Table of contents :

Epidemiology Pathogenesis Symptoms & signs Laboratory examinations Therapy  Prognosis Web resources

Epidemiology : average onset at age 70
Pathogenesis :

• chromosome 3 anomalies :
• t(3;14)(q27;q32)
• t(2;3)(p11-12;q27)
• t(3;22)(q27;q11)
FOXP1 (3p13) is highly expressed in a proportion of DLBCL and is recurrently targeted by chromosome translocations. The genomic rearrangement of FOXP1 was identified by FISH in 3 cases with a t(3;14)(p13;q32) involving the immunoglobulin heavy chain (IGH) locus, and in one case with a variant t(2;3) affecting sequences at 2q36. These aberrations were associated with strong expression of FOXP1 protein in tumor cells, as demonstrated by IHC. The cases with t(3p13) were diagnosed as DLBCL ( x 1), gastric MALT lymphoma ( x 1) and B-cell NHL, NOS ( x 2). Further IHC and FISH studies performed on 98 cases of DLBCL and 93 cases of extranodal MZL showed a high expression of FOXP1 in approximately 13 and 12% of cases, respectively. None of these cases showed, however, FOXP1 rearrangements by FISH. However, over-representation of the FOXP1 locus found in 1 additional case of DLBCL may represent another potential mechanism underlying an increased expression of this gene^ref.
• mutations in BCL2 (30-50%)
• t(14;18) (15-20%)
• GCB-type DLBCL :
• mutation in BCL6 (30-40%)^ref, thereby preventing PRDM1/BLIMP-1–driven differentiation => aberrant BCL6 expression at the post germinal center stage takes place where normal counterpart cells down-regulate BCL6 expression
• > 40% express the differentiation antigen Ki67
• ABC-type DLBCL :
• PR (PRDI-BF1-RIZ) domain zinc finger protein 1 (PRDM1) / BLIMP-1 (6q21) is a transcription repressor with a pivotal role in plasma-cell differentiation. Clonal inactivating mutations in PRDM1 in the DLBCL cell line OCI-Ly3 and in 8 of 35 de novo clinical DLBCL samples. The mutational spectrum consists predominantly (7 cases) of single-nucleotide mutations affecting consensus splice donor sites, some of which are recurrent, that lead to splicing aberrations and premature translation termination. In 2 of these cases, point mutations appear to be caused by RNA editing with G-to-A and U-to-G conversions. Other mutations include frame-shift deletion and chromosomal inversion. Except for one mutant, which may act as a dominant-negative, all mutations are associated with either deletion or silencing of the paired PRDM1 allele. This study identifies PRDM1 inactivation as a recurrent genetic defect in DLBCL cells and establishes PRDM1 as a potential tumor suppressor gene in DLBCL. Moreover, it implies inhibition of terminal differentiation as a pathogenetic pathway in DLBCL, particularly for the activated B-cell–like DLBCL. It also demonstrates for the first time the potential role of RNA editing in lymphomagenesis^{ref1, ref2, ref3}
• typically, cells of DLBCLs express Ig of a single isotype, but there may be accompanying cells that express alternative isotypes : there was apparent clustering of mutational patterns into either an IgMD/IgG₃/IgA set or an IgG1/IgA set, indicating that the switch to IgA can occur by different routes^ref. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease^ref

• bone marrow trephine biopsy (BMTB) (positive in 12-31%^{ref1, ref2, ref3, ref4}) : bone marrow involvement in DLBCL patients portends a poorer prognosis^{ref1, ref2}. Morphological features that have been reported to influence prognosis in patients with DLCL with marrow involvement include the pattern and extent of marrow infiltration^{ref1, ref2, ref3} and the presence of histological discordance between the primary site of involvement and bone marrow^{ref1, ref2, ref3, ref4}. Debate has existed for many years over the value of unilateral versus bilateral bone marrow aspirates in DLCL, with recent publications^{ref1, ref2} suggesting that overall trephine length may be more important than the number of sites sampled. Examination of serial sections of the trephine biopsy would allow a greater area of bone marrow to be assessed with no extra morbidity to the patient, while potentially increasing the likelihood that any marrow involvement would be detected. The international working party convened by the NCI to standardise response criteria for NHL recommended that a minimum of 20 mm of trephine biopsy be examined for bone marrow involvement^ref. More recently, Bain^ref suggested a minimum trephine length of 16 mm, but stated that more focal lesions would be detected with larger biopsies. Other authors have recommended examination of at least five well-preserved marrow spaces^ref, > 5 high-power microscope fields^ref, or evaluation of a total marrow area of 150 mm^2ref. However, there is little direct evidence in the literature to support any of these recommendations. In an audit of bone marrow biopsy in 767 patients with malignant tumours conducted at a single institution, the rate of bone marrow involvement was related to trephine length, with a plateau occurring at 16 mm^ref. Because bone marrow involvement in DLCL is often focal, a number of authors have emphasised their preference for bilateral bone marrow biopsy in order to optimise the rate of positive results^{ref1, ref2}. Morphological bone marrow involvement in DLBCL is optimally demonstrated by a 20-mm long trephine biopsy from a single site which is examined at multiple levels (4 or more). This obviates the need for bilateral sampling, thereby reducing patient morbidity from the procedure^ref.
• immunophenotype : CD5^-10 / CALLA^+/-19⁺20⁺22⁺30 / Ki-1 Ag^+/-45^+/-71⁺79a⁺, sIg⁺, HLA-DR⁺. CD5 is expressed in most cases of chronic lymphocytic leukemias (CLLs) and mantle cell lymphomas (MCLs) and in some cases of DLBCL^{ref1, ref2, ref3}. Among the DLBLs, the CD5⁺ DLBCLs have been recognized as Richter's syndrome as a consequence of a morphologic transformation of CLL^ref. However, some patients with CD5⁺ DLBCL have no past history of lymphoproliferative disease (including CLL), and their DLBCL is considered to arise de novo. Patients with de novo CD5⁺ DLBCL lacked cyclin D1 gene overexpression, a critical characteristic of MCL^ref. Bcl-6 rearrangement, which is not seen in DLBL associated with Richter's syndrome, was exhibited in half of the cases of de novo CD5⁺ DLBCL examined^ref. These findings indicate that de novo CD5⁺ DLBCL is distinct from other CD5⁺ B-cell lymphomas such as the CLLs and MCLs^ref. De novo CD5⁺ DLBCL cells are derived from a B-1 subset distinct from those of CLL or MCL.
• histology : nucleus larger than macrophage nucleus. WHO variants (2001) :
• centroblastic
• immunoblastic
• T-cell/histiocyte-rich
• anaplastic
• plasmablastic
• DLBCL with expression of full-length anaplastic lymphoma kinase (ALK) : only 33 currently reported cases. cytoplasmic ALK-1, CD138, VS38 (3/3), monoclonal cytoplasmic light chain, CD45, EMA, CD4, and CD57 (2/3), and were negative for CD3, CD30, CD56, and TIA-1. Two showed variable CD79a expression, and one had rare CD20⁺ cells. 2 of 3 cases exhibited rare CD43⁺ reactivity. One case showed scattered cytokeratin⁺ cells, which could possibly lead to a misdiagnosis of carcinoma. After CHOP and radiotherapy, 2 stage I patients were free of disease at 58 and 36 months, whereas a stage IV patient was dead of disease at 22 months^ref
• organ involvement : lymph nodes, G-I tract, skin, CNS, bones, testes, lungs
• primary mediastinal large B-cell lymphoma (PMBL / PMLBCL) / mediastinal (thymic) large B cell lymphomas (MLBCLs) (4-8%^{ref1, ref2}) :

Epidemiology : patients tend to be young, with a median age of 30–35 yr at diagnosis, which contrasts with other DLBCL patients who have a median age of >60 yr at diagnosis. 53% of the patients predicted to be PMBL were younger than 35 at diagnosis, and in this age group women outnumbered men 1.8 to 1^ref. In western countries, especially France and Italy, studies of > 100 PMLBL patients have been reported^{ref1, ref2}, whereas only sporadic case reports have been published from Japan^{ref1, ref2, ref3}, suggesting a much lower incidence of PMLBL in Japan
Symptoms & signs : in many patients given this diagnosis, the only site of lymphoma involvement is the mediastinum (a bulky mediastinal tumor with local invasion to neighboring structures, including the superior vena cava, chest wall, and pleuropericardial cavities^ref), but the lymphoma can extend locally to involve other thoracic structures and occasionally disseminate to distinctive extranodal sites such as the kidney and the brain^ref but not extrathoracic sites typical of other DLBCLs
Laboratory examinations :
• while serum LDH tends to elevate, serum b₂m remains low^{ref1, ref2}
• histology : pathologically, PMLBL is similar to DLBCL in other sites, however, the presence of cells with a clear cytoplasm and high sclerosis is frequently observed (Bankes PM, Warnke RA. Mediastinal (thymic) large B cell lymphoma. In: Jaffe WA, Harris NL, Stein H, eds.; World Health Organization Classification of Tumours. Tumours of Haematopoietic and Lymphoid Tissues. Lyon, IARC Press, 2001; pp. 175–176). Immunohistochemical analysis has confirmed the B cell origin of this lymphoma, although the expression of immunoglobulin genes is notably low. PMBLs have low expression of CD10, a marker of the germinal center stage of B cell differentiation, prompting speculation that PMBL may be distinct from the GCB DLBCL subgroup^ref
• cytogenetics : amplifications on chromosome 9p^{ref1, ref2}, the low prevalence of bcl-6 rearrangements and lack of bcl-2 rearrangements^ref, and the frequent expression of the MAL gene^ref
• comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) analyses have supported the notion that PMBL is a pathogenetically distinct subgroup of DLBCL. Gains of chromosome arm 9p have been detected in over half of the PMBL cases, and this karyotypic abnormality is only occasionally detected in other DLBCLs^{ref1, ref2}. Chromosome 9p gains can also be accompanied by amplification of the JAK2 gene, and interestingly, both 9p gains and JAK2 amplification have also been detected in Hodgkin lymphoma^ref. Some patients with Hodgkin lymphoma (HL) have been noted to develop PMBL within 1 yr after treatment, and some "grey zone" lymphomas can have histological features that are intermediate between HL and PMBL^{ref1, ref2}. PMBL clinically resembles the nodular sclerosis subtype of classical Hodgkin lymphoma (cHL) and gene expression profiles show that both have low levels of expression of multiple components of the BcR signaling cascade^ref. These observations have led to speculation that PMBL and HL may be pathogenetically related^ref (Jaffe, E.S., and K. Muller-Hermelink. 1999. Relationship between Hodgkin's disease and non-Hodgkin's lymphomas. Hodgkin's Disease. P.M. Mauch, J.O. Armitage, V. Diehl, R.T. Hoppe, and L.M. Weiss, editors. Lippincott Williams & Wilkens, Philadelphia, PA. 181–191). Currently, no molecular tests are routinely available for the diagnosis of PMBL. 2 genes, MAL and FIG1, are expressed frequently in PMBLs, but these markers may not identify all PMBL cases, and FIG1 is also expressed in some DLBCLs^{ref1, ref2}. 34% of the PMBL signature genes are also characteristically expressed in HL (including high expressionof MAL, JAK2, STAT1, TRAF1, FIG1, PDL2 / B7DC, and LFA3) and these 2 B-cell malignancies have a similar clinical presentation. PDL2 / B7DC overexpression might allow a malignant B cell to co-exist with T lymphocytes in the thymus, where this lymphoma arises. The NF-kB pathway is activated in almost all cases of PBML and this might be the mechanism by which PMBL and HL cells resist apoptosis. Despite these similarities, several mature B-cell genes are only detectable in the PMBL cells^{ref1, ref2}. Gene expression profiling (GEP) strongly supported a relationship between PMBL and HL: over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in HL cells. PDL2 / B7DC, which encodes a regulator of T cell activation, was the gene that best discriminated PMBL from other DLBCLs and was also highly expressed in HL cells. The genomic loci for PDL2 and several neighboring genes were amplified in over half of the PMBLs and in HL cell lines. 2 of the PMBL distinction genes, CD30 and CCL17 / TARC, are both characteristically expressed in the malignant HRS cells of HL^{ref1, ref2}. 2 general possibilities could account for the striking gene expression similarities between PMBL and HL :
• the PMBL signature genes that are also expressed by HL may be the downstream targets of a signaling pathway or transcription factor that is active in both lymphoma types. Many of these genes are known to be activated by the NF-kB or interferon signaling pathways, but the actual pathways responsible for their expression in these lymphomas remain to be elucidated.
• PMBL and some forms of HL may originate from a thymic B cell^{ref1, ref2, ref3}. Indeed, 60% of HLs involve the mediastinum at presentation and can involve the thymus (Jaffe, E.S., N.L. Harris, H. Stein, and J.W. Vardiman. 2001. Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon, France. 351 pp). In this scenario, the gene expression program that is shared by PMBL and HL might reflect the normal characteristics of thymic B cells^{ref1, ref2, ref3} or might reflect an oncogenic mechanism that is required for a malignant B cell to develop in this anatomical site.
It is intriguing that the chromosome 9p region that is gained/amplified in PMBL and HL contains 2 regulators of T cell responses, PDL1 and PDL2. These genes encode members of the B7 family that are ligands for the PD-1 receptor on T cells^{ref1, ref2, ref3, ref4, ref5} but may also bind other T cell surface molecules^ref. PDL1 and PDL2 have been reported to have both negative and positive effects on T cell responses^{ref1, ref2, ref3, ref4, ref5, ref6}, which may be explained by the existence of more than one receptor for these ligands on T cells^ref. Expression of PDL1 on tumor cells has been shown to inhibit tumor immunity^{ref1, ref2}, and therefore PDL1 expression might allow a malignant B cell arising in the thymus to evade T cell recognition. Alternatively, the ability of PDL2 to costimulate T cells might lead to local cytokine production that is beneficial to the tumor cells. It is important to emphasize that PDL1 and PDL2 were highly expressed in most PMBLs, even those that lacked evidence of gains or amplifications of this genomic region. Thus, elevated transcription of these genes appears to be a characteristic feature of PMBL that may be augmented by genomic copy number gains. Besides PDL1 and PDL2, the amplicon on chromosome 9p in PMBL includes JAK2, which encodes a tyrosine kinase, and SMARCA2, which encodes a putative chromatin regulator. Functional studies will be needed to elucidate the relative contributions of each of these chromosome 9p genes to the pathogenesis of PMBL and HL. Despite these striking parallels between PMBL and HL, there are important differences. A subset of the PMBL signature genes was not expressed highly in Hodgkin lymphoma cell lines, and the down-regulation of mature B cell genes that is characteristic of HL was not observed in PMBLs. In addition, the intense immune cell infiltration that is characteristic of HL is not observed in most PMBLs. Interestingly, "grey zone" lymphomas have been described that have histological features of both PMBL and HL^ref (Jaffe, E.S., and K. Muller-Hermelink. 1999. Relationship between Hodgkin's disease and non-Hodgkin's lymphomas. Hodgkin's Disease. P.M. Mauch, J.O. Armitage, V. Diehl, R.T. Hoppe, and L.M. Weiss, editors. Lippincott Williams & Wilkens, Philadelphia, PA. 181–191). Such cases may indicate that there is a spectrum of lymphomas between PMBL and HL, a prospect that can be tested by gene expression profiling and other molecular analyses^ref.
Therapy : CHOP followed by consolidation radiation therapy up to 30-40 Gy with localized fields seems to be the standard of management for PMLB^ref.
Prognosis : PMBL is an aggressive lymphoma, and its relative responsiveness to treatment is controversial^ref. Some studies concluded that PMBL patients have a relatively poor prognosis^{ref1, ref2}, but another study showed a 5-yr overall survival rate of 46% with anthracycline-based chemotherapy, similar to that of other DLBCLs^ref. A more recent study that combined chemotherapy with radiotherapy reported an 82% overall survival at 3 yr, a rate much higher than in other DLBCLs^ref. Imprecision in the diagnosis of PMBL may account for some of the heterogeneity in reported clinical responses. In particular, other DLBCLs that may originate by chance in the mediastinal region may be confused with PMBL. PMBL patients had a relatively favorable clinical outcome, with a 5-yr survival rate of 64% compared with 46% for other DLBCL patients^ref. IPI^ref is not useful to differentiate prognostic groups, while modified tumor score (mTS)^ref is reported to reflect prognosis better^{ref1, ref2}.
MHC II average expression was lower in PMBCL than in certain subtypes of DLBCL however, only 12% had complete loss of MHC II expression. Poor patient survival in PMBCL correlated with incremental decreases in MHC II expression. Although overall survival was better, survival of the lowest 10% of MHC II expressers was similarly poor in DLBCL and PMBCL^ref.
• primary central nervous system lymphoma (PCNSL)
• mediastinal gray zone lymphomas (MGZL), with features transitional between cHL nodular sclerosis (NS) and primary mediastinal large B-cell lymphoma (MLBCL). There is accumulating evidence that MLBCL and cHL are related entities. Further support for a relationship between MLBCL and cHL-NS is provided by composite and metachronous lymphomas in the same patient, as well as the existence of MGZL with transitional morphology and phenotype^ref
• intravascular lymphoma (IVL) is a very rare subtype of extranodal diffuse large B-cell lymphoma (DLBCL). This entity was initially considered as an endothelial neoplasm, but with the advent of immunohistochemical studies, the lymphoid origin of this peculiar disease was established, imposing its reclassification as angioendotheliotropic lymphoma (Kiel), angiotropic large cell lymphoma (Lukes–Collins), and unclassified large B-cell lymphoma in the Revised European American Lymphoma (REAL) Classification^{ref1, ref2, ref3}. In the current World Health Organization (WHO) classification, IVL is recognized as a unique disease entity^ref. It is an extremely rare extranodal lymphoma of B-cell origin, proliferating in the lumina of small vessels, particularly capillaries, while the parenchyema of the involved organs is spared. In some cases, fibrin thrombi may be seen in the vascular lumen. In view of the rarity of the disease, epidemiologic data are only available through collaborative study group registries^ref. It usually affects middle-aged or elderly subjects and follows a wide pattern of dissemination in extranodal sites, while nodal involvement is considered rare. Noteworthy is the tropism of IVL for the central nervous system (CNS) and the skin^ref. Virtually any organ can be involved, including the kidneys, adrenals, heart, liver, spleen, gastrointestinal tract, lungs, and genitourinary tract^{ref1, ref2, ref3, ref4, ref5}, while the bone marrow is infiltrated in a third of cases^ref. Thus the occlusion of small vessels by tumor cells results in clinical manifestations that are highly variable, including pyrexia, dementia, cutaneous nodules or plaques, and occasionally breathlessness, hypertension, adrenal failure, hematologic abnormalities (autoimmune hemolytic anemia, leukopenia, pancytopenia, disseminated intravascular coagulation), and markedly elevated lactate dehydrogenase (LDH)^{ref1, ref2, ref3, ref4}. Interestingly, a "cutaneous variant," limited exclusively to the skin, has been described and included in the recent WHO-European Organization for Research and Treatment of Cancer (EORTC) classification for cutaneous lymphomas^ref. This variant is mainly confined to woman and has a better prognosis than disseminated disease^ref. In view of the variety of the clinical manifestations and the rarity of the disease, it is frequently misdiagnosed. This is the reason why the majority of cases have been diagnosed postmortem and reports studying the clinical course and outcome of the disease following chemotherapy are limited to retrospective data from study groups^ref. From a population of 700 newly diagnosed patients with NHL who were registered and followed up at our unit between 1990 and 2005, three cases (0.4%) have been classified as IVL. Among the patients, there were 2 men and one woman, with a median age of 52 years. All patients presented with systemic symptoms and disseminated disease. All patients received anthracycline-based chemotherapy + rituximab (immunochemotherapy). CR was achieved in all 3 patients, and currently all remain progression free with a follow-up of 24-45 months. In conclusion, anthracycline-based immunochemotherapy induces durable remissions in patients with IVL, an ultimately fatal disease, suggesting that the clinical course of this disease may be altered with immunochemotherapy^ref
• multiple mechanisms lead to downregulation of b₂m and concomitant loss of HLA class I expression in DLBCL^ref

• advanced DLBCL : at the completion of the initial staging evaluation, bulky stage II, stage III or stage IV disease is documented in approximately 75% of all DLCL-B patients. Therefore, chemotherapy is the mainstay of treatment. Although the standard chemotherapy regimen has not significantly changed over 25 years, the relatively recent incorporation of monoclonal antibody therapy into the standard treatment program represents an improvement in overall survival for the majority of patients with DLCL-B. The study that defined the chemotherapy standard was an Intergroup trial conducted by Southwest Oncology Group (SWOG) and Eastern Cooperative Oncology Group (ECOG). In this study, previously untreated patients with stages II bulky, III, and IV disease with intermediate- or high-grade histology were randomized to one of 4 treatment arms: CHOP (cyclophosphamide, hydroxydaunomycin, Oncovin, prednisone), m-BACOD, ProMACE-CytaBOM, or MACOP-B^ref. Each of the regimens was administered exactly as had been described in the prior Phase II studies. The median age of patients was 54 years, with 25% of the patients being older than 64. Actual 5-year failure-free survival varied between 33% and 38% and 5-year overall survival ranged between 45% and 46% (unpublished data). None of those differences among the treatment arms are significant. However, when fatal and life-threatening reactions were combined, significant differences were found between regimens, with CHOP and ProMACE-CytaBOM being less toxic than m-BACOD and MACOP-B (P < .001). Clinical trials comparing CHOP versus m-BACOD, CHOP versus ProMACE-CytaBOM and CHOP versus MACOP-B reached similar conclusions. These results, along with the fact that CHOP was cheaper and easier to administer than the other regimens, established CHOP as the standard therapy throughout the world. However, with a projected disease-free survival rate of 36%, it is obvious that it is far from ideal therapy, and there is clearly a need for better treatment approaches. How could such disparate results occur between the single institution Phase II studies and the subsequent national Phase III trials? One key explanation is that DLCL, as identified in the current classification schemes, is not a uniform disease. The International Non-Hodgkin’s Lymphoma Prognostic Factors Index (IPI) utilized pretreatment prognostic factors in a sample of over 5000 patients to develop a predictive model of outcome for DLBCL^ref. The majority of patients had received doxorubicin-based chemotherapy regimens. 5 pretreatment characteristics were found to be independent predictors of death:
• age ( 60 vs > 60)
• tumor stage I or II (localized) vs. III or IV (advanced)
• number of extranodal sites of involvement ( 1 vs > 1),
• patient ECOG performance status (0 or 1 [ambulatory] vs  2 [not ambulatory])
• serum lactate dehydrogenase (LDH) level (<= 1 times normal vs > 1 times normal).
Each of the individual factors had comparable relative risks and thus could be summed together. The resulting model identified 4 risk groups with associated 5-year survival rates: low risk (0–1, risk factor), 73%; low intermediate risk (2 risk factors), 51%; high intermediate risk (3 risk factors), 43%; and high risk (4–5 risk factors), 26%. The increased risk of death was due to both a lower rate of complete responses and a higher rate of relapse from complete response. The percentage of patients with favorable (low) IPI scores in the National Cancer Institute original trial of ProMACE-CytaBOM (44%) was almost twice that in the subsequent SWOG-8516 Phase III study (22%). The differences in outcome caused by these imbalances in patient prognostic factors can easily exceed any treatment differences. Currently, attempts are being made to define the prognosis of biologically defined subsets of patients with DLCL-B. The Leukemia-Lymphoma Molecular Profiling Project (LLMPP) has used cDNA microarray techniques to demonstrate two distinct subpopulations of DLBCL with different prognoses and different genetics. Patients with a GC B cell–like signature have a more favorable course than those with an activated B cell–like profile^ref. These differences exist within each risk group identified by the IPI. Lossos et al subsequently demonstrated that they could develop a new predictor of survival of DLBCL based on the expression of 6 genes assayed by quantitative real-time PCR^ref. Knowledge of the genetics of each group may permit the future use of therapy targeted against critical cell pathways^ref. The mAb has been combined with CHOP chemotherapy in an attempt to improve the therapeutic results. The GELA group randomized 399 previously untreated patients with DLBCL, 60 to 80 years old, to receive either 8 cycles of CHOP every 3 weeks or 8 cycles of CHOP + rituximab given on day 1 of each cycle^ref. Complete response rates of 76% and 63% (P = 0.005) and 2-year overall survivals of 70% and 57% (relative risk for CHOP-R versus CHOP: 0.64, P = 0.007) were achieved by CHOP-R and CHOP, respectively. The incidence of severe or serious side effects was similar in the 2 treatment arms. In patients with a low-risk, age-adjusted IPI (0 or 1 adverse prognostic factors), 1-year event-free survival (EFS) was 81% and 57% for the CHOP-R and CHOP arms, respectively (P < 0.001). For those with high-risk disease (2 or 3 adverse factors), 1-year EFS was 61% and 47% for the CHOP-R and CHOP arms, respectively (P = 0.01). These results suggest that addition of rituximab therapy to standard CHOP may lead to significant prolongation of event-free and overall survival in elderly patients with both high-risk and low-risk disease with no increase in toxicity. In an early analysis, CHOP-R appeared to be more effective than CHOP in bcl-2–positive, but not in bcl-2–negative, patients, suggesting that the benefit of addition of rituximab might overcome bcl-2 associated chemotherapy resistance. A larger (N = 632) intergroup US study randomized a similar population of elderly patients to receive initial therapy with either CHOP or CHOP with rituximab. The rituximab was given on a differently than the GELA study and used a schedule initially described in follicular lymphoma^ref. Responding patients then were randomized to receive either rituximab maintenance therapy (4 doses q 6 months x 2 years) or no maintenance. Preliminary results suggest a PFS benefit for the group initially randomized to CHOP + rituximab (P = 0.059); however, no OS benefit was apparent. One obvious difference between this trial and the GELA trial that might account for the failure to show a survival benefit is the fact that approximately 40% of patients on the CHOP induction arm received maintenance rituximab. While rituximab maintenance therapy benefited all patients, a subset analysis demonstrated that there was no benefit of maintenance rituximab for patients who received initial treatment with CHOP + rituximab. The trial is therefore difficult to analyze for OS because of this demonstrable interaction between the induction and maintenance therapies. When a weighted analysis was performed to mathematically model 2 groups being treated with CHOP alone or CHOP + rituximab, an OS benefit was apparent when induction therapy consisted of CHOP combined with rituximab. Thus this study shows a benefit of combining rituximab with CHOP chemotherapy as either induction therapy or maintenance therapy but not both. Ongoing analysis of this trial is being conducted to determine whether the benefit of rituximab is restricted to bcl-2⁺ patients as suggested in the GELA trial. Results of CHOP with or without rituximab in elderly DLBCL :

study	therapy	2-year TTF	2-year OS
SWOG-85161	CHOP	49%	60%
GELA6	CHOP	40%	60%
	CHOP-rituximab	58%	72%
ECOG-44947	CHOP	55%	65%
	CHOP-rituximab	65%	71%

However, until recently, there were no Phase III data addressing the value of rituximab in younger patients with DLBCL. Preliminary results from the MInT Trial (Mab Thera International Trial), a trial evaluating the combination of CHOP and rituximab in patients younger than age 60, were presented in 2004 (Pfreundschuh MG, Trumper L, Ma D, et al. Randomized intergroup trial of first line treatment for patients < = 60 years with diffuse large B-cell non-Hodgkin’s lymphoma with a CHOP-like regimen with or without the anti-CD20 antibody rituximab - early stopping after the first interim analysis. Proc ASCO; 2004:556a). Eligibility criteria included CD20⁺ DLBCL, 18–60 years, IPI 0 or 1, stages II–IV or stage I with bulk. Patients received 6 cycles of any one of several CHOP-like regimens followed by radiation therapy (30–40 Gray to bulky disease or E lesions). Rituximab was administered in the same schedule used by the previously described GELA trial. The preliminary report analyzed the first 326 patients. Median age of patients was 48 years, and 50% had bulky disease. Stage distribution was as follows: stage I, 19%, stage II, 57%; stage III, 12%, and stage IV, 12%. Thus, this trial mixed early and advanced stage DLCL-B patients. In fact, since almost 80% of patients had stage I and II disease and only 50% had bulky disease, one can estimate that almost 30% of the patients had low bulk, early-stage disease, i.e., they had an excellent prognosis. Patients receiving rituximab with chemotherapy had a significantly longer 2-year time to treatment failure (81% vs 58%) compared with those receiving chemotherapy alone. In addition, the 2-year OS significantly favored chemotherapy + rituximab (95% vs 85%, P = 0.0026). This is the first randomized trial supporting the use of rituximab in younger patients, albeit a selected subset of younger patients; it did not deal with the poor-prognosis subset of younger patients. Based largely upon the published results from GELA, CHOP with rituximab therapy (all therapy administered on day 1) has emerged to become the standard initial treatment for advanced stage DLBCL in the US. Ongoing research will better define groups of patients with large cell lymphoma, if any, who do not benefit from the addition of monoclonal antibody therapy to chemotherapy. A second concept aimed at improving the treatment of advanced stage DLBCL is dose intensity. 2 recent studies suggest possible benefit to dose intensification strategies. The SWOG conducted a pilot study evaluating dose-intensified CHOP (CHOP-DI: cyclophosphamide 1,600 mg/m², doxorubicin 65 mg/m², and vincristine 1.4 mg/m²) with filgrastim support, every 14 days for 6 planned courses^ref. Treatment with CHOP-DI was safely administered in the cooperative group setting and resulted in survival 14% better compared with historical SWOG controls. Moreover, the NHL-B1 trial from Germany randomized patients to 6 cycles of CHOP-21, CHOP-14, CHOEP-21 (CHOP plus etoposide 100 mg/m² d1-d3), or CHOEP-14 in a 2 x 2 factorial study design (Pfreundschuh M, Truemper L, Kloess M, et al. 2-weekly vx. 3-weekly CHOP with and without etoposide for patients > 60 years of age with aggressive non-Hodgkin’s lymphoma (NHL): Results of the completed NHL-B-2 trial of the DSHNHL. Ann Oncol. 2002;13 (supp 2)). Patients in the 2-weekly regimens received G-CSF starting from day 4. Patients in this trial also received radiotherapy (36 Gy) to sites of initial bulky disease and extranodal disease. In patients older than age 60, 5-year OS rates were 40.6% for CHOP-21 and 53.3% for CHOP-14, suggesting a benefit to intensified therapy in this group of patients. There is no question that toxicity is also significantly increased in these dose-intensive regimens. A modification of this approach combines infusional therapy with dose escalation to maximal patient tolerance^ref. While additional trials incorporating mAb therapy into intensified programs are ongoing, the routine use of dose-intensive regimens is not recommended outside of a clinical trial setting.
HSCT :
• autologous HSCT
• as part of the initial treatment regimen : not all patients with aggressive lymphoma benefit when autoHSCT is incorporated into their initial treatment strategy compared with patients who are treated with conventional strategy of initial chemotherapy followed by stem-cell transplant at first relapse. Thus there seems to be no indication to add ASCT to the initial combination chemotherapy treatment for all patients with aggressive lymphoma. However, all trials that retrospectively have shown a benefit to ASCT in subset analyses have incorporated a standard course of induction therapy (rather than an abbreviated course) prior to consolidative transplantation and focused on patients with high clinical risk. An international consensus conference reached the conclusion that high-dose therapy and autotransplantation in patients with high-risk IPI scores seemed to provide benefit, and this is the subject of an ongoing intergroup randomized trial in the US^ref. At the present time, we do not recommend routine use of ASCT as consolidative therapy for newly diagnosed large cell lymphoma outside a clinical trial. Additionally, it is important to note that, regardless of the progress that has been made to date, a significant number of patients are still not cured of this disease. Multiple new agents are being considered for incorporation into the initial treatment regimens of patients with advanced stage DLBCL. These include radioimmunotherapy (RIT), PKCb inhibitors, anti-CD22 (epratuzumab), gallium nitrate, Bcl-2 antisense, and anti-VEGF agents. Hopefully, these more targeted agents will not only increase the cure rate but also decrease toxicity. The initial step in planning salvage chemotherapy is to determine the goal of treatment. Some patients who fail to achieve an initial remission or relapse from complete remission can be cured. This is less likely in elderly patients, those with extensive disease, and those with a poor performance status. In such patients less intensive, palliative systemic treatments, with single agent vincristine, cytarabine, alkylating agents, or anthracyclines might be better pursued. Responses to single agent rituximab occur approximately 30% of the time, and are generally of brief duration^ref. Radiotherapy can also be used to alleviate the symptoms at a particular site of involvement in patients with relapsed DLBCL. Most younger patients receive second-line combination chemotherapy regimens. These regimens usually incorporate drugs such as cisplatin, ifosfamide, etoposide, and cytarabine, often in combination with rituximab.
• in relapsed DLCL : in the international randomized PARMA trial, 109 patients who had relapsed from CR and responded to 2 cycles of DHAP (dexamethasone, cytarabine, cisplatin) were randomly allocated to high-dose chemotherapy or continued treatment with DHAP. Bone marrow transplantation was associated with a superior FFS (51% vs 12% at 5 years) and OS (53% vs 32% at 5 years). This trial enrolled only young patients at first relapse who remained chemosensitive. Thus salvage ABMT, as currently utilized will result in survival of approximately 50% of all patients who actually receive transplants; however, only a minority of all patients meet all the strict selection criteria for ideal outcome following transplantation. For these patients, however, high-dose therapy and autologous bone marrow transplantation are the treatments of choice^ref. The outcomes of autologous HSCT^ref are better in patients with chemosensitive relapse: those with a longer duration of first remission (>12 month) and those with an age-adjusted low-risk IPI at relapse. Several high-dose regimens with or without TBI have been used with similar outcomes. Relapse remains the most common cause of treatment failure, and thus the use of radioimmunotherapy (RIT) in the high-dose regimens and incorporation of rituximab in the transplant setting have been explored. Several studies have shown that RIT both at conventional dose and at high dose can be given in combination with high-dose chemotherapy regimens without additional toxicity or delay in hematopoietic recovery after ASCT. Additional studies using RIT in combination with high-dose chemotherapy and ASCT are ongoing, and preliminary results suggest that these approaches may be superior to conventional high-dose regimens. Since rituximab is an effective therapy for B-NHL and given its limited toxicity, rituximab has been incorporated into HDT and ASCT for DLCL-B as in vivo purging, as part of high-dose regimens, and as maintenance therapy to prevent relapse. Preliminary results suggested that rituximab during ASCT and as maintenance therapy post-transplant reduces the risk of relapse and improves survival; however, these results need to be confirmed in phase III randomized trials. The role of ASCT during first remission as consolidative therapy in patients with DLCL-B remains controversial and should not be performed outside of the clinical trial setting^ref
Higher pre-transplant levels of DC1 cells and total DCs were significantly associated with improved survival : similarly, greater post transplant levels of total DCs and both subsets were significantly associated with survival^ref. 4-course HDT with escalated CHOP + etoposide (MegaCHOEP) including autologous HSCT after courses 2, 3, and 4 is feasible and effective treatment of patients with aggressive NHL. OS at 5 years was 67.2%, freedom from treatment failure (FFTF) was 62.1%, and TRM was 4.5%. There was a trend to better FFTF at dose level 2 (DL2) compared to DL1 (66.9% versus 54.2%). Dose level 2 (DL2) of this therapy is being used in an ongoing phase 3 study^ref.
• allogeneic HSCT has been used less frequently for patients with DLBCL. While occasional patients failing autologous HSCT can have prolonged survival with allogeneic HSCT, overall results have favored autologous HSCT, due to toxicity associated with allogeneic HSCT. Ongoing studies in high-risk patients are evaluating RIC allogeneic HSCT. Allogeneic HSCT for patients with relapsed DLCL-B is associated with significant toxicity and should be reserved for patients who relapse after autologous HSCT or those with persistent marrow involvement. Innovative approaches are needed for primary refractory and chemoresistant relapsed DLCL-B since these patients have very poor outcomes after autologous HSCT^ref.

• currently, the prognosis of patients with DLBCL is estimated using the clinical parameters of the International Prognostic Index (IPI)^ref. However, these clinical parameters reflect a mixture of underlying biologic or genetic differences. In an attempt to elucidate these underlying factors, the prognostic value of numerous individual proteins has been studied by immunoperoxidase and molecular techniques^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11, ref12, ref13, ref14, ref15, ref16, ref17, ref18, ref19, ref20, ref21, ref22, ref23, ref24, ref25, ref26, ref27, ref28, ref29, ref30, ref31}. However, these studies have yielded conflicting results, and none have been validated in a large prospective trial. Therefore, in contrast to the IPI, these individual markers are generally not used in clinical practice for selecting therapy or predicting prognosis.
• using a cDNA microarray, DLBCL can be divided into prognostically significant subgroups with^{ref1, ref2} :

germinal center B-cell–like (GCB)

activated B-cell–like (ABC)

type 3 gene expression profiles was subsequently added to reflect the heterogeneity of DLBCL in gene expression^{ref1, ref2}

The GCB group has a significantly better survival than the ABC group. The type 3 group is heterogeneous and not well defined, but has a poor outcome similar to the ABC group. Another study using an oligonucleotide array has demonstrated that DLBCL can be divided into 2 molecularly distinct populations (cured and fatal/refractory)^ref. Because this technology is expensive and not generally available, a simpler, more widely available method to subclassify DLBCL into molecularly distinct and prognostically significant groups using IHC would have wide applicability and practical utility in routine clinical practice. A few studies have used the immunohistochemical expression of CD10, bcl-6, or MUM1 to classify cases of DLBCL into GCB and non-GCB groups^{ref1, ref2, ref3, McClintock S, 244a}. However, the resulting data are conflicting, with 2 studies showing a significantly better survival for the GCB group^{ref1, McClintock S, 244a}. whereas 2 others have found no difference in survival between the GCB and non-GCB groups^{ref1, ref2}. None of these studies had cDNA microarray gene expression data to correlate with their immunohistochemical findings. All of the cases included in our study were previously evaluated by cDNA microarray technology^{ref1, ref2}. The goal of this study was to evaluate the use of immunoperoxidase staining for predictive markers to accurately subdivide DLBCL into these prognostically relevant subgroups using the cDNA microarray results as a gold standard. Gene expression studies using cDNA microarrays have identified prognostic subgroups in DLBCL^{ref1, ref2, ref3}. Although as few as 13 to 17 genes can be used to identify prognostic subgroups^ref1,ref2, gene expression technology is not currently available for routine clinical use. Furthermore, this technology requires fresh or frozen tissue with an adequate amount of RNA. With the frequent use of radiologically directed needle biopsies, adequate tissue for routine histology is sometimes difficult to obtain. Therefore, the ability to identify subgroups of DLBCL using immunohistochemistry would have great practical utility. The GCB and non-GCB subtypes of DLBCL can be accurately predicted using a panel of only 3 immunostains. The expression pattern of CD10, bcl-6, and MUM1 can be used to classify DLBCL into GCB and non-GCB subtypes. The antibodies selected for this study recognize molecules whose mRNA expression was highly associated with the GCB and non-GCB groups in cDNA microarray studies^{ref1, ref2}, and are reactive in formalin-fixed, paraffin-embedded tissue. Compared with the cDNA microarray, this immunostain panel reproduced the gene expression results in 71% of GCB and 88% of non-GCB cases and predicted for survival in a similar manner. Immunohistochemical staining is already a widely used method and allows for the direct visualization and, therefore, evaluation of the neoplastic cells. On the other hand, gene expression methods require a fresh or frozen tumor sample, which is not available in many cases, and sometimes fail to yield results (6.6% of our cases). In addition, unless microdissection is performed, the tissue submitted for cDNA analysis contains not only tumor but also the associated nontumor tissue. If there is a significant amount of nontumor tissue present, the cDNA expression data may not reflect the gene expression profile of the tumor. In the current study, 22 cases were classified as GCB by the cDNA microarray but as non-GCB by the TMA. However, these 22 patients had a median survival of only 2.7 years and, therefore, behaved similarly to the other non-GCB cases. This finding suggests that those patients actually belong in the non-GCB group. The cDNA classification may have been inaccurate due to the presence of normal lymph node tissue or normal germinal centers in the cDNA sample, which could have biased the cDNA results. In addition, the amount of stromal tissue present in the cDNA sample may also have influenced the gene expression results. In these 22 discordant cases, the TMA classification appears to predict survival more accurately, which was likely due to our ability to directly visualize immunostaining of the tumor cells. Actual protein expression in the tumor cells is more likely to be predictive of outcome than mRNA expression, because these 2 parameters may not always correlate and the expression of protein is the ultimate effector of gene expression. However, because these findings need to be confirmed, we are unable to conclude at this time that the TMA classification is a better predictor than the cDNA microarray. Recently, the cDNA results from most of our cases were reclassified using an alternative algorithm^ref. This alternative method defined 3 groups designated as GCB, ABC, and unclassified. Of our 139 cases analyzed using this algorithm, 70 were classified as GCB, 44 as ABC, and 25 unclassified. The TMA classification had a sensitivity of 70% for the GCB group and 87% for the non-GCB group when using this alternative method, and the positive predictive value of the TMA classification was 84% for the GCB group and 74% for the non-GCB group. The survival outcomes of the cases were similar to those shown in Figures 3 and 4, thus reaffirming the predictive value of our immunostain panel. The TMA is a cost-effective tool that allows the rapid evaluation of immunohistochemical staining of multiple tumors in a single tissue section^ref. Although the TMA is a research tool, TMA immunostaining results agree with whole tissue section staining in 86% to 100% of cases and, as the number of core samples increases, the level of agreement also increases^{ref1, ref2}. By using 4 core samples from each case, the agreement between TMA and whole section staining is 97% to 100%^ref. Furthermore, when compared with whole section immunohistochemistry, there is better consistency in the immunostaining between cases because most cases are located on the same TMA section. Quantitation of the staining results is also easier because each tissue core can be completely viewed under one intermediate-power microscopic field, and each case can be evaluated in a matter of seconds. Furthermore, use of a TMA preserves the tissue in the paraffin blocks for future studies. CD10 is a membrane-associated, neutral endopeptidase that is expressed in a variety of human tissues, but has a restricted expression in the germinal center cells of reactive lymphoid tissues^ref. Previous studies using flow cytometry have suggested that CD10 expression in DLBCL may predict for inferior survival^{ref1, ref2}, especially in conjunction with bcl-2 expression^ref. However, these studies included only a limited number of patients with short clinical follow-up. Another report of CD10 expression using flow cytometry or immunostaining in DLBCL found that patients with low IPI scores and CD10 expression had a significantly better OS^ref, whereas other studies have found no difference in outcome for patients with DLBCL that express CD10^{ref1, ref2, McClure RF, 244a}. However, in the study by Colomo and colleagues^ref, the CD10⁺ cases were significantly more likely to have advanced-stage disease, which may have negated any predictive value of CD10 expression. Some studies using immunohistochemical methods have found CD10 expression to be associated with significantly improved OS^{ref1, ref2, ref3, McClintock S, 244a}. Like-wise, CD10 expression predicts for better OS. However, given the variability of outcomes in these retrospective studies, it is doubtful that CD10 alone can be used to predict survival in DLBCL. Bcl-6 is a zinc-finger protein that acts as a transcriptional repressor^ref56 and is expressed in germinal center B cells and a subset of CD4⁺ T cells^{ref1, ref2, ref3, ref4}. Gene rearrangements involving bcl-6 have been detected in 16% to 37% of DLBCL, but most studies have found no difference in outcome^{ref1, ref2, ref3, ref4, ref6}. One study found bcl-6 rearrangement to predict for better OS^ref, whereas 2 other studies have found it to predict for worse OS^{ref1, ref2}. However, studies of bcl-6 rearrangements do not identify all of the DLBCL cases that overexpress bcl-6, because some mutations of the bcl-6 gene may also result in overexpression^{ref1, ref2}.59,60 Immunohistochemical studies of bcl-6 expression and its relationship to outcome in DLBCL are limited in number. A recent study reported no difference in OS related to bcl-6 expression^ref, whereas another study found bcl-6 expression to be associated with a better EFS but not OS.18 Other studies have reported that bcl-6 expression predicts for better OS^{ref1, ref2, ref3}. Bcl-6 expression by immunohistochemistry predicts for both better OS and EFS. Furthermore, bcl-6, in conjunction with CD10 and MUM1, is a useful marker to identify the GCB phenotype. However, some cases of DLBCL that express bcl-6 and MUM1 have an ABC gene expression pattern. Although these cases express bcl-6, the outcome is most likely to be that of the ABC subtype, and this may explain why there are discrepancies in outcome prediction when using bcl-6 expression alone. The cases in this study were investigated using a polyclonal anti–bcl-6 antibody. More recently, a monoclonal antibody (clone PG-B6p), which is also suitable for detection of the bcl-6 molecule in routine biopsies, has become commercially available. The monoclonal antibody should facilitate future tissue microarray studies because of its high specificity, absence of background staining, and the good reproducibility of immunostaining results between different centers^ref. MUM1/IRF-4 is a lymphoid-specific member of the interferon regulatory factor family of transcription factors^ref. MUM1 is normally expressed in plasma cells and a minor subset of germinal center cells, and has been reported in 50% to 77% of DLBCLs^{ref1, ref2, ref3}. Although a recent study found no association between MUM1 expression and OS^ref, we found that expression of MUM1 in at least 30% of tumor cells is associated with a significantly worse OS and EFS. Others have also found MUM1 to be predictive of worse survival^{McClintock S, 244a, Trzpuc T, 267a}. Expression of MUM1 may denote the final step of germinal center B-cell differentiation with subsequent B-cell maturation toward plasma cells^ref. Given this biologic function, it appears that MUM1 has potential to be a marker of the non-GCB phenotype. Indeed, when used in conjunction with CD10 and bcl-6, we found that MUM1 identified cases of the non-GCB phenotype. The prognostic value of bcl-2 expression in DLBCL is controversial. By Southern blot analysis, the presence of bcl-2 gene rearrangement does not appear to be predictive of survival^{ref1, ref2, ref3, ref4, ref6, ref7, ref8, ref9, ref10} with only one study reporting worse survival^ref. In fact, some studies suggest that patients with bcl-2 gene rearrangements have better survival^{ref1, ref2} Multiple studies have looked at the expression of bcl-2 using immunostains and most have found no difference in OS^{ref1, ref2, ref3, ref4, ref6, ref7, ref8, ref9, ref10}. Some studies have found that bcl-2 expression is associated with a significantly worse OS^{ref1, ref2, ref3, ref4, ref6, ref7, ref8}. However, one of these studies included T-cell lymphomas13 and, in 2 studies, the bcl-2⁺ group had higher stage disease^{ref1, ref2}. A number of studies have found bcl-2 expression to correlate with worse EFS^{ref1, ref2, ref3, ref4, ref6}. In the cDNA study^ref, a 4-fold increase of bcl-2 mRNA was more common in the ABC group (71%) compared with the GCB group (29%), but we did not find a significant difference in the expression of bcl-2 protein between the GCB and non-GCB groups in this study. However, mRNA expression does not always translate to protein expression. Other studies using an immunohistochemical panel that classified DLBCL into GCB and non-GCB groups have also found no difference in the expression of bcl-2 protein between these 2 groups^{ref1, ref2}. These studies reported bcl-2 expression in 50% to 67% of GCB and 45% to 62% of non-GCB cases^{ref1, ref2}. Similarly, we found that bcl-2 was expressed in 59% of the GCB group and 43% of the non-GCB group. Bbcl-2 expression by itself had no prognostic effect on OS or EFS. However, when used in conjunction with the TMA subclassification, expression of bcl-2 was associated with a significantly worse outcome in the non-GCB group, but not in the GCB group. FOXP1 is a winged-helix transcription factor that acts as a transcriptional repressor^{ref1, ref2}. It is expressed in a wide variety of normal and neoplastic tissues, including a subset of DLBCL^ref. In reactive lymphoid tissues, FOXP1 is seen in a variable proportion of B cells, both within and outside the germinal centers, but it does not stain plasma cells^ref. One group has reported an inverse correlation between FOXP1 and bcl-6 expression in DLBCL^{Jones M, 332a}. By immunohistochemical staining, we found FOXP1 expression in 61% of DLBCL. Within the TMA subgroups, FOXP1 was expressed more often in the non-GCB group (71% of cases) than in the GCB group (48% of cases). However, FOXP1 expression had no effect on OS or EFS when evaluated alone or within the context of IPI scores or the TMA subclassification. Cyclin D2 is a cell cycle regulatory protein that is reported to be expressed in 27% of DLBCLs by immunohistochemical staining^ref. However, another study found no evidence of genomic amplification of the cyclin D2 gene in 24 cases of DLBCL^ref. No studies to date have evaluated the prognostic significance of cyclin D2 expression in DLBCL. Expression of cyclin D2 was found in 14% of DLBCLs. However, all of these patients were in the non-GCB subgroup and they had a very poor clinical outcome with a median survival of only 1 year. Only 2 of the 19 patients were alive at 5 years. Additional studies evaluating the prognostic value of cyclin D2 expression in DLBCL are needed to confirm our preliminary findings. A recent study similar to ours used the coexpression of CD10 and bcl-6 to determine the "GC phenotype," and found that it was predictive of better OS^ref. However, by using this more limited approach, some GCB cases that mark with either CD10 or bcl-6 alone would be inaccurately classified as non-GCB. In our study, this method would have misclassified 28 GCB patients as non-GCB, because these cases were positive for either CD10 or bcl-6, but not both. Although it may be useful to identify patients who will perform better with current therapy, it is perhaps more important to accurately identify those patients who will do poorly in order to provide more aggressive therapy at the time of diagnosis. To accurately subdivide DLBCL, markers predictive of both the GCB and non-GCB phenotypes should be used. Another study similar to ours using an immunostain panel of CD10, bcl-6, MUM1, and CD138 was recently published,31 but did not find a survival difference between the GCB and non-GCB patients. However, the cases expressing CD10 were significantly more likely to have advanced stage (Ann Arbor stage III or IV) disease compared with those who were negative for CD10. This clinical difference may account for their inability to find a survival difference between CD10⁺ and CD10^– cases and, in turn, GCB and non-GCB cases. In addition, 14% of the patients included in that study did not receive an anthracycline-containing chemotherapy regimen. Recently, another group reported that the combination of CD10, bcl-6, MUM1, and CD138 could identify 2 prognostic subgroups in DLBCL^{McClintock S, 244a}. Similarly, we found that CD10, bcl-6, and MUM1 expression could subclassify DLBCL into GCB and non-GCB groups. In addition to the IPI score, our immunostain panel was an independent predictor of survival in multivariate analysis. The expression of CD10, bcl-6, MUM1, bcl-2, and cyclin D2 are each predictive of survival in DLBCL, and that the results for CD10, bcl-6, and MUM1 can be combined to divide DLBCL into GCB and non-GCB subgroups with an outcome similar to that predicted by cDNA microarray analysis. In fact, this latter panel of immunostains predicted the cDNA classification in 71% of GCB and 88% of ABC or type 3 cases. However, these findings need to be confirmed and more studies comparing gene expression and protein expression are clearly needed. Currently, the number of immunohistochemical markers available for delineating prognostic groups is rather limited. As new antibodies for GCB- and ABC-specific proteins or other markers are developed for immunohistochemistry, additional studies would be of interest. If such studies are confirmatory, a panel of immunohistochemical stains could be used to stratify patients for risk-adjusted therapies. In particular, patients with the non-GCB phenotype, especially those with high IPI scores or with tumors expressing bcl-2 or cyclin D2, could be identified prospectively for more aggressive or experimental therapies^ref.
A multivariate model based on the RNA expression of 6 genes - LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2 - predicts survival in DLBCL patients. Since LMO2 emerged as the strongest predictor of superior outcome, a monoclonal anti-LMO2 antibody was arrangeed in order to study its tissue expression pattern. IHC of over 1200 normal and neoplastic tissue and cell-lines showed that LMO2 protein is expressed as a nuclear marker in normal GC B-cells, GC-derived B-cell lines and in a subset of GC-derived B-cell lymphomas. LMO2 was also expressed in erythroid and myeloid precursors and in megakaryocytes, and also in lymphoblastic and acute myeloid leukemias. It was rarely expressed in mature T, NK and plasma cell neoplasms and was absent from non-hematolymphoid tissues except for endothelial cells. Hierarchical cluster analysis of immunohistologic data in DLBCL demonstrated that the expression profile of the LMO2 protein was similar to that of other GC-associated proteins (HGAL, BCL6 and CD10) but different from that of non-GC proteins (MUM1/IRF4 and BCL2)^ref
63 cases of DLBCL in pediatric patients analyzed by FISH and IHC showed a striking predominance of GCB subtype, which might explain the good clinical outcome in these lymphomas : interestingly, FISH applied to 50 of these cases, as well as conventional cytogenetics available in 3 cases, revealed absence of the translocation t(14;18) involving the BCL2 gene, which is present in about 15% of adult GCB subtype DLBCL^ref.
• the recently completed National Cancer Institute (NCI)-organized Leukemia and Lymphoma Molecular Profiling Project (LLMPP) used DNA microarrays to study molecular diagnosis and clinical outcome prediction in DLBCL^ref. The LLMPP project analyzed 240 de novo DLBCLs on a 12 196-element DNA array, the Lymphochip (National Institutes of Health, Bethesda, MD), which identified 608 genes that had predictive value. These most predictive genes were categorized into 4 biologic groupings called gene-expression "signatures." The 4 signatures consisted of genes associated with :

proliferation

MHC class II

lymph node (host response)

germinal center differentiation

2 of these signatures (MHC class II and lymph node) suggested that the host response to lymphoma cells may be a crucial determinant of survival. The MHC class II gene-expression signature in particular suggested that antigen presentation to the immune system has a role in therapeutic responses, since loss of MHC class II expression was strikingly correlated with poor patient outcome. These results extended previous immunohistochemical studies of protein expression in aggressive lymphomas (DLBCL and Burkitt) in which several groups of investigators related loss of cellular adhesion molecules, such as ICAMs, and LFA-1, LFA-3^{ref1, ref2, ref3}, and loss of MHC class II proteins^{ref1, Rybski JA, 31-38, ref3} to poor outcome. Some of these studies and others related poor outcome to loss of immunosurveillance^{ref1, ref2, ref3, ref4}. In particular, some authors have described loss of tumor-infiltrating T cells (T-TILs), including CD8⁺ T cells, further suggesting that aberrant loss of CAM and/or MHC class II expression equated with deficient host response and tumor containment^{ref1, ref2, ref3, ref4, ref5}. Other investigators have successfully used a microarray study with supervised learning classification techniques to subdivide DLBCL into prognostic groups not recognized by the clinical IPI scoring system. The LLMPP database is one of the first large, multi-institutional, publicly available microarray databases of gene expression in any type of lymphoma^{ref1, ref2}. As such, it is an extremely valuable resource for detailed investigation. Loss of MHC class II gene expression is an independent prognostic variable, even after adjusting for the IPI score, and the poor survival of patients lacking MHC class II gene expression cannot be explained by differences in HIV status, tumor location, or histologic subtype. Losses of MHC class II genes, including HLA-DRA, are continuous variables related in a nonlinear fashion to the hazard risk of death. IHC protein stains correlated with the microarray gene expression data in 20 of 22 MHC class II⁺ and MHC class II^- cases tested, indicating a good correlation between gene expression and protein presence. In 2 of the 10 MHC class II^- cases, localization of the HLA-DR protein within the cell cytoplasm, as opposed to the usual cell membrane location, was observed. Since the storage, antigenic loading, and shuttling of MHC class II proteins to the cell surface is a complex process involving invariant chain, lysozomes, and cathepsin S, this apparent inconsistency may indicate aberrant structure, function, or trafficking of the proteins in these cases^{ref1, ref2, ref3}. Possibly most important from a pathologic standpoint, in all 10 of the cases with low HLA-DRA expression by microarray, including the 2 with unusual protein staining, the % of CD8⁺ tumor-infiltrating lymphocytes assessed by IHC were extremely low, suggesting diminished immunosurveillance. This relationship between low TILs and negative HLA-DRA status is in agreement with previous studies of aggressive non-Hodgkin lymphoma^{ref1, ref2, ref3}. In the immune response, cytotoxic T cells (CD8⁺) react to antigens presented by MHC class I molecules found ubiquitously on nearly every nucleated cell type. MHC class II antigens are present on only a few types of antigen-presenting cells (including B cells, macrophages, and dendritic cells) and provoke a T-helper (CD4⁺) cell response. The T-helper response in turn assists the T-cytotoxic response. While assessment of CD4⁺ cell response may be a more proximal measure of loss of immune surveillance by MHC class II-deficient tumor cells, assessment of CD8⁺ cells reflects the final common pathway of cell-mediated tumor cell death. Furthermore, the T-helper population consists of several subtypes, and CD4 is present on macrophages, making quantification in tissue sections more problematic. The area of fewest T-TILs in the tissue sections should be quantified, with the reasoning that if any portion of the tumor escaped immunosurveillance, there would be an increased risk of progression or relapse : decreased numbers of CD8⁺ T-TILs are highly predictive of patient survival. Several models of human lymphoma have been established in immunodeficient mice (either SCID or irradiated) with the use of immortalized cell lines, primary tumor samples, or peripheral blood mononuclear cells from EBV-seropositive donors^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. T cells derived from autologous, allogeneic, or CTL lines have been effective in eradicating a variety of human-SCID mouse xenografts of lymphoma, carcinoma, and melanoma^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. The activity of human CTLs against specific tumors can be augmented in vitro, prior to reintroduction to the mouse-human xenograft, by repeated stimulation with tumor cells or IL-2^{ref1, ref2, ref3}. CTLs stimulated by exposure to tumor were shown to act in an HLA-restricted fashion in a human-mouse lymphoblastic leukemia model^ref. Examination of the tumor mass demonstrated infiltration by CD8⁺ T cells in human-mouse xenografts of colon cancer^ref. Importantly, T-cell subset depletion studies demonstrated that CD8⁺ cells were the critical subset in tumor suppression in head and neck carcinoma^ref. These studies indicate that the antitumor activity of cytotoxic CD8⁺ T cells is dependent on appropriate HLA expression and can be a significant pathway in tumor containment for in vivo mouse models. Work by other authors examined sequential biopsies from 9 patients with various tumors, including 1 NHL. After treatment of these tumors with IL-2, a stimulant of HLA-DR, these authors reported increased numbers of CD8⁺ cytotoxic T cells in tumor samples from patients responding to therapy but not in the nonresponders. No increase in numbers of NK cells or CD4⁺ helper cells was found. Interestingly, in 4 of the 5 responders, tumor cells were positive for HLA-DR before therapy, and the fifth responder became positive during treatment, whereas all nonresponders were HLA-DR^- before and after therapy^ref. Increased numbers of tumor-infiltrating CTLs have a direct relationship to response to cytokine therapy that is aimed at up-regulating tumor immunogenicity. Genetic deletions of MHC genes appear to be common in extranodal lymphomas at immune-privileged sites, in particular the CNS and testes. In one study, 60% of immune-privileged site lymphomas had a total loss of MHC class I genes as compared with 10% of intranodal cases. Additionally, MHC class II expression was lost in 56% of immune-privileged lymphomas compared with only 5% of nodal lymphomas^ref. Loss of MHC class II in these particular types of lymphomas is due to both homozygous and heterozygous deletions as well as point mutations^{ref1, ref2}. Of importance, none of the cases in this group of the lowest 10% of MHC class II expression were from immune-privileged sites. The mechanisms of lost MHC class II expression in NHL from nonimmune privileged sites, such as those primarily studied in this work, are not known^ref
• subdividing DLBCL with either a GCB or non-GCB immunophenotypic profile was not of prognostic significance. Nevertheless, CD10 expression was a predictor of favorable outcome, whereas high bcl-2 expression and BCL6 rearrangement were adverse predictors of disease-free survival. Interestingly, large cleaved DLBCL was clearly associated with a GCB immunophenotypic profile, CD10 expression, BCL2 rearrangement, age younger than 60 years, and low to low/intermediate International Prognostic Index risk, but was not of prognostic significance. In contrast, immunoblastic morphology was associated with a non-GCB profile and was a significant predictor of unfavorable DFS^ref.
• R-CHOP prevented from progression or relapse in both bcl-2⁺ and bcl-2^- patients without increasing the risk of death in complete remission^ref.

Copyright © 2001-2005 Daniele Focosi. All rights reserved  |  Terms of use  | Legal notices
About this site  |  Site map  |  Acknowledgements | Open forum  |  Tell a friend about this site  |  Current link partners
 Abbreviations and acronyms  |  Medical terminology  |  Add a link  |  Translate this site into your favourite language  |  Softwares

Rate Us:10 - The Best987654321 - The Poorest

---

---

Search EntrezNCBI Web Site-------------PubMedGeneLocusLinkTaxonomyProteinNucleotideStructureGenomeBooksCancerChromosomesConserved Domains3D DomainsGEO ProfilesGEO DatasetsHomoloGeneJournalsMeSHNLM CatalogOMIMPMCPopSetPubChem BioAssayPubChem CompoundPubChem SubstanceSNPUniGeneUniSTS for

Search AboutAltaVistaAsk JeevesDictionaryDirectHitExciteFindWhatGoogleGoogle GroupsHotBotLookSmartLycosMetacrawlerMetaLocateNorthern LightOpen DirectorySnapThesaurusWebcrawlerYahoo for

Search Medical Dictionary  for