The feasibility of gene expression profiling generated in fine-needle aspiration specimens from patients with follicular lymphoma and diffuse large B-cell lymphoma.

Lymphoma of germinal center cell (GC) origin generally is an indolent malignancy that transforms progressively into a more aggressive disease. According to the World Health Organization classification, lymphomas of follicular center cell origin are classified as either large B-cell lymphoma (LBCL) or follicular lymphoma (FL). The authors tested the feasibility of performing gene expression profiling using amplified RNA from fine-needle aspirates (FNA) obtained from lymph nodes. Twenty-four samples from patients with a diagnosis of FL or LBCL were obtained after Institutional Review Board-approved informed consent was obtained. The diagnoses were confirmed by 2 pathologists and were classified into 2 groups (10 LBCL samples and 14 FL samples) by using conventional morphology and immunophenotyping. One hundred nanograms of total RNA were subjected to 2 cycles of standard, double-stranded complementary DNA synthesis and in vitro transcription for target amplification using a small-sample target-labeling protocol. The biotinylated cRNA from each sample was hybridized to gene chips. Gene expression profiling results were analyzed first by principal-component analysis (PCA) by using a list of 146 probe sets that represented 62 genes that are characteristic of an activated B-cell (ABC) signature or a GC signature. The analysis identified 5 LBCL samples with an ABC cell signature. Using a list of 207 probe sets that represented 113 genes involved in FL transformation, PCA analysis identified 2 overlapping clusters corresponding to FL and GC-diffuse LBCL. To improve this classification further, the authors generated a list of 72 genes that were expressed differentially between FL and GC-LBCL. Using this list of genes, PCA analysis demonstrated a clear separation between FL and GC-LBCL. However, five FL samples clustered as an intermediate group between FL and GC-DLBCL. These samples were characterized morphologically by a mixed cell pattern with relatively fewer large, noncleaved lymphocytes and more small, cleaved lymphocytes. The results support the feasibility of FNA-based transcription profiles in patients with FL or LBCL, which, in combination with morphology and immunophenotyping, can help in the subtyping of these entities.

Phase II study of proteasome inhibitor bortezomib in relapsed or refractory B-cell non-Hodgkin's lymphoma.

PURPOSE: Evaluate efficacy and toxicity of bortezomib in patients with relapsed or refractory B-cell non-Hodgkin's lymphoma. PATIENTS AND METHODS: Patients were stratified, based on preclinical data, into arm A (mantle-cell lymphoma) or arm B (other B-cell lymphomas) without limitation in number of prior therapies. Bortezomib was administered as an intravenous push (1.5 mg/m2) on days 1, 4, 8, and 11 every 21 days for a maximum of six cycles. RESULTS: Sixty patients with a median number of prior therapies of 3.5 (range, one to 12 therapies) were enrolled; 33 patients were in arm A and 27 were in arm B, including 12 diffuse large B-cell lymphomas, five follicular lymphomas (FL), three transformed FLs, four small lymphocytic lymphomas (SLL), two Waldenstrom's macroglobulinemias (WM), and one marginal zone lymphoma. In arm A, 12 of 29 assessable patients responded (six complete responses [CR] and six partial responses [PR]) for an overall response rate (ORR) of 41% (95% CI, 24% to 61%), and a median time to progression not reached yet, with a median follow-up of 9.3 months (range, 1.7 to 24 months). In arm B, four of 21 assessable patients responded (one SLL patient had a CR, one FL patient had a CR unconfirmed, one diffuse large B-cell lymphoma patient had a PR, and one WM patient had a PR) for an ORR of 19% (95% CI, 5% to 42%). Grade 3 toxicity included thrombocytopenia (47%), gastrointestinal (20%), fatigue (13%), neutropenia (10%), and peripheral neuropathy (5%). Grade 4 toxicity occurred in nine patients (15%), and three deaths from progression of disease occurred within 30 days of withdrawal from study. CONCLUSION: Bortezomib showed promising activity in relapsed mantle-cell lymphoma and encouraging results in other B-cell lymphomas. Future studies will explore bortezomib in combination with other cytotoxic or biologic agents.

Establishment and characterization of a new mantle cell lymphoma cell line M-1.

A new mantle cell lymphoma cell line, M-1, was established from peripheral blood mononuclear cells of a patient with a diagnosis of blastoid variant of mantle cell lymphoma in leukemic phase. This cell line showed cell surface antigens identical to the original tumor and demonstrated the profile of a mature B-cell phenotype typical of mantle cell lymphoma: positive for CD5, CD19, CD20, sIgM and FMC7, and negative for CD3, CD10 and CD23. Cytogenetically, the M-1 cell line showed chromosomal alterations similar to the initial clinical specimen, among which a translocation t(11;14) (q13;q32) resulting in the overexpression of cyclin D1 as well as additional abnormalities involving chromosomes 3, 9 and 10. This cell line was used as a model to investigate the activity of the three drugs doxorubicin, cyclophosphamide and vincristine, commonly used in the treatment of mantle cell lymphoma patients. The effect of the drugs was evaluated by a 24 h cytotoxicity test and a 7-days anti-proliferation test using a microculture tetrazolium-based assay (MTT). Both assays indicated a higher sensitivity of the cell line to vincristine when compared to doxorubicin and cyclophosphamide. The characterization of a new mantle cell lymphoma cell line is a unique tool for studying the biology of this subtype of lymphoma for which only a few cell lines have been established.

Signal transducer and activator of transcription-3 activation contributes to high tissue inhibitor of metalloproteinase-1 expression in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma.

The tissue inhibitor of metalloproteinase-1 (TIMP1) is expressed in a subset of malignant lymphomas and can inhibit tumor spread and promote cell survival. Recent data suggest that TIMP1 expression may be regulated by signal transducer and activator of transcription (STAT)-3. Thus, we tested the hypothesis that TIMP1 expression is related to STAT3 activation in lymphomas, with a focus on anaplastic large cell lymphomas (ALCLs), which are known to express high levels of phosphorylated/active STAT3 (pSTAT3). Specific inhibition of STAT3 with a dominant-negative construct led to concentration-dependent down-regulation of TIMP1 expression in two anaplastic lymphoma kinase (ALK)(+) ALCL cell lines, Karpas 299 and SU-DHL-1. Using cDNA microarrays, ALK(+) ALCL cell lines consistently expressed the highest TIMP1 level among 29 lymphoma cell lines of various subtypes. The association between TIMP1 expression and high level of STAT3 activation was validated by Western blots and immunostaining using antibodies specific for pSTAT3 and TIMP1. We further evaluated the relationship between TIMP1 expression and STAT3 activation in 43 ALCL tumors (19 ALK(+) and 24 ALK(-)) using immunohistochemistry and a tissue microarray. The TIMP1(+) group had a mean of 64% pSTAT3(+) cells as compared to 23% pSTAT3(+) cells in the TIMP1(-) group (P = 0.002). As expected, TIMP1 positivity was higher in the ALK(+) group (15 of 19, 79%) compared with the ALK(-) group (5 of 24, 21%; P = 0.0002) because NPM-ALK restricted to ALK(+) tumors was previously shown to activate STAT3. In conclusion, STAT3 directly contributes to the high level of TIMP1 expression in ALK(+) ALCL, and TIMP1 expression correlates with high level of STAT3 activation in ALCL. TIMP1, as a downstream target of STAT3, may mediate the anti-apoptotic effects of STAT3.

Update on the proteasome inhibitor bortezomib in hematologic malignancies.

The ubiquitin-proteasome system plays a crucial role in eukaryotic cells in maintaining protein homeostasis. Through the disruption of a variety of pathways and cell cycle checkpoints, proteasome inhibition leads to apoptosis and in experimental models can overcome chemoresistance. Bortezomib is the first of its class of proteasome inhibitors tested in humans that showed promising activity in several tumor types, and especially in hematologic malignancies, in phase I studies. The remarkable results obtained in phase II studies in multiple myeloma (MM) led to its fast-track approval by the US Food and Drug Administration in May 2003 for relapsed MM. More recent observation also revealed promising activity in non-Hodgkin's lymphoma. This review will explore the rationale for the use of bortezomib in hematologic malignancies as well as provide an update on the results of ongoing studies and future directions for the use of this new agent in hematologic malignancies. The mechanism of action of bortezomib and its nonoverlapping toxicity profile make it a very appealing drug for combination with other chemotherapeutic or biologic agents. Bortezomib represents an excellent example of how progress in understanding the biology of cancer cells can impact clinical practice and lead toward a new era of rational therapeutics.

Prognostic significance of MUC-1 expression in systemic anaplastic large cell lymphoma.

Systemic anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) and overexpresses anaplastic lymphoma kinase (ALK). MUC-1, a highly glycosylated transmembrane protein, is detected in normal and malignant epithelial cells and has been associated with a poorer patient survival in various human malignancies. We have shown previously that MUC-1 is expressed as a consequence of t(1;14)(q21;32) in a subset of diffuse large B-cell lymphomas. ALCLs are known to express MUC-1, but its clinical significance is undefined. For this study, eligible patients with ALCL were HIV negative, received anthracycline-containing regimens, and had pretreatment archival tissue. Expression of MUC-1 and ALK was determined immunohistochemically after heat-induced antigen retrieval. A 10% cutoff for MUC-1 positivity was used. We identified 63 patients with systemic ALCL (22 ALK+, 41 ALK-) with a median age of 47 years, and 41 were male. MUC-1 was detected in 16 of 22 (73%) ALK-positive and 20 of 41 (49%) ALK-negative ALCL (P = 0.06, chi(2) test). MUC-1 expression was not associated with apoptotic rate as detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay or proliferation index as evaluated by MIB-1 antibody. For 48 patients with ALCL (16 ALK+, 32 ALK-) and complete clinical follow-up, 5-year progression-free survival (PFS) was 39.7% for patients with MUC-1-positive tumors versus 75.2% (P = 0.027 by Log-rank) for patients with MUC-1-negative tumors. For the ALK-negative ALCL group of 32 patients, the 5-year PFS was 26 versus 70.8% for patients with MUC-1-positive versus MUC-1-negative tumors (P = 0.0096 by Log-rank). For the ALK-positive ALCL group of 16 patients, the 5-year PFS was 52 versus 100% for patients with MUC-1-positive versus MUC-1-negative tumors (P, not significant). In summary, MUC-1 is frequently expressed in systemic ALCL, and its expression is associated with significantly inferior outcome in patients untreated previously with ALK-negative tumors. Future studies should explore the underlying molecular mechanisms of MUC-1 expression in these tumors and its role as a target for novel therapeutic strategies.

MUC-1 mucin protein expression in B-cell lymphomas.

We have recently shown that MUC1, mapped to the chromosomal band 1q21, is rearranged or amplified in 15% of B-cell lymphomas and that rearrangement led to over-expression of MUC-1 mucin in a case of diffuse large B-cell lymphoma (DLBCL). To determine the incidence of MUC-1 mucin expression and its clinical significance in B-cell lymphomas, we investigated a panel of 113 cases by immunohistochemistry (IHC). MUC-1 mucin expression was detected in the majority of cases (92.9%), with moderate to high levels noted in 50.4% of all histologic subsets comprising DLBCL (82 cases), follicular lymphoma (FL) (15 cases), FL with transformation to DLBCL (4 cases), and other B-cell lymphomas (12 cases). No statistically significant correlation was found between MUC-1 mucin expression and MUC1 genomic status (amplification/rearrangement) evaluated by Southern blot analysis, and 1q21 abnormality by karyotypic analysis. For all cases, MUC-1 mucin expression correlated with a previous history of lymphoma (p=0.003).