.3 and CDK4 in 12q14.1, which have been previously involved in B-cell maligancies^{ref1, ref2}. In previous studies, trisomy of chromosome 7 has been identified as a marker of progression from indolent to aggressive FCL^{ref1, ref2}. According to our new data, the minimal region of gain relies between 7q11.2 and 7q22.1, including one amplification site on 7q21.3. Potential candidate genes located into this area and found overexpressed are CYP51 and CUTL1 (CCATT displacement protein, cux/CDP). This gene regulates normal B lymphopoiesis, and its alteration is correlated with lymphoid abnormalities in mice^ref. In addition, several genomic changes not previously reported in association with FCL transformation were identified, including overrepresentation on 6p12.3-p21. This chromosomal site is frequently targeted by translocation and genomic gain that include rearrangement of dominant oncogenes such as PIM1 and CCDN3 in B-NHL and multiple myeloma^{ref1, ref2}. In classical FCL, one case exhibited high-level amplification of 6p21^ref, whereas a more centromeric amplicon was identified on 6p12.3. The HLA-DRB1 gene, which is highly expressed in normal B cells, was found to be overexpressed in one case. Gain of 9p is common in primary mediastinal B-cell lymphoma and classical Hodgkin lymphoma^{ref1, ref2, ref3}, and according to our data may also have a role in FCL transformation. Other putative targeted gene in FCL transformation was NEM1, located in a novel region of gain in 17q21.33. This gene is a transcription factor and nucleoside diphosphate kinase that is up-regulated by MYC and has a role in the transcriptional regulation of MYC expression and has been involved in human malignancies^ref. Several areas of chromosomal deletion also were identified in association with FCL transformation. The deletion of the long arm of chromosome 11 is a common change in mantle cell lymphoma and chronic lymphocytic leukemia, but in the majority of cases this deletion involves band 11q22 where the ATM gene is frequently the targeted gene^{ref1, ref2}. However, the region of deletion delineated here is far telomeric and may target a novel tumor suppressor gene on 11q24-q25. The deletion of the telomeric portion of 1p36.3 included the site of the tumor-suppressor gene TP73, which may be altered by deletion in B-NHL^ref. Other areas of genomic loss described in the study will require further analysis to identify putative inactivated tumor suppressor genes^ref.

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