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Introduction: A number of novel therapies are under investigation in relapsed or refractory peripheral T-cell lymphoma (PTCL); however, their relative impact on outcome is unknown. We examined the survival of patients with PTCL after relapse or progression in the absence of hematopoietic stem-cell transplantation and explored factors influencing survival. The three most common subtypes encountered in North America were evaluated: PTCL not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large-cell lymphoma (ALCL; anaplastic lymphoma kinase [ALK] positive and ALK negative., Patients and Methods: After exclusions, 153 patients were analyzed (PTCL-NOS, n = 79 [52%]; AITL, n = 38 [25%]; ALK-positive ALCL, n = 11 [7%]; ALK-negative ALCL, n = 27 [16%; including ALK status unknown, n = 1])., Results: Median time from initial diagnosis to relapse or progression after primary therapy was 6.7 months, and median age at relapse was 66 years (ALK-positive ALCL, 39 years). Median overall survival (OS) and median progression-free survival (PFS) after relapse or progression (second PFS) were 5.5 and 3.1 months, respectively, and were only marginally better in patients who received chemotherapy at relapse (n = 89 [58%]; 6.5 and 3.7 months, respectively). Patients with good performance status (PS) of 0 or 1 (n = 47) at relapse who received chemotherapy had a more favorable OS (P < .001; median OS, 13.7 months) and PFS (P = .006; median second PFS, 5.0 months), which remained significant in multivariate analysis (OS: hazard ratio [HR], 2.09; P = .002; second PFS: HR, 1.66; P = .030)., Conclusion: Most patients with relapsed or refractory PTCL have poor outcomes with short survival. Select patients with good PS have more favorable outcomes with standard chemotherapy.

Purpose: The International Prognostic Score (IPS) is the most widely used risk stratification index for Hodgkin's lymphoma (HL). It is based on patients treated before 1992 and predicts 5-year freedom from progression (FFP) and overall survival (OS) ranging from 42% to 84% and 56% to 89%, respectively. The IPS has not been validated in a recently treated population in which outcomes have improved compared with historic results., Patients and Methods: By using the British Columbia Cancer Agency Lymphoid Cancer Database, we identified all patients age ≥ 16 years newly diagnosed with advanced-stage HL (stage III to IV, or stage I to II with "B" symptoms or bulky disease ≥ 10 cm) from 1980 to 2010, treated with curative intent with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) or an ABVD-equivalent regimen with complete clinical information., Results: In all, 740 patients were identified. Five-year FFP and OS were 78% and 90%, respectively. The IPS was prognostic for both FFP (P < .001) and OS (P < .001), with 5-year FFP ranging from 62% to 88% and 5-year OS ranging from 67% to 98%. Analysis limited to patients age 16 to 65 years (n = 686) demonstrated a narrower range of outcomes, with 5-year FFP ranging from 70% to 88% and 5-year OS ranging from 73% to 98%., Conclusion: The IPS remains prognostic for advanced-stage HL, but the range of outcomes has narrowed considerably. This improvement in outcome with ABVD should be acknowledged before consideration of alternate initial therapies and when comparing results from current trials with those of historic controls.

The Lunenburg Lymphoma Biomarker Consortium (LLBC) evaluated the prognostic value of IHC biomarkers in a large series of patients with diffuse large B-cell lymphoma (DLBCL). Clinical data and tumor samples were retrieved from 12 studies from Europe and North America, with patients treated before or after the rituximab era. Using tissue microarrays from 1514 patients, IHC for BCL2, BCL6, CD5, CD10, MUM1, Ki67, and HLA-DR was performed and scored according to previously validated protocols. Optimal cut points predicting overall survival of patients treated in the rituximab era could only be determined for CD5 (P = .003) and Ki67 (P = .02), whereas such cut points for BCL2, BCL6, HLA-DR, and MUM1 could only be defined in patients not receiving rituximab. A prognostic model for patients treated in the rituximab era identified 4 risk groups using BCL2, Ki67, and International Prognostic Index (IPI) with improved discrimination of low-risk patients. Newly recognized correlations between specific biomarkers and IPI highlight the importance of carefully controlling for clinical and biologic factors in prognostic models. These data demonstrate that the IPI remains the best available index in patients with DLBCL treated with rituximab and chemotherapy.

Purpose: In diffuse large B-cell lymphoma (DLBCL), prior studies suggest that concordant bone marrow involvement with DLBCL portends a poorer prognosis, whereas discordant bone marrow involvement with small B-cell lymphoma does not. We examined the significance of bone marrow involvement in patients treated in the current era of therapy including rituximab., Patients and Methods: We performed a retrospective analysis of the prognostic impact of bone marrow involvement in an unselected population of patients with newly diagnosed DLBCL treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone in British Columbia and Auckland, New Zealand, with complete clinical information and evaluable staging bone marrow biopsies., Results: In total, 795 patients were identified. Six hundred seventy (84.3%) of 795 had a negative bone marrow, 67 patients (8.4%) had concordant and 58 (7.3%) had discordant involvement. Median follow-up was 41 months (range, 1 to 115). Progression-free survival (PFS) was inferior in those with concordant (P < .001) and discordant (P = .019) involvement while overall survival (OS) was inferior in those with concordant involvement (P < .001) only. In a multivariate analysis controlling for the International Prognostic Index (IPI) score, concordant involvement remained an independent predictor of PFS (P < .001) and OS (P = .007). Discordant involvement was associated with older age, elevated lactate dehydrogenase, advanced stage, and increased number of extranodal sites and was not a negative prognostic factor independent of the IPI score., Conclusion: The negative prognostic impact of discordant involvement is adequately represented by the IPI score, while the risk with concordant involvement is greater than that encompassed by this predictor. The results emphasize the need for accurate staging assessment of bone marrow involvement in DLBCL.

The addition of rituximab (R) to standard CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy has altered the significance of previously recognized prognostic factors. We sought to re-examine the prognostic utility of (1) the number of extranodal sites of disease involvement, and (2) a primary extranodal presentation in patients with DLBCL treated with immunochemotherapy. We retrospectively analyzed all patients with DLBCL diagnosed between January 1979 and May 2006 who were treated with an anthracycline-based therapy with curative intent. In all, 1781 patients were identified, of whom 513 (29%) received R-CHOP. In the R-CHOP group, extranodal involvement as defined by the International Prognostic Index (>or=2 sites) was not prognostic on multivariate analysis, but the presence of any extranodal involvement (>or=1 site) was associated with decreased progression-free survival (HR 1.6, 95% CI 1.1-2.4, p = 0.024) and overall survival (HR 1.8, 95% CI 1.1-2.7, p = 0.011). A total of 133 (26%) R-CHOP treated patients presented with primary extranodal DLBCL. There was no difference in outcome between patients with primary extranodal and nodal DLBCL, and no primary site of involvement was associated with an inferior outcome. In patients with DLBCL treated with R-CHOP, the presence of extranodal disease remains prognostic, whereas a primary extranodal presentation did not affect outcome.

Purpose: To prospectively investigate the prognostic significance of p21 and p53 expression in diffuse large B-cell lymphoma in the context of the U.S. Intergroup trial comparing conventional cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy to rituximab-CHOP (R-CHOP) induction, with or without maintenance rituximab., Experimental Design: Immunohistochemical staining of 197 paraffin-embedded biopsy specimens was scored by an independent panel of experts., Results: The cyclin-dependent kinase inhibitor, p21, was expressed in 55% of cases examined. In a multivariable analysis adjusting for International Prognostic Index score and BCL2 status, p21 expression was a significant, independent, favorable predictive factor for failure-free survival (relative risk, 0.3; P = 0.001) and overall survival (relative risk, 0.3; P = 0.003) for patients treated with R-CHOP. Expression of p21 was not predictive of outcome for CHOP-treated patients. Only p21-positive cases benefited from the addition of rituximab to CHOP. Among p21-positive patients, treatment with R-CHOP was associated with a higher failure-free survival rate at 5 years compared with CHOP (61% versus 24%; P = 0.01). In contrast, no significant differences were detected in failure-free survival according to treatment arm for p21-negative patients. Expression of p53, alone or in combination with p21, did not predict for outcome in univariable or multivariable analyses., Conclusions: In this study, p21 protein expression emerged as an important independent predictor of a favorable clinical outcome when rituximab was added to CHOP therapy. These data suggest that rituximab-related effects on lymphoma survival pathways may be functionally linked to p21 activity.

CD19 and CD20 are B cell-specific antigens whose expression is heterogeneous when analyzed by flow cytometry (FCM). We determined the association between CD20 expression and clinical outcome in patients with diffuse large B-cell lymphoma (DLBCL). The mean fluorescence intensity of CD20 and CD19 was determined by FCM, and the cytoplasmic expression of CD20 was determined by immunohistochemistry (IHC) on 272 diagnostic DLBCL samples. Exon 5 of the MS4A1 gene coding for the extracellular component of the CD20 antigen was sequenced in 15 samples. A total of 43 of 272 (16%) samples had reduced CD20 expression by FCM; of these, 35 (13%) had bright CD19 expression. The latter had a markedly inferior survival when treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or rituximab-CHOP (R-CHOP; median survival of 1.2 and 3.0 years vs not reached for the others, P < .001 and P = .001), independent of the International Prognostic Index. A total of 41 of 43 samples with reduced CD20 expression by FCM had strong staining for CD20 by IHC. There were no mutations in exon 5 of the MS4A1 gene to explain the discrepancy between FCM and IHC. CD20 and CD19 expression by FCM should be determined on all biopsies of patients with DLBCL because reduced CD20 expression cannot be reliably detected by IHC.

The International Peripheral T-Cell Lymphoma Project is a collaborative effort designed to gain better understanding of peripheral T-cell and natural killer (NK)/T-cell lymphomas (PTCLs). A total of 22 institutions in North America, Europe, and Asia submitted clinical and pathologic information on PTCLs diagnosed and treated at their respective centers. Of the 1314 eligible patients, 181 had anaplastic large-cell lymphoma (ALCL; 13.8%) on consensus review: One hundred fifty-nine had systemic ALCL (12.1%) and 22 had primary cutaneous ALCL (1.7%). Patients with anaplastic lymphoma kinase-positive (ALK(+)) ALCL had a superior outcome compared with those with ALK(-) ALCL (5-year failure-free survival [FFS], 60% vs 36%; P = .015; 5-year overall survival [OS], 70% vs 49%; P = .016). However, contrary to prior reports, the 5-year FFS (36% vs 20%; P = .012) and OS (49% vs 32%; P = .032) were superior for ALK(-) ALCL compared with PTCL, not otherwise specified (PTCL-NOS). Patients with primary cutaneous ALCL had a very favorable 5-year OS (90%), but with a propensity to relapse (5-year FFS, 55%). In summary, ALK(-) ALCL should continue to be separated from both ALK(+) ALCL and PTCL-NOS. Although the prognosis of ALK(-) ALCL appears to be better than that for PTCL-NOS, it is still unsatisfactory and better therapies are needed. Primary cutaneous ALCL is associated with an indolent course.

BCL-2 protein expression correlates with shorter survival in patients with diffuse large B cell lymphoma (DLBCL) who are treated with CHOP chemotherapy. We report a retrospective analysis of the prognostic significance of BCL-2 status in patients who received CHOP with the addition of rituximab (CHOP-R) for DLBCL. Patients over 15 years of age with de novo, HIV negative DLBCL, without CNS involvement, and known BCL-2 protein status were identified from the BCCA Lymphoid Cancer Database. BCL-2 tumour positivity was defined as over 50% of tumour cells with BCL-2 protein expression. 140 patients who received CHOP-R were analysed. The majority (59%) of patients were over 60 years of age. Disease stage distribution was limited (22%) and advanced (78%). BCL-2 protein expression was observed in 90 (64%) cases. IPI score was similar in both BCL-2 positive and negative cases. Median follow-up time for living patients is 40 months. BCL-2 status did not predict for either progression-free or overall survival. IPI score was predictive for progression-free survival but not overall survival. The addition of rituximab to CHOP chemotherapy negates the adverse prognostic influence of BCL-2 protein expression on progression free and overall survival in DLBCL.

Purpose: The results of immunohistochemical class prediction and prognostic stratification of diffuse large B-cell lymphoma (DLBCL) have been remarkably various thus far. Apart from biologic variations, this may be caused by differences in laboratory techniques, scoring definitions, and inter- and intraobserver variations. In this study, an international collaboration of clinical lymphoma research groups from Europe, United States, and Canada concentrated on validation and standardization of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL., Patients and Methods: Sections of a tissue microarray from 36 patients with DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM1, and MIB-1 according to local methods. The study was performed in two rounds firstly focused on the evaluation of laboratory staining variation and secondly on the scoring variation., Results: Different laboratory staining techniques resulted in unexpectedly highly variable results and very poor reproducibility in scoring for almost all markers. No single laboratory stood out as uniformly poor or excellent. With elimination of variation due to staining, high agreement was found for CD20, HLA-DR, and CD10. Poor agreement was found for bcl-6 and Ki-67. Optimization of techniques and uniformly agreed on scoring criteria improved reproducibility., Conclusion: This study shows that semiquantitative immunohistochemistry for subclassification of DLBCL is feasible and reproducible, but exhibits varying rates of concordance for different markers. These findings may explain the wide variation of biomarker prognostic impact reported in the literature. Harmonization of techniques and centralized consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity, with patients exhibiting a wide range of outcomes. The addition of rituximab to CHOP chemotherapy (R-CHOP)has led to a marked improvement in survival and has called into question the significance of previously recognized prognostic markers. Since randomized controlled trials of R-CHOP in DLBCL have included select subgroups of patients, the utility of the International Prognostic Index (IPI) has not been reassessed. We performed a retrospective analysis of patients with DLBCL treated with R-CHOP in the province of British Columbia to assess the value of the IPI in the era of immunochemotherapy. The IPI remains predictive, but it identifies only 2 risk groups. Redistribution of the IPI factors into a revised IPI (R-IPI) provides a more clinically useful prediction of outcome. The R-IPI identifies 3 distinct prognostic groups with a very good (4-year progression-free survival [PFS] 94%, overall survival [OS] 94%), good (4-year PFS 80%, OS 79%), and poor (4-year PFS 53%, OS 55%) outcome, respectively (P < .001). The IPI (or R-IPI) no longer identifies a risk group with less than a 50% chance of survival. In the era of R-CHOP treatment, the R-IPI is a clinically useful prognostic index that may help guide treatment planning and interpretation of clinical trials.

Bcl-6 protein expression, a marker of germinal center origin, has been associated with a favorable prognosis in diffuse large B-cell lymphoma (DLBCL). To determine the prognostic significance of this marker when rituximab (R) was added to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy, we prospectively studied Bcl-6 protein expression by immunohistochemical staining of 199 paraffin-embedded specimens from patients enrolled in the US Intergroup phase 3 trial comparing R-CHOP to CHOP with or without maintenance R. In Bcl-6(-) patients, failure-free survival (FFS) and overall survival (OS) were prolonged for those treated with R-CHOP alone compared to CHOP alone (2-year FFS 76% versus 9%, P < .001; 2-year OS 79% versus 17%, P < .001). In contrast, no differences in FFS and OS were detected between treatment arms for Bcl-6(+) cases. In the multivariate analysis, treatment arm (CHOP versus R-CHOP) was the major determinant of both FFS (P < .001) and OS (P < .001) for the Bcl-6(-) subset, whereas the International Prognostic Index risk group was the only significant predictor of outcome among Bcl-6(+) cases. Bcl-2 protein expression was not predictive of outcome in either group. In this study, we observed a reduction in treatment failures and death with the addition of R to CHOP in Bcl-6(-) DLBCL cases only. Our finding that Bcl-6(+) cases did not benefit from the addition of R to CHOP requires independent confirmation.

The objectives of this study were foremost to further characterize pre-existing cell lines containing the t(11;14)(q13;q32) translocation. This translocation along with cyclin D1 overexpression is characteristic of Mantle Cell Lymphoma (MCL), an aggressive B cell neoplasm. Considerable variation in the abundance of cyclin D1 expression was observed. mRNA levels were examined by RT-PCR as differences in cyclin D1 mRNA abundance have been shown to synergize with INK4A/Arf deletions to dictate proliferation rate and survival in MCL patient samples. In this study, the cell lines, Z-138 and HBL-2, which exhibited the fastest growth rates and the shortest survival times in Rag2-M mice, had high expression of either one or both cyclin D1 mRNA isoforms and had negligible expression of p16. On the other hand, NCEB-1 and JVM-2 had low expression of both mRNA isoforms, retained p16 expression, and had slower growth rates and exhibited longer survival times in Rag2-M mice. Furthermore, JVM-2, which was found to have the lowest expression of cyclin D1, was the only cell line that expressed cyclin D2. The results of the characterization of Z-138, HBL-2, NCEB-1 and JVM-2 reveal that this group of cell lines represents both classic and variant features of MCL.

Purpose: To review the clinical, radiologic, serologic, histopathologic, immunohistochemical, and molecular genetic features of patients having Sjögren's syndrome (SS) with lacrimal gland enlargement., Design: Retrospective case series review., Participants: Fourteen patients histopathologically diagnosed with SS with lacrimal enlargement. Twenty-three age- and gender-matched controls were used for comparison on radiologic analysis., Methods: Clinical and serologic data were determined directly or by chart review. Computed tomography images from patients were compared with those from the control group. Histopathologic sections were reviewed and graded using the Chisholm-Mason scale, and quantitative immunohistochemical analysis was applied., Main Outcome Measures: Clinically, patients were assessed for age, gender, onset, symptoms and signs, systemic features, treatment, and outcome. Existing histologic specimens were reviewed according to the Chisholm-Mason scale, and then the percentages of plasma cells containing immunoglobulin (Ig) A, IgG, and IgM were determined. Imaging was assessed for lacrimal gland thickness, prolapse, density, and margin contour; extraocular muscle size; orbital tissue displacement; and proptosis., Results: Clinical and histopathological data fulfilled the revised American-European criteria for primary SS in 79% of patients. Compared with other large series of primary SS patients, similarities were found with age, xerophthalmia, parotidomegaly, and articular involvement. Differences included a lower incidence of autoantibodies, xerostomia, and extraglandular features and a higher male-to-female ratio. In almost all patients (93%), the percentage of plasma cells positive for IgA was less than 70%, consistent with SS. Compared with controls, the lacrimal glands were enlarged significantly (P<0.0001) and prolapsed (P<0.001). Involved glands had blurred margins (P<0.007), caused displacement of adjacent tissues (P = 0.03), and were associated with hyperdense fat (P = 0.007). Lymphocytic infiltration of orbital fat was present in all patients for whom fat biopsy results were available. Three patients had monoclonal infiltrates, and 1 patient experienced subsequent extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue type, 4 years after presentation., Conclusions: The criteria used to diagnose primary SS are controversial, but both diagnostic and quantitative immunohistochemical criteria suggest that these patients, with lacrimal gland enlargement resulting from lymphocytic infiltration, represent a new subtype of primary SS. This is clinically important in view of the increased risk of lymphoma associated with SS, compared with idiopathic nonspecific lacrimal inflammation.

Purpose: For more than two decades, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) has been the standard therapy for diffuse large B-cell lymphoma (DLBCL). The addition of rituximab to CHOP has been shown to improve outcome in elderly patients with DLBCL. We conducted a population-based analysis to assess the impact of this combination therapy on adult patients with DLBCL in the province of British Columbia (BC)., Methods: We compared outcomes during a 3-year period; 18 months before (prerituximab) and 18 months after (postrituximab) institution of a policy recommending the combination of CHOP and rituximab for all patients with newly diagnosed advanced-stage (stage III or IV or stage I or II with "B" symptoms or bulky [> 10 cm] disease) DLBCL., Results: A total of 292 patients were evaluated; 140 in the prerituximab group (median follow-up, 42 months) and 152 in the postrituximab group (median follow-up, 24 months). Both progression-free survival (risk ratio, 0.56; 95% CI, 0.39 to 0.81; P = .002) and overall survival (risk ratio, 0.40; 95% CI, 0.27 to 0.61, P < .0001) were significantly improved in the postrituximab group. After controlling for age and International Prognostic Index score, era of treatment remained a strong independent predictor of progression-free survival (risk ratio, 0.59; 95% CI, 0.41 to 0.85; P = .005) and overall survival (risk ratio, 0.43; 95% CI, 0.29 to 0.66; P < .001). The benefit of treatment in the postrituximab era was present regardless of age., Conclusion: The addition of rituximab to CHOP chemotherapy has resulted in a dramatic improvement in outcome for DLBCL patients of all ages in the province of BC.

Background: The objective of this study was to evaluate the clinical outcome of a population-based cohort of immunocompetent patients with primary central nervous system lymphoma (PCNSL) treated with 3 different strategies over 13 years., Methods: One hundred twenty-two consecutive patients (median age, 66 years) with PCNSL were identified. Three treatment strategies were employed: 1) whole-brain irradiation with (from January, 1990, to June, 1991) or without (from April, 1995, to December, 1999) cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-type chemotherapy (n=50 patients); 2) combined-modality therapy, including 1 g/m2 methotrexate plus whole-brain irradiation (from July, 1991, to March, 1995; n=34 patients); and 3) 8 g/m2 methotrexate alone (from January, 2000, to March, 2003) with whole-brain irradiation reserved for those with progressive disease (n=38 patients). Treatment failure was defined as progressive disease, disease recurrence, death from toxicity or lymphoma, or toxicity that necessitated a change in primary treatment., Results: The median failure-free survival was 7 months, and the median overall survival (OS) was 17 months. The median OS was similar in all 3 eras. In this population-based analysis, one-third of patients did not receive the treatment strategy of the era. Therefore, the data also were analyzed by treatment received. On multivariate analysis (including era of treatment), 3 factors-age > 60 years, lactate dehydrogenase > normal, and omission of methotrexate-were associated significantly with poorer OS (hazard ratio: 2.3, 2.2, and 2.3, respectively)., Conclusions: Outcomes for a general population with PCNSL remained constant despite different treatment strategies over three eras. For the two-thirds of patients who could receive potentially curative treatment, age, lactate dehydrogenase level, and receipt of > or = 1 g/m2 methotrexate appeared to be important determinants of OS., (2005 American Cancer Society.)

Regional deletions of 6q are frequent karyotypic alterations in malignant lymphoma and are associated with an adverse clinical outcome. One such region of recurrent deletion is 6q16-q21; however, the specific genes affected have not been identified. Our objective in this study was to identify cases with deletion of 6q16-q21 in follicular lymphoma and to define a minimal region of deletion. A physical map of 6q16.2-q21 was constructed using map information from both sequence-based and bacterial artificial chromosome (BAC) fingerprint-based maps. Forty-three BAC clones spanning a 6-Mb region of 6q16.2-q21 were identified and obtained from the RP-11 library. Selected BACs were fluorescence-labeled and hybridized to a series of 34 follicular lymphomas with a regional 6q deletion detected by G banding. Twenty-four cases with deletion of the 6q16.3 region were detected. A minimal deletion of 2.3 Mb was defined. Our study has identified a limited region of deletion of 6q16.3 that may implicate four known genes in follicular lymphoma and possibly in other cancers. A BAC contig spanning a 6-Mb region has been anchored to the 6q16.2-q21 region. This map represents a useful resource for gene identification in this region, not only in lymphoma but also in other neoplasms with 6q alterations., (Copyright 2004 Wiley-Liss, Inc.)

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

In anaplastic large cell lymphoma, the ALK gene at 2p23 is known to be fused to NPM, TPM3, TPM4, TFG, ATIC, CLTC, MSN, and ALO17. All of these translocations result in the expression of chimeric ALK transcripts that are translated into fusion proteins with tyrosine kinase activity and oncogenic properties. We report a case showing a restricted cytoplasmic staining pattern of ALK and a novel chromosomal abnormality, t(2;22)(p23;q11.2), demonstrated by fluorescence in situ hybridization analysis. The result of 5' RACE analysis showed that the ALK gene was fused in-frame to a portion of the non-muscle myosin heavy chain gene, MYH9. Nucleotide sequence of the MYH9-ALK chimeric cDNA revealed that the ALK breakpoint was different from all those previously reported. It is localized in the same exonic sequence as MSN-ALK, but 6 bp downstream, resulting in an in-frame fusion of the two partner proteins. In contrast to the previously reported ALK fusion proteins, MYH9-ALK may lack a functional oligomerization domain. However, biochemical analysis showed that the new fusion protein is tyrosine phosphorylated in vivo but seems to lack tyrosine kinase activity in vitro. If further investigations confirm this latter result, the in vivo tyrosine phosphorylation of MYH9-ALK protein could involve mechanisms different from those described in the other ALK hybrid proteins., (Copyright 2003 Wiley-Liss, Inc.)

The clinical and pathological features, including karyotype data and BCL2 protein expression pattern, of follicular lymphoma without a t(14;18)(q32;q21) have not been well defined. We have identified and conducted a detailed analysis of 50 cases with follicular lymphoma who lack the t(14;18). Fluorescent in situ hybridization (FISH) analysis was used to exclude cases with a cryptic IGH/BCL2 rearrangement. BCL2 protein expression level was assessed by immunohistochemistry. The karyotypes were assessed for recurrent sites of structural rearrangement, duplications and deletions on a band-by-band basis, and compared with a large cohort of cases with t(14;18). A distinct pattern of chromosomal alterations was identified in the cases without t(14;18). BCL2 protein overexpression was detected in 33% of 49 tested cases. In this minority, the karyotypes frequently showed increased copies of chromosome 18. The majority of cases (67%) did not show BCL2 overexpression and were characterized prominently by the presence of t(3;14)(q27;q32), implying a role for BCL6. Follicular lymphomas that lack a t(14;18) were segregated into two subgroups with distinct cytogenetic, phenotypic and possibly clinical features: one with BCL2 protein overexpression not related to an IGH/BCL2 rearrangement and a second without BCL2 overexpression. Objective identification of BCL2 expression level as well as BCL2 and BCL6 status by cytogenetic or FISH analysis has potential clinical utility and may yield insights into alternative genetic mechanisms associated with B-cell lymphomas with a follicular growth pattern.

Anaplastic large cell lymphoma (ALCL) is an aggressive lymphoma that is frequently associated with the t(2;5)(p23;q35), resulting in expression of a fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which can be detected by either monoclonal or polyclonal antibodies to the ALK protein. The clinical features of adults with ALCL are incompletely described, and the prognostic factors that are useful for predicting survival remain unclear. This report describes the clinical and laboratory findings in 70 adults with systemic ALCL who were treated with curative intent. We attempted to identify the clinical and pathological factors of prognostic importance, including the International Prognostic Index (IPI), immunophenotype, and expression of the ALK protein. The median age of the patients was 49 years (range, 15 to 75). There were 26 women and 44 men with a median follow-up of 50 months for living patients. Advanced stage was present in 56% and B symptoms were noted in 70% of the patients. Immunostains showed that 46% of the cases had a T-cell phenotype, 36% a null phenotype, and 18% a B-cell phenotype. The expression of ALK protein was found in 51% of the cases. The IPI factors were evenly distributed between the ALK+ and ALK- groups, except that the ALK+ patients were younger (median age, 30 v 61 years; P <.002). The ALK+ cohort included cases with null (44%), T-cell (42%), and B-cell (14%) phenotypes. All 10 cases with cytogenetic or molecular evidence of a t(2;5) were ALK+. The 5-year overall survival (OS) of the entire cohort was 65%. The 5-year OS of the ALK+ and ALK- cases was 79% and 46%, respectively (P <.0003). Analysis of only the T-cell/null cases (n = 57) showed a 5-year OS of 93% for the ALK+ cases and only 37% for the ALK- cases (P <.00001). Univariate analysis of the clinical features showed that age

We report two cases of CD30 and t(2;5) positive lymphomas with peripheral blood (PB) involvement. Case one demonstrated the histological appearance of a diffuse large cell lymphoma with disease in the bone marrow (BM) and PB. Immunoperoxidase stains of the BM for CD30 proved to be of value in detecting disease. RT-PCR for the t(2;5) translocation product was positive in the PB, BM and lymph node. Case two had a typical anaplastic large cell lymphoma (ALCL) morphology, with a suboptimal BM biopsy, but abnormal circulating cells in the PB showing the presence of the NPM/ALK fusion product demonstrated by RT-PCR. The first case demonstrates that not all CD30 positive and t(2;5)-associated lymphomas have an anaplastic appearance. Routine staining for CD30 and EMA in BM biopsies is useful for pathological staging. The significance of the t(2;5) in defining a specific histological subtype is unclear. RT-PCR for the t(2;5) is a more sensitive test to detect disease in PB and BM as compared with light microscopy. The clinical significance of molecular staging for patients with ALCL using RT-PCR needs to be evaluated.

The Caenorhabditis elegans cell death gene, Ced-3, encodes a protein homologous to mammalian interleukin-1beta-converting enzyme (ICE), a cysteine protease implicated in programmed cell death (PCD). CPP32, also known as Yama, apopain, and Caspase-3, is a member of this family, has substrate specificities similar to Ced-3, and has been shown to have an active role in PCD. Evidence suggests that these proteases act downstream of inhibitors of PCD such as Bcl-2 and Bcl-x(L), which are frequently expressed in Reed-Sternberg (RS) cells of Hodgkin's disease (HD). To date there have been no studies examining the role of the ICE/Ced-3 family of proteins, in particular CPP32, in HD. We examined 24 cases of HD with a classical immunophenotype and 6 cases of nodular lymphocyte predominant HD (NLPHD) for the expression of CPP32 in the RS cells and lymphohistiocytic (L&H) cells as detected by immunohistochemistry. Twenty two of 24 cases (92%) of HD expressed the protein in the RS cells, whereas the L&H cells in all 6 cases of NLPHD lacked expression of CPP32. These results provide further evidence that NLPHD is a phenotypically different disease distinct from classical forms of HD. The differential expression of the cell death protein CPP32 may be an important factor contributing to the apparently different clinical behaviour of NLPHD in contrast to classical HD. The lack of expression of CPP32 in NLPHD shares similarities with low-grade B-cell non-Hodgkin's lymphomas and may explain their common clinical course. Further studies are required to elucidate the significance of CPP32 in HD.

We conducted this study to assess the clinical utility of heteroduplex analysis of polymerase chain reaction (PCR) products for Tgamma gene rearrangements in the diagnosis of T-cell lymphoproliferative disorders (LPDs) using a single set of consensus primers. This method was used to study 103 cases using fresh or frozen tissue. These included 62 cases of T-cell LPDs, 25 cases of B-cell LPDs, 3 cases of anaplastic large cell lymphomas of undefined lineage, and 13 cases of reactive disorders. The latter group served as the negative control group. For all cases of T-cell LPDs, the results of Tbeta Southern analysis (SA) and heteroduplex PCR analysis were compared. We also studied 10 cases of postthymic T-cell lymphoma using paraffin-embedded tissue. Heteroduplex Tgamma PCR was positive in 51 (89%) of 57 cases that were rearranged by Tbeta SA. Eight of the 10 cases of T-cell lymphoma analyzed using paraffin-embedded tissue were positive. There were no false-positive results. These results demonstrate that this method is an effective screening test in the clinical laboratory. A positive result obviates the need to perform SA. Preliminary results suggest this method is useful using paraffin-embedded tissue.

Immunohistochemical analysis of the apoptosis-effector protease CPP32 (Caspase-3) in normal lymph nodes, tonsils, and nodes affected with reactive hyperplasia (n = 22) showed strong immunoreactivity in the apoptosis-prone germinal center B-lymphocytes of secondary follicles, but little or no reactivity in the surrounding long-lived mantle zone lymphocytes. Immunoblot analysis of fluorescence-activated cell sorted germinal center and mantle zone B cells supported the immunohistochemical results. In 22 of 27 (81%) follicular small cleaved cell non-Hodgkin's B-cell lymphomas, the CPP32-immunopositive germinal center lymphocytes were replaced by CPP32-negative tumor cells. In contrast, the large cell component of follicular mixed cells (FMs) and follicular large cell lymphomas (FLCLs) was strongly CPP32 immunopositive in 12 of 17 (71%) and in 8 of 14 (57%) cases, respectively, whereas the residual small-cleaved cells were poorly stained for CPP32 in all FLCLs and in 12 of 17 (71%) FMs, suggesting that an upregulation of CPP32 immunoreactivity occurred during progression. Similarly, cytosolic immunostaining for CPP32 was present in 10 of 12 (83%) diffuse large cell lymphomas (DLCLs) and 2 of 3 diffuse mixed B-cell lymphomas (DMs). Immunopositivity for CPP32 was also found in the majority of other types of non-Hodgkin's lymphomas studied. Plasmacytomas were CPP32 immunonegative in 4 of 12 (33%) cases, in contrast to normal plasma cells, which uniformly contained intense CPP32 immunoreactivity, implying downregulation of CPP32 in a subset of these malignancies. All 12 peripheral blood B-cell chronic lymphocyte leukemia specimens examined were CPP32 immunopositive, whereas 3 of 3 small lymphocytic lymphomas were CPP32 negative, suggesting that CPP32 expression may vary depending on the tissue compartment in which these neoplastic B cells reside. The results show dynamic regulation of CPP32 expression in normal and malignant lymphocytes.

A case is reported in which a congenital posterolateral diaphragmatic hernia of the Bochdalek type was the cause of sudden unexpected death in a 22-month-old girl. This case shows that not all late-presenting diaphragmatic hernias have a favorable prognosis.