The proliferation gene expression signature is a quantitative integrator of oncogenic events that predicts survival in mantle cell lymphoma.

We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.

Verification that common variation at 2q37.1, 6p25.3, 11q24.1, 15q23, and 19q13.32 influences chronic lymphocytic leukaemia risk.

A recent genome wide association study of chronic lymphocytic leukaemia (CLL) provided evidence that common variation at 2q13 (rs17483466), 2q37.1 (rs13397985), 6p25.3 (rs872071), 11q24.1 (rs735665), 15q23 (rs7176508) and 19q13.32 (rs11083846) affects CLL risk. To verify and further explore the relationship between these variants and CLL risk we genotyped case-control datasets from Spain and Sweden (824 cases, 850 controls). Combined data provided statistically significant support for an association between genotypes at rs13397985, rs872071, rs735665, rs7176508 and rs11083846 and CLL risk. CLL risk increased with increasing numbers of risk alleles (P(trend) = 1.40 x 10(-15)), consistent with a polygenic model of disease susceptibility. These data validate the relationship between common variation and risk of CLL.

Molecular diagnosis of Burkitt's lymphoma.

BACKGROUND: The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is crucial because these two types of lymphoma require different treatments. We examined whether gene-expression profiling could reliably distinguish Burkitt's lymphoma from diffuse large-B-cell lymphoma. METHODS: Tumor-biopsy specimens from 303 patients with aggressive lymphomas were profiled for gene expression and were also classified according to morphology, immunohistochemistry, and detection of the t(8;14) c-myc translocation. RESULTS: A classifier based on gene expression correctly identified all 25 pathologically verified cases of classic Burkitt's lymphoma. Burkitt's lymphoma was readily distinguished from diffuse large-B-cell lymphoma by the high level of expression of c-myc target genes, the expression of a subgroup of germinal-center B-cell genes, and the low level of expression of major-histocompatibility-complex class I genes and nuclear factor-kappaB target genes. Eight specimens with a pathological diagnosis of diffuse large-B-cell lymphoma had the typical gene-expression profile of Burkitt's lymphoma, suggesting they represent cases of Burkitt's lymphoma that are difficult to diagnose by current methods. Among 28 of the patients with a molecular diagnosis of Burkitt's lymphoma, the overall survival was superior among those who had received intensive chemotherapy regimens instead of lower-dose regimens. CONCLUSIONS: Gene-expression profiling is an accurate, quantitative method for distinguishing Burkitt's lymphoma from diffuse large-B-cell lymphoma.

Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells.

BACKGROUND: Patients with follicular lymphoma may survive for periods of less than 1 year to more than 20 years after diagnosis. We used gene-expression profiles of tumor-biopsy specimens obtained at diagnosis to develop a molecular predictor of the length of survival. METHODS: Gene-expression profiling was performed on 191 biopsy specimens obtained from patients with untreated follicular lymphoma. Supervised methods were used to discover expression patterns associated with the length of survival in a training set of 95 specimens. A molecular predictor of survival was constructed from these genes and validated in an independent test set of 96 specimens. RESULTS: Individual genes that predicted the length of survival were grouped into gene-expression signatures on the basis of their expression in the training set, and two such signatures were used to construct a survival predictor. The two signatures allowed patients with specimens in the test set to be divided into four quartiles with widely disparate median lengths of survival (13.6, 11.1, 10.8, and 3.9 years), independently of clinical prognostic variables. Flow cytometry showed that these signatures reflected gene expression by nonmalignant tumor-infiltrating immune cells. CONCLUSIONS: The length of survival among patients with follicular lymphoma correlates with the molecular features of nonmalignant immune cells present in the tumor at diagnosis.

The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.

BACKGROUND: The survival of patients with diffuse large-B-cell lymphoma after chemotherapy is influenced by molecular features of the tumors. We used the gene-expression profiles of these lymphomas to develop a molecular predictor of survival. METHODS: Biopsy samples of diffuse large-B-cell lymphoma from 240 patients were examined for gene expression with the use of DNA microarrays and analyzed for genomic abnormalities. Subgroups with distinctive gene-expression profiles were defined on the basis of hierarchical clustering. A molecular predictor of risk was constructed with the use of genes with expression patterns that were associated with survival in a preliminary group of 160 patients and was then tested in a validation group of 80 patients. The accuracy of this predictor was compared with that of the international prognostic index. RESULTS: Three gene-expression subgroups--germinal-center B-cell-like, activated B-cell-like, and type 3 diffuse large-B-cell lymphoma--were identified. Two common oncogenic events in diffuse large-B-cell lymphoma, bcl-2 translocation and c-rel amplification, were detected only in the germinal-center B-cell-like subgroup. Patients in this subgroup had the highest five-year survival rate. To identify other molecular determinants of outcome, we searched for individual genes with expression patterns that correlated with survival in the preliminary group of patients. Most of these genes fell within four gene-expression signatures characteristic of germinal-center B cells, proliferating cells, reactive stromal and immune cells in the lymph node, or major-histocompatibility-complex class II complex. We used 17 genes to construct a predictor of overall survival after chemotherapy. This gene-based predictor and the international prognostic index were independent prognostic indicators. CONCLUSIONS: DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.

[The value of positron emission tomography/computed tomography (PET/CT) in the staging of diffuse large B-cell lymphoma]. / Utilidad de la tomografía por emisión de positrones/ tomografía computarizada (PET/TC) en el estudio de extensión en pacientes con linfoma B difuso de células grandes.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the role of positron emission tomography/computed tomography (PET/CT) in improving the staging and changing the management of aggressive lymphoma patients in comparison with the conventional imaging modalities (CT, and 67Ga scintigraphy). PATIENTS AND METHOD: Forty consecutive patients with diffuse large B-cell non Hodgkin lymphoma, were prospectively evaluated. All 40 patients underwent a whole body FDG PET/CT and conventional staging techniques (chest and abdomen CT, 67Ga scintigraphy) were studied before therapy. Sixty minutes after the intravenous administration of 370 MBq FDG, a whole body PET/CT was acquired. We hypothesize that PET/CT improves the diagnostic staging of lymphoma and changes the clinical management of patients. RESULTS: PET/CT and CT were concordant in 28 patients (65%). However, PET/CT detected more lesions than CT in 11 patients (27.5%). Only in one patient, CT revealed more extensive disease than PET/CT. Additional information of PET/CT had lead to a change in staging (upstaging) in 6 patients (15%), in turn leading to a change in treatment strategy in 1 patient. PET/CT and 67Ga scintigraphy were concordant in 23 patients (60.5%). PET/CT detected more lesions than 67Ga scintigraphy in 14 patients (42%). PET/CT results changed staging (upstaging) in 4 patients (15%), leading to a change of treatment strategy in one patient. CONCLUSIONS: The impression is that PET/CT detected more lesions than conventional examination, but this rarely translates into changes of staging and treatment strategy in aggressive lymphoma.

Genomic p16 abnormalities in the progression of chronic myeloid leukemia into blast crisis: a sequential study in 42 patients.

OBJECTIVE: The molecular abnormalities involved in the progression of chronic myeloid leukemia (CML) are poorly understood. Genetic alterations of the INK4A/ARF locus have been implicated in the lymphoid blast crisis (BC), but sequential studies are not available. The aim of this study was to contribute to a better knowledge of the status of such locus in the different phases of CML and to analyze the prognostic significance of its inactivation. MATERIALS AND METHODS: Sequential assessment by quantitative real-time polymerase chain reaction (PCR) and conventional semiquantitative PCR of p16 exon 2 deletions was performed in 42 CML patients in whom paired DNA samples from the chronic phase and the BC were available. Samples of 10 healthy donors and 30 patients with nonleukemic myeloproliferative syndromes served as controls. The methylation status of the promoter region of the p16 gene was also studied by methylation-specific PCR. RESULTS: The concordance rate between the two PCR techniques was 97.8% (87/89). By real-time PCR, homozygous p16 deletions were found in 6 of 21 patients (29%) with lymphoid BC, whereas they were not observed in chronic-phase CML nor in 21 myeloid BC patients. Hypermethylation of the p16 gene was not detected in any of the lymphoid BC. No specific clinical profile was associated with homozygous p16 deletions. Therapeutic response and survival did not significantly differ in p16-deleted and p16 germline lymphoid BC patients. CONCLUSION: P16 gene deletions are detected in a substantial proportion of lymphoid BC of CML by quantitative real-time PCR analysis, but this is not associated with any clinico-hematological feature other than lymphoid phenotype and does not influence the patients' outcome. Such technique is simple and reliable to assess the p16 gene status.

Elevated levels of soluble CD44 are associated with advanced disease and in vitro proliferation of neoplastic lymphocytes in B-cell chronic lymphocytic leukaemia.

PURPOSE: Increased expression of the adhesion molecule CD44 has been associated with an unfavourable clinical outcome in lymphomas. We evaluated the prognostic value of soluble CD44 in B-cell chronic lymphocytic leukaemia (B-CLL) and analysed the source and regulation of CD44 secretion in B-CLL clones in vitro. PATIENTS AND METHODS: Levels of soluble CD44 standard (sCD44s) and of the soluble variant isoform CD44v6 (sCD44v6) were analysed by enzyme linked immuno sorbent assay. Highly purified B-CLL cells (98% CD19 + CD3 - cells) were stimulated in vitro by different combinations of thioredoxin (Trx), Staphylococcus aureus Cowan strain 1 (SAC), IL-2, IL-4, IL-10, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by anti-CD40 mAbs presented on irradiated CD32L cells. RESULTS: Serum levels of sCD44s and of sCD44v6 are significantly elevated in B-CLL patients (n = 90) in comparison with normal persons (n = 44) (P < 0.001). Elevated levels of sCD44s and sCD44v6 are associated with an advanced disease as reflected by an extended lymph node involvement (P < 0.02), an advanced Binet (P < 0.03) and Rai stage (P < 0.04) and chemotherapy requirement (P < 0.02). High levels of sCD44s are associated with high leukocyte counts (P < 0.04) and increased sCD44v6 is significantly associated with splenomegaly (P < 0.002). In B-CLL sCD44s as well as sCD44v6 is shed from leukaemia cells as shown by in vitro cultures. Stimulation of B-CLL clones results in a proliferation-associated increased secretion of sCD44s (rho = 0.7; P = 0.0001) and of sCD44v6 (rho = 0.5; P = 0.005). B-CLL clones from advanced stage patients are characterised by an increased capacity for proliferation and CD44 production in comparison with early stage patients. CONCLUSIONS: Both sCD44s and sCD44v6 represent a reliable prognostic marker in B-CLL and may be involved in the pathogenesis of B-CLL.

Long-term disease-free survival in patients with angioimmunoblastic T-cell lymphoma after high-dose chemotherapy and autologous stem cell transplantation.

BACKGROUND AND OBJECTIVES: Patients with angioimmunoblastic T-cell lymphoma (AIL) have a poor prognosis with conventional treatment. DESIGN AND METHODS: We initiated an EBMT-based survey studying the impact of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation in patients with AIL. Data on 29 patients, who were transplanted between 1992 and 1998 in 16 transplant centers, were collected on standardized documentation forms. RESULTS: The median age at transplantation was 53 years. HDCT was given as part of 1st-line therapy (N=14; 48%) or 2nd/3rd-line therapy (N=15; 52%). Regimens for the mobilization of peripheral blood stem cells (PBSC) included VIPE (N=7; 26%), DexaBEAM (N=6; 22%), CHOP-like regimens (N=6; 22%), other regimens (N=5; 19%) or alternatively growth factor alone (N=3; 11%). The median yield of PBSC was 3.8x106 CD34+cells/kg. Two patients received autologous bone marrow. The HDCT consisted of BEAM-type regimens in 16 patients, ICE-type regimens in 7, and other regimens in 6 patients. There was one treatment-related death. The rate of complete remissions increased from 45% before HDCT to 76% after HDCT. As of January 2003, after a median observation time of living patients of 5 years (range 2.5 to 10 years), 14 patients have died (13 from progressive disease), and 15 patients are alive. The probability of 5-year overall and event-free survival was 44% (95% CI, 22% to 66%) and 37% (95% CI, 17% to 57%), respectively. Long-term disease-free survival was observed in patients transplanted during 1st-line treatment as well as in the context of 2nd/3rd-line therapy. INTERPRETATION AND CONCLUSIONS: There is evidence that AIL is susceptible to high-dose chemotherapy. HDCT and autologous stem cell transplantation should be considered in selected patients with AIL.

Clinico-biological characterization and outcome of primary nodal and extranodal diffuse large B-cell lymphoma in the rituximab era.

To study the main clinico-biological characteristics and the outcome of patients with diffuse large B-cell lymphoma (DLBCL) according to the primary site (nodal vs. extranodal), we included 262 patients consecutively diagnosed with DLBCL in a single institution, 5 years before and after immunochemotherapy was considered as the standard treatment. Altogether 116 patients received CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone) and 146 rituximab plus CHOP (R-CHOP). The primary site was the lymph node in 140 patients (53%), Waldeyer's ring (WR) in 22, gastrointestinal (GI) in 33, and other extranodal in 67. The addition of rituximab significantly improved the CR rate in nodal, but not in extranodal, lymphomas. Patients receiving R-CHOP showed higher OS than those treated with CHOP alone (5-year OS: 71% vs. 48%). This difference was maintained in primary nodal (5-year OS: 69% vs. 37%, p < 0.0001), but was not observed in primary extranodal (75% vs. 65%, p = 0.45) lymphomas. The IPI, treatment, and primary site were the main variables for OS in multivariate analysis. In nodal cases, IPI and treatment maintained value, whereas only IPI predicted OS in extranodal cases. In conclusion, immunochemotherapy treatment dramatically improved the outcome of patients with nodal DLBCL; however, its effect was less in primary extranodal cases, so the prognosis of patients with nodal and extranodal lymphomas has been equalized in the rituximab era.

Common variants at 2q37.3, 8q24.21, 15q21.3 and 16q24.1 influence chronic lymphocytic leukemia risk.

To identify new risk variants for chronic lymphocytic leukemia (CLL), we conducted a genome-wide association study of 299,983 tagging SNPs, with validation in four additional series totaling 2,503 cases and 5,789 controls. We identified four new risk loci for CLL at 2q37.3 (rs757978, FARP2; odds ratio (OR) = 1.39; P = 2.11 x 10(-9)), 8q24.21 (rs2456449; OR = 1.26; P = 7.84 x 10(-10)), 15q21.3 (rs7169431; OR = 1.36; P = 4.74 x 10(-7)) and 16q24.1 (rs305061; OR = 1.22; P = 3.60 x 10(-7)). We also found evidence for risk loci at 15q25.2 (rs783540, CPEB1; OR = 1.18; P = 3.67 x 10(-6)) and 18q21.1 (rs1036935; OR = 1.22; P = 2.28 x 10(-6)). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy.

Point mutations and genomic deletions in CCND1 create stable truncated cyclin D1 mRNAs that are associated with increased proliferation rate and shorter survival.

A gene expression signature of tumor proliferation rate in mantle cell lymphoma (MCL) is an overriding molecular predictor of the length of survival following diagnosis. Many strongly proliferative MCL tumors have exceptionally high cyclin D1 mRNA levels and preferentially express short cyclin D1 mRNA isoforms. We demonstrate here that these short mRNAs are cyclin D1a isoforms with truncated 3'UTRs, not alternatively spliced cyclin D1b mRNA isoforms. Among 15 MCL tumors with truncated cyclin D1 mRNAs, 7 had genomic deletions in the CCND1 3'UTR region. In 3 others, CCND1 contained point mutations that created premature polyadenylation signals, giving rise to 1.5-kb mRNAs lacking most of the 3'UTR. Both types of genomic alteration created transcripts lacking mRNA destabilization elements present in the wild-type cyclin D1a mRNA. Premature polyadenylation due to a 3'UTR mutation also was present in the Z-138 MCL cell line, which expressed both truncated and full-length cyclin D1a mRNAs. In these cells, the half-life of the short cyclin D1a mRNA was much longer than that of the full-length mRNA. We conclude that alterations of CCND1 3'UTR structure can significantly increase its oncogenic effect and worsen the clinical course of MCL patients.

Myelofibrosis with myeloid metaplasia following essential thrombocythaemia: actuarial probability, presenting characteristics and evolution in a series of 195 patients.

Myelofibrotic transformation is a known complication of essential thrombocythaemia (ET), but information on its incidence, presenting features and evolution is scarce. In a series of 195 patients with ET followed for a median of 7.2 years (range: 1.9-24), evolution into myelofibrosis with myeloid metaplasia (MMM) occurred in 13 cases, a median of 8 years (range: 3.6-20.2) from diagnosis. The actuarial probability of this complication was 2.7% (95% CI: 2.4-2.9) at 5 years, 8.3% (95% CI: 7.8-8.9) at 10 years, and 15.3% (95% CI: 6.1-24.5) at 15 years. Four patients had not been treated before developing MMM. The main features indicating this condition were the appearance of immature myeloid precursors in the peripheral blood, a decrease in the Hb value not related to treatment and increased serum lactate dehydrogenase levels, followed by a progressive decrease in the platelet count, increasing leucocytosis and progressive splenomegaly. No patient had constitutional symptoms, and none of five evaluable cases showed chromosome abnormalities in bone marrow or unstimulated blood. After a median the myelofibrotic transformation, three patients have died and four have not required treatment for MMM as yet.

Imatinib mesylate (STI571) treatment in patients with chronic-phase chronic myelogenous leukaemia previously submitted to autologous stem cell transplantation.

Imatinib mesylate (STI571) is a highly effective and well-tolerated treatment for patients with chronic-phase chronic myeloid leukaemia (CML), but information on its efficacy and tolerance in intensively pretreated patients is scarce. Thirty-three chronic-phase CML patients who were resistant or intolerant to interferon (IFN) and had been previously submitted to autologous stem cell transplantation were treated with imatinib for a median of 14 months (range: 6-19 months). Seven patients were in haematological response (HR) at the start of treatment; the remaining 26 attained a HR at a median of 3 weeks (range: 1-4 weeks). Major cytogenetic response rates at 3, 6 and 12 months were 42%, 45% and 55%, respectively, including 21%, 24% and 33% complete responses. Grade 3-4 neutropenia, thrombocytopenia and anaemia developed in 33%, 27% and 12% of patients respectively. Non-haematological toxicity included superficial oedema (21% of patients), gastrointestinal symptoms (18%), muscle cramps (15%), skin rash and liver enzyme increase (3% each). These results were not significantly different from those in 65 chronic-phase CML patients, resistant or intolerant to interferon without a previous ASCT, who were included in the same protocol. Imatinib mesylate is effective and safe in chronic-phase CML patients with a previous history of intensive treatment.

Clonally unrelated Hodgkin's disease following autologous stem cell transplant for B-cell lymphoma.

Lymphoproliferative disorders after autologous stem cell transplantation (SCT) are rare. We describe two cases of Hodgkin's disease (HD) as a late secondary neoplasia following autologous SCT for mantle cell lymphoma and B-cell chronic lymphocytic leukaemia respectively. Both HD cases were of mixed cellularity type, showed Epstein-Barr virus (EBV) positivity and followed an aggressive course. Clonal analysis of rearranged immunoglobulin genes from the primary B-cell neoplasm and the secondary HD provided evidence of separate clonal origins of the two tumours in both patients, thus excluding secondary transformation of the original B-cell clone through EBV as the causative event for development of HD.