Blastoid Mantle Cell Lymphoma Research Articles

Assessment of Microrna Expression in Mantle Cell Lymphoma Using High Throughput Techniques

Mantle cell lymphoma (MCL) accounts for 3–10% of all lymphomas and demonstrates a poor clinical response to current therapeutic approaches, with a median survival of 3–5 years. The natural history of MCL is heterogeneous and not well-defined by standard clinical markers as patients may die within months of diagnosis or experience long-term survival. There remains a need for reliable biomarkers of MCL prognosis. To date, global gene expression signatures have not been determined for formalin-fixed, paraffin-embedded (FFPE) MCL samples due to difficulties isolating full-length mRNA transcripts from FFPE tissues. Examining microRNA expression in FFPE samples may circumvent this problem, as this population of RNAs remains intact during processing of FFPE samples. MicroRNAs (miRs) are small, non-coding RNAs, which regulate gene expression by inhibiting mRNA translation. Although miR expression signatures have been derived for other hematological malignancies, assessment of miR expression in patients with MCL has yet to be undertaken. We hypothesize that different pathological subtypes of MCL have unique miR expression signatures, distinct from miR expression profiles of other B-cell non-Hodgkin lymphomas. Our objectives were two-fold:To determine and validate miR expression in different pathological subtypes of MCL; and,To compare miR expression in MCL to other B-cell non-Hodgkin lymphomas.Total RNA was extracted from FFPE samples [17 conventional MCL, 11 blastoid MCL, 4 follicular lymphomas (Grades 1, 2, 3a, and 3b), 1 nodal marginal zone lymphoma, 1 small lymphocytic lymphoma (SLL/CLL) and 3 benign, reactive lymph nodes (normal controls)] using the RecoverAll kit for FFPE tissues (Ambion). RNA was subjected to quantitative real-time PCR (qRT-PCR) for 365 miRs and 3 endogenous control small nucleolar RNAs to obtain miR expression profiles using the TaqMan Low Density Array (TLDA) v1.0 platform (MicroFluidic card, Applied Biosystems). TLDAs were run on the ABI7900 HT analyzer with TLDA upgrade and analysed with RQ Manager software provided by Applied Biosystems. Expression profiles were correlated to pathological subtype, and hierarchical clustering, principal component analysis (PCA), and ANOVA were performed using Partek software [Partek Genomics Suite for Gene Expression Data]. Results indicate that miR expression profiles differ between B-cell, non-Hodgkin lymphoma and MCL samples. Clustering analysis and PCA both demonstrated different profiles between MCL and B-cell, non-Hodgkin lymphomas. Although PCA did not demonstrate significant differences between the conventional and blastoid MCL samples, a set of fifteen miRs may be able to distinguish these two groups of MCL, since these 15 miRs are relatively upregulated in blastoid MCL in comparison to conventional MCL. In addition, PCA revealed five (3 conventional MCL samples and 2 blastoid samples), which did not cluster with their respective groups. These five samples were from patients known to have progressive disease, indicating that such patients may have different miR expression profiles compared to patients with non-progressive disease. We conclude that high-throughput miR expression profiles can be generated from FFPE samples in B-cell non-Hodgkin lymphomas and that miR expression profiles for MCL samples differ from those of other B-cell non-Hodgkin lymphomas. Blastoid and conventional MCL samples may not have significantly differing profiles, however a set of 15 miRs appears to be able to distinguish between these two groups. Of note, samples from patients with known progressive disease have significantly different profiles from those with non-progressive disease.

Dose Escalated Radioimmunotherapy with Yttrium-90-Ibritumomab Tiuxetan as Part of a Reduced Intensity Conditioning Regimen for Allogeneic Hematopoietic Cell Transplantation in Patients with Advanced Aggressive Non-Hodgkin Lymphoma: An Interim Analysis of a Phase I/II Study

Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a potential curative therapy to patients with advanced aggressive NHL. RIC HCT induces potent graft-versus-lymphoma effects with best results in patients with low tumor burden at time of HCT. Combined use of radioimmunotherapy (RIT) with RIC may increase anti-lymphoma activity of RIC. In addition to the graft versus lymphoma effect, HCT provides rescue from hematologic toxicity of RIT which may allow dose escalation of RIT. A multicenter phase I/II dose escalation study of RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20, Zevalin®) at two dose levels (0,6 (22 MBq) and 0,8 (30 MBq) mCi/kg) in 10 patients each, combined with RIC for treatment of patients with aggressive NHL has been initiated. Patients received dosimetry with In111-CD20 on day −21 and RIT with an escalated dose of Y90–CD20 (0,6–0,8 mCi (22–30 MBq)/kg) on day −14 followed by RIC using fludarabine (30 mg/m2 day −8 to −4), melphalan (140 mg/m2 day −3) and alemtuzumab (20–30 mg day −3 to −2). For postgrafting immunosuppression cyclosporine is administered. Primary objective of this study was the evaluation of treatment related mortality and engraftment after RIT-RIC HCT. Secondary objectives were disease response, relapse rate, disease free and overall survival, GVHD and immune reconstitution.Twenty patients have been enrolled on the study. Diagnoses were diffuse large B-cell lymphoma (n=13), transformed chronic lymphocytic leukaemia (n=4), blastoid mantle cell lymphoma (n=2) and follicular lymphoma grade 3 (n=1). Median age was 51 (range, 30–69) years. PBSC grafts were either from matched related (n=5) or matched unrelated donors (n=15). All patients were “high risk” with refractory disease or relapse after preceding autologous HCT. Disease status at time of HCT was CR=4, PR=15 and SD=1. No additional toxicity due to RIT was observed in comparison to our previous experience with the same RIC even after dose escalation to 0.8 mCi (30 MBq)/kg. Engraftment was rapid and sustained with no graft rejections. Median time to >500 granulocytes/μL was 14 (range, 9–32) days and to >20000 platelets/μL 11 (range, 9–56) days. TRM at day +100 was 0% and overall 10% with two deaths due to infection. Incidence of grade II–IV GVHD was 45% (II=8, III=1). To date, chronic extensive GVHD occurred in 3 patients. Causes of death were relapse=6, suspected suicide=1 and infection=2. 11/20 patients (55%) are alive with a median follow-up of 119 (range, 28–363) days. Disease status of patients alive is CR=8, PR=2 and Relapse=1.In conclusion, dose escalation of RIT and combined use with RIC is feasible with no additional toxicity due to RIT and with stable engraftment in all patients. Preliminary response data suggest that this strategy may improve early post-transplant disease control, but long-term disease-free survival remains to be determined.

Translocations targeting CCND2, CCND3, and MYCN do occur in t(11;14)-negative mantle cell lymphomas

AbstractThe genetics of t(11;14)(q13;q32)/cyclin D1–negative mantle cell lymphoma (MCL) is poorly understood. We report here 8 MCL cases lacking t(11;14) or variant CCND1 rearrangement that showed expression of cyclin D1 (2 cases), D2 (2 cases), and D3 (3 cases). One case was cyclin D negative. Cytogenetics and fluorescence in situ hybridization detected t(2;12)(p11;p13)/IGK-CCND2 in one of the cyclin D2-positive cases and t(6;14)(p21;q32)/IGH-CCND3 in one of the cyclin D3-positive cases. Moreover, we identified a novel cryptic t(2;14)(p24;q32) targeting MYCN in 2 blastoid MCLs: one negative for cyclin D and one expressing cyclin D3. Interestingly, both cases showed expression of cyclin E. Notably, all 3 blastoid MCLs showed a monoallelic deletion of RB1 associated with a lack of expression of RB1 protein and monoallelic loss of p16. In sum-mary, this study confirms frequent aberrant expression of cyclin D2 and D3 in t(11;14)-negative MCLs and shows a t(11;14)-independent expression of cy-clin D1 in 25% of present cases. Novel findings include cyclin E expression in 2 t(11;14)-negative MCLs characterized by a cryptic t(2;14)(p24;q32) and identification of MYCN as a new lymphoma oncogene associated with a blastoid MCL. Clinically important is a predisposition of t(11;14)-negative MCLs to the central nervous system involvement.

Ki67 and PIM1 expression in aggressively treated mantle cell lymphoma (MCL): A Cancer and Leukemia Group B (CALGB) 59909 correlative science study

8560 Background: Gene expression profiling has suggested that the proliferation signature is an important predictor of outcome in MCL. Retrospective studies have suggested that the proliferative index, measured by mitoses or Ki67, is an important prognostic indicator. This has not been validated in a US cooperative group setting or in the context of aggressive therapy. We assessed Ki67 and PIM1 (a cell cycle-related gene upregulated in blastoid MCL) expression in a prospective phase 2 cooperative group study. Methods: CALGB 59909 examined the efficacy of aggressive chemotherapy followed by autologous stem cell transplantation (ASCT) in untreated MCL patients less than 70 years of age. Cases with available material were studied for Ki67 and PIM1 expression by immunohistochemistry. Ki67 was quantitated by image analysis (IA Ki67) and manually as # positive cells/1000x hpf (Ki67-count). PIM1 staining was scored semi-quantitatively (0–3+). Progression-free survival (PFS) and disease-free survival (DFS) were t...

Ki67 and PIM1 expression predict outcome in mantle cell lymphoma treated with high dose therapy, stem cell transplantation and rituximab: a Cancer and Leukemia Group B 59909 correlative science study

The proliferation index in mantle cell lymphoma (MCL) has not been validated in the context of aggressive therapy regimens in the rituximab era. We assessed Ki67 and PIM1 (a cell cycle-related gene upregulated in blastoid MCL) expression by immunohistochemistry in a phase II study Cancer and Leukemia Group B 59909 of aggressive chemotherapy and rituximab followed by autologous stem cell transplantation plus rituximab in untreated MCL patients <70 years of age. As a continuous variable or using a cutoff of 35%, higher image analysis (IA Ki67, n = 52) was associated with shorter progression free survival (PFS) (P ≤ 0.030) and event free survival (EFS) (P ≤ 0.017). PIM1 expression (n = 50) was associated with PFS (P = 0.033) and EFS (P = 0.043). Bivariate Cox models showed IA Ki67 and PIM1 were independent of clinical factors. High Ki67 (>35%) is an important independent prognostic marker in aggressively treated MCL in the rituximab era. PIM1 expression predicts poor outcome and, given its potential role as a therapeutic target, deserves further study.

In-111 Ibritumomab Scintigraphy (Planar and SPECT) and FDG PET/CT Before Y-90 Ibritumomab Treatment in a Patient With Mantle Cell Lymphoma

We report the case of a 75-year-old man, in whom Y-90 ibritumomab was requested because of relapse of blastoid variant mantle cell lymphoma diagnosed in 1995. Before Y-90 ibritumomab treatment, FDG PET and In-111 ibritumomab scintigraphy with planar views at 24 hours, 48 hours, and 5 days, including SPECT, were performed. Discordant information between both examinations was observed as, in addition to the lesions detected by In-111 ibritumomab imaging, FDG PET detected lesions that did not take up the ibritumomab. The discrepancy shown by both radiotracers has to be kept in mind before planning treatment with Y-90 ibritumomab, and for the correct evaluation of treatment response.

Epstein-Barr Virus-Positive Blastoid Variant of Mantle Cell Lymphoma in an Adult with Recurrent Infectious Mononucleosis-Like Symptoms: A Case Report

Epstein-Barr virus (EBV) is closely associated with several lymphomas, such as Burkitt lymphoma, natural killer/T-cell lymphoma, peripheral T-cell lymphoma, and Hodgkin's lymphoma; however, whether EBV is implicated in mantle cell lymphoma (MCL) has not been established. We report the case of an adult with recurrent infectious mononucleosis (IM)-like symptoms who developed an EBV-positive blastoid variant of MCL. A 54-year-old Japanese man presented with fever, swelling of the oral mucosa and tongue, dispersed pulmonary infiltrations, systemic lymphadenopathy, and splenomegaly. He had a history of recurrent IM-like symptoms (prolonged fever and cervical lymphadenopathy) for at least 1 year. MCL was diagnosed by biopsy of the cervical lymph node. The anti-EBV antibody titer indicated a reactivation of chronic infection with this virus. EBV was detected in most of the lymphoma cells and in the peripheral blood. EBV might have played some role in the tumorigenesis of blastoid MCL.

Blastoid Mantle Cell Lymphoma Occurring in a Patient in Complete Remission of Chronic Myelogenous Leukemia

The development of a de novo lymphoma in patients affected by chronic myelogenous leukemia (CML) is a rare event. The introduction of new molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH), allows a correct differential diagnosis between lymphoid blastic crisis and a blastoid variant of mantle cell lymphoma (MCL), which shows an aggressive behavior and some molecular characteristics detectable by cytogenetics and immunohistochemistry. We report a case of a blastoid variant of MCL that developed in a patient with CML who achieved complete cytogenetic and molecular response to imatinib mesylate treatment.

Expression of centrosome-associated gene products is linked to tetraploidization in mantle cell lymphoma

In mantle cell lymphoma (MCL), a blastoid variant with a striking tendency to harbor chromosome numbers in the tetraploid range has been identified. Centrosome aberrations have recently been implicated in the induction of aneuploidy in many human malignancies including MCL by malsegregation of chromosomes during anaphase of mitosis. Recently, we showed that centrosome aberrations occur more frequently in tetraploid MCL as compared to their diploid counterparts. To test the hypothesis of an association between tetraploidization and expression of genes coding for centrosomal proteins in MCL, tumor RNA of 33 MCL samples was hybridized to custom-made cDNA microarrays, representing 4,628 distinct human gene-specific fragments, with particular enrichment for cancer-relevant (n = 2,440) and centrosome-associated genes (n = 359). Notably, 4 of the 6 most significant genes (CAMKK2, PCNT2, TUBGCP3, TUBGCP4) discriminating between diploid and near-tetraploid MCL code for centrosomal proteins. As confirmed by quantitative RT-PCR analysis, calcium/calmodulin-dependent protein kinase II (CAMKK2), pericentrin (PCNT2) and gamma-tubulin complex associated protein 3 (TUBGCP3) were all found to be significantly higher expressed in near-tetraploid than in diploid MCL samples. In conclusion, we describe a comprehensive expression signature of a set of genes associated with tetraploidization in MCL. The high expression level of centrosome-associated gene products in blastoid MCL matches the description of more frequent centrosome aberrations in this MCL variant.

Sequence analysis proves clonal identity in five patients with typical and blastoid mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is typically composed of small irregular lymphoid cells. Blastoid variants, composed of lymphoblast-like (classic type) or large (pleomorphic type) cells, arise de novo or in patients with typical MCL. Although it has been assumed that blastoid variant represents histologic transformation of typical MCL, the clonal relationship between the two tumors has rarely been assessed at the molecular level. We identified five patients with typical MCL who subsequently developed the blastoid variant. There were two men and three women with a median age of 65 years (range, 34–70) at diagnosis of typical MCL involving lymph nodes. The median interval between typical and blastoid MCL was 36 months (range, 11–103). Subsequent blastoid variant MCL involved soft tissue (two), lymph node (one), ileum (one), or rectum (one). All typical and blastoid neoplasms were positive for CD20, cyclin D1, and monotypic surface immunoglobulin light chain, and all typical cases were positive for CD5. Two blastoid neoplasms lost CD5 expression, one of which aberrantly expressed CD10. Immunostaining for Ki-67 showed a median proliferative fraction of 20% in typical and 70% in blastoid neoplasms. Sequence analysis of the VDJ regions of the rearranged IgH allele proved clonal identity in each set of paired samples in all five patients. These results support the concept that blastoid MCL arising in patients with typical MCL represents histologic transformation of the original neoplastic clone.

Blastoid Mantle Cell Lymphoma (MCL): 5-yr Failure-Free Survival (FFS) Rate of 50%, without Failures after 2.5 Years Following Treatment with Rituximab (R)-HyperCVAD Alternating with R-Methotrexate/Cytarabine (M/A).

Blastoid MCL has a very poor prognosis, with a median survival of 16–20 months after treatment with CHOP-like chemotherapy regimens. We recently described an intense regimen where R-HyperCVAD is alternated with R-M/A for 6–8 cycles, capable of achieving 87% complete remission (CR) rates and an overall 3-year FFS rate of 64% in a group of 97 patients with newly diagnosed aggressive MCL treated under an institutionally approved clinical trial from March 1999 to March 2001 (J Clin Oncol 23:7013–7023).Fourteen of these patients presented with the blastoid cytologic variant and the following clinical features were compared with those without a blastoid cytology: age > 65 years, beta 2 microglobulin ≥ 3 gm/dL, elevated serum lactic dehydrogenase (LDH), and high international prognostic score (IPI). Only serum LDH was significantly different, being higher for those with blastoid presentation (p = 0.03). Rates of complete remission were lower for blastoid when compared to non-blastoid cytology (79% vs 89%, respectively) but not statistically significant (p = 0.72). With a median follow up of 57 months, there is a plateau in the FFS curve for blastoid MCL as shown below. At 57 months the median overall survival has not been reached. Contrary to what we had expected, the improved outcome in this group of patients with blastoid cytology suggests a potential for long term remission after intense, non-myeloablative chemotherapy. [Display omitted]

Clinicopathologic Subtypes of Mantle Cell Lymphoma (MCL) Show Distinct Patterns of Genetic Copy Number Alteration.

Introduction: MCL is an aggressive NHL characterized by a t(11;14)(q13;q32). However this translocation alone cannot initiate lymphomagenesis and multiple secondary genomic alterations are necessary to induce the neoplastic phenotype. In addition, three distinct clinicopathologic subtypes of MCL (classic, blastoid, and leukemic) have been identified with differing prognosis. Secondary genomic alterations associated with each subtype have been poorly defined. ExperimentalDesign: Genomic profiles from a panel of MCL samples comprised of 20 classic, 5 leukemic (no nodal involvement), and 12 blastoid cases, were created using a tiling-resolution BAC array CGH platform. Alignment of profiles to identify regions of recurrent alteration was accomplished using a custom developed software package (SeeGH). In addition, specific genes were analyzed for copy number status. Statistically significant distributions were determined using Chi square and Fisher's exact analyses.Results: Twenty two regions of recurrent alteration were identified that were statistically different between subtypes (partial data in Table 1). As expected chromosome 9 losses were more prevalent in blastoid MCL. 13q34 losses were prevalent within classic and blastoid MCL, as opposed to leukemic MCL, which showed gains of this region. Analysis of biological pathways revealed that apoptotic signaling is not disrupted via copy number in a large portion of MCL cases (21%), while chromosomal instability genes show copy number disruption in 67% of MCL cases. Surprisingly, the glioblastoma pathway was preferentially disrupted in blastoid MCL (p=1.09x10−5), affecting all cases; whereas the p53 pathway was disrupted via copy number more often in leukemic MCL (p=0.0207).Conclusions: Multiple genomic regions and cancer related genes show a differential pattern of altered copy number distribution between MCL subtypes. These regions and genes likely contribute to the different clinicopathologic subtypes of MCL cases.Differential regions in MCL subtypes+/−chr. bandsize (Mb)clas%leuk%blast%enriched indeficient in−1q25.2-q31.215.5002033blast (p=0.03)class (p=0.01)−2q26.32.915033blast (p=0.03)+3cent-p12.14.1806025leuk (p=0.02)class (p=0.005)+7q11.233.240033blast (p=0.008)class (p=0.04)−9p21.31.02102083blast (p=0.00001)class (p=0.002)−9q21.131.1054075blast (p=0.0003)class (p=0.0002)−9q21.2-q31.122.8754067blast (p=0.001)class (p=0.0007)−9q31.10.8304067blast (p=0.0004)class (p=0.00006)−9q34.12-qter7.2702042blast (p=0.009)class (p=0.005)+10p12.2-p12.310.635042blast (p=0.009)−13q341.6940075blast (p=0.03)leuk (p=0.05)−17p13.3-pter2.2856025leuk (p=0.04)−17p13.11.2156025leuk (p=0.04)

Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma

To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt and multiple downstream targets in primary MCL cases and cell lines. Akt was phosphorylated in 12 of 12 aggressive blastoid MCL variants and in 4 of 4 MCL cell lines. In contrast, phosphorylated Akt was present in only 5 of 16 typical MCL, 3 at comparable levels to the blastoid cases, and 2 at low levels. The presence of p-Akt was accompanied by the phosphorylation of p27(kip1), FRKHL-1, MDM2, Bad, mTOR, and p70S6K. Inhibition of the PI3K/Akt pathway in the MCL cell lines abrogated or reduced the phosphorylation of Akt, p27(kip1), FRKHL-1, MDM2, Bad, mTOR, GSK-3beta, IkappaB, and led to cell-cycle arrest and apoptosis. Six MCL cases (5 with activated Akt and 1 with inactive Akt) and 3 of 4 cell lines showed loss of PTEN expression. PIK3CA mutations were not detected. We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL and preferentially occurs in blastoid variants. One possible mechanism of activation is loss of PTEN expression. These data suggest that PI3K/Akt inhibitors may be effective in the treatment of Akt-activated MCL.

Constitutive Activation of AKT Contributes to the Pathogenesis and Survival of Blastoid Variants of Mantle Cell Lymphoma.

The serine/threonine protein kinase AKT [protein kinase B (PKB)] plays a pivotal role in the tumorigenesis of many human malignancies, including hematopoietic neoplasms. AKT mediates cancer cell survival and cell growth through the phosphorylation of key regulatory proteins such as FRKHL-1, MDM2 and p27. We wished to determine whether the AKT signaling pathway might be involved in the pathogenesis of mantle cell lymphoma (MCL).The activated form of AKT (pAKT) and phosphorylation of several AKT targets, including FRKHL-1, MDM2 and p27 as well as the upstream modulators PTEN and TCL-1 were analyzed by immunoblotting in 31 MCL cases (16 classic MCL and 15 MCL of the blastoid variant) and in four MCL cell lines (Granta 519, NCEB, REC-1 and Z138). PI3KCA mutation analysis at two hotspot regions (Exons 9 and 20) were completed for all cases and cell lines. Subsequently, inhibition of the PIK3/AKT-pathway with LY294002, Wortmannin and AKT-inhibitor was performed in the four cell lines. pAKT was expressed in all 15 blastoid variants of MCL and in the 4 cell lines, with high expression in 13/15 cases and low expression in 2/15 cases. In contrast, only 2/16 typical MCL expressed pAKT, at low levels. Furthermore, pAKT expression in the blastoid variants and cell lines was accompanied by the phosphorylation of multiple downstream targets of activated AKT, including the cell cycle regulatory proteins p27, FRKHL-1, MDM-2 and the apoptosis associated protein bad. 42% of the blastoid MCL cases showed loss of PTEN, while only 2 (12%) of the typical cases showed diminished PTEN expression.TCL-1 expression levels were not correlated with AKT activation and no mutations of PI3KCA were detected. Functional studies of all four MCL lines demonstrated that the phosphorylation of the downstream effectors were regulated by activated AKT:Inhibition of the PI3K/AKT pathway by both 20 μM LY294002 and 8 μM Wortmannin or 40 μM AKT-inhibitor abrogated the phosphorylation of AKT, p27, FRKHL-1, bad and MDM2 after 24 to 48 hours of treatment, and resulted in G1 arrest after 24 hours of treatment and measurable apoptosis by 48 hours. We conclude that constitutive activation of AKT contributes to the pathogenesis of the blastoid variants of MCL. One possible mechanism of AKT activation is the loss of PTEN expression. The inhibition of the PIK3/AKT pathway in MCL cell lines leads to induction of cell cycle arrest and apoptosis, raising the possibility that PI3K/AKT inhibitors may be effective in the treatment of aggressive (blastoid) variants of MCLs.

IgVH mutations in blastoid mantle cell lymphoma characterize a subgroup with a tendency to more favourable clinical outcome

Mantle cell lymphoma (MCL) is associated with a very unfavourable clinical course. This is particularly true for mantle cell lymphoma of the blastoid subtype (MCL-b). In order to define prognostic factors, we analysed the impact of immunoglobulin heavy chain variable (IgV H) gene somatic hypermutations on clinical outcome in a series of 21 cases of morphologically, phenotypically, and genotypically well-characterized MCL-b. Testing and estimation were performed using log-rank statistics and displayed on Kaplan-Meier graphs. Thirteen of 21 cases of MCL-b revealed a homology rate of > or = 99% compared to IgV H germ-line sequences in the databases and were scored as non-mutated. Eight of 21 cases (38%) of MCL-b were mutated. In MCL-b the mutation frequency was usually low and the mutation pattern was only rarely antigen-selected, in contrast to a control group of 11 cases with morphologically almost identical, but phenotypically and genotypically clearly distinguishable, diffuse large B cell lymphoma, derived, most likely, from germinal centre B cells. In our series of 21 MCL-b, positive IgV H mutational status, irrespective of varying homology thresholds, had no statistically significant prognostic impact on event-free or overall survival. However, mutated MCL-b tended to present more frequently at an earlier stage and without bone marrow involvement and to show lower rates of relapse and death, resulting in a more favourable clinical outcome.

Checkpoint kinase 1 (CHK1) protein and mRNA expression is downregulated in aggressive variants of human lymphoid neoplasms

CHK1: gene encodes for a serine/threonine kinase involved in the regulation of cell cycle progression and DNA damage checkpoints. To determine the role of CHK1 in the pathogenesis of lymphoid neoplasms and its relationship to other DNA damage response genes, we have analyzed the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid tissues. CHK1 protein and mRNA expression levels were very low in both reactive tissues and resting lymphoid cells, whereas tumor samples showed a variable pattern of expression related to their proliferative activity. However, seven aggressive tumors showed a dissociate pattern of extremely low or negative protein expression in spite of a high proliferative activity. Four of these tumors were diffuse large B-cell lymphomas (DLCLs) with concordant reduced levels of mRNA, whereas one blastoid mantle cell lymphoma (B-MCL) and two DLCLs had relatively normal levels of mRNA. No gene mutations, deletions, or hypermethylation of the promoter region were detected in any of these cases. In all these tumors ATM, CHK2, and p53 genes were wild type. These findings suggest that CHK1 inactivation in NHLs occurs by loss of protein expression in a subset of aggressive variants alternatively to ATM, CHK2, and p53 alterations.

The Leukemic Presentation of Mantle-cell Lymphoma: Disease Features and Prognostic Factors in 58 Patients

Mantle-cell lymphoma (MCL) is a B-cell malignancy with distinct molecular genetics and pathological features. Peripheral blood involvement has been reported with variable frequency, but information on the natural history of cases presenting with leukemia is lacking. This study aimed to determine the clinical and prognostic features of such cases. We studied clinical features, tumor characteristics, prognostic factors and outcome in 58 patients with leukemic presentation of MCL. Diagnosis was based on morphology, immunophenotype, presence of t(11;14), histology and cyclin D1 expression. The median age was 62 years and male:female 2.4:1. Presenting features included splenomegaly (74%), lymphadenopathy (45%), hepatomegaly (17%) and, in a minority, gastro-intestinal involvement or involvement of Waldeyer's ring; 10% had lymphocytosis alone. Six patients developed central nervous system disease. Median lymphocyte count was 58 x 10(9)/l, 55% had anemia and 17% had thrombocytopenia. Morphology of peripheral blood showed small-cell MCL in 15% of cases, typical MCL in 46% and blastoid MCL in 39%. Immunological markers showed a typical phenotype (CD5+ CD23 -) in 68%, and atypical phenotypes, CD5- CD23- in 17% or CD5+ CD23+ in 15%. CLL scores were 0, 1 or 2 in 96%. Median overall survival was 36 months. Good response to first-line treatment (P = 0.0008) and splenomegaly (P = 0.03) were favorable prognostic factors, while other features including morphology and CD38 expression had no impact on survival or treatment response. This analysis demonstrates that except for splenomegaly, survival of MCL patients presenting with leukemia is not significantly influenced by clinical or tumor characteristics. Splenectomy is a useful treatment option in this group of patients.

Coexistence of BCL1/CCND1 and CMYC aberrations in blastoid mantle cell lymphoma: a rare finding associated with very poor outcome

A patient with mantle cell lymphoma (MCL) of the pleomorphic blastoid subtype is reported. The disease was clinically aggressive and refractory to chemotherapy, and the patient survived only 2 months. Cytogenetically, a t(11;19;14)(q13;q13;q32) was found. Fluorescent in situ hybridization (FISH) and molecular analyses demonstrated involvement of the BCL1/CCND1 locus in a three-way translocation. In addition, subclonal abnormalities of the region 8q24 manifested either as a t(8;22)(q24;q11)/CMYC rearrangement or trisomy 8 were identified. The pathogenetic impact of this very uncommon association of BCL1/CCND1 and CMYC rearrangements in MCL is discussed and the literature is reviewed.

Nuclear Survivin Expression in Mantle Cell Lymphoma Is Associated with Cell Proliferation and Survival

Survivin is a member of the inhibitor of apoptosis protein family that is expressed in G2/M phase. Survivin is overexpressed and associated with parameters of poor prognosis in different human tumors. The role of survivin in the pathogenesis of mantle cell lymphoma (MCL) was examined in a series of typical and blastoid tumors. Survivin was detected as a nuclear pattern in a variable number of tumor cells. Mitotic figures were always positive with a strong delineation of the chromosomes. Western blot analysis confirmed the presence of survivin only in nuclear fractions. Protein expression detected by immunohistochemistry correlated with mRNA levels analyzed by quantitative real-time reverse transcription-polymerase chain reaction (P < 0.0001). Survivin expression levels were higher in blastoid MCL variants (P < 0.0001) and were associated with the proliferative activity (P = 0.001), but not with the ploidy status of the tumors. The number of apoptotic cells was independent of survivin or Ki-67 expression. Overall survival was significantly shorter in patients with high survivin expression. However, in a multivariate analysis, proliferative index was a better predictor of survival than survivin score. These findings indicate that survivin is commonly expressed in MCL with a nuclear and mitotic pattern. The expression levels are strongly associated with the proliferative activity of the tumors and the survival of the patients, suggesting a potential role in cell cycle regulation and tumor progression.

Cytogenetic findings in blastoid mantle cell lymphoma

A subset of mantle cell lymphoma (MCL) tumors has blastoid morphology, and a number of morphologic variants of blastoid MCL have been described in the literature. In this report, we document the cytogenetic findings in 27 cases of blastoid MCL. Conventional cytogenetic analyses were performed on bone marrow aspirates involved by MCL from 27 patients. There were 14 men and 13 women with a median age of 63 years (range, 40–79 years). Diagnostic tissue biopsy and bone marrow specimens were reviewed, and cases were divided into 2 morphologic groups: classic (12 cases) and pleomorphic (15 cases), as defined in the World Health Organization classification. All tumors had an immunophenotype compatible with MCL, were positive for cyclin D1, and carried the t(11;14). Twenty-four cases had complex karyotypes with 3 or more chromosomal abnormalities in addition to the t(11;14). In classic blastoid MCL, abnormalities of chromosomes 13, 18, and 8 were most common. In pleomorphic blastoid MCL, abnormalities of chromosomes 13, 17, and 3 were most frequent. Chromosome 22 abnormalities were detected exclusively in the pleomorphic group. Tumors in which the neoplastic cells showed prominent nucleoli had a significantly higher frequency of chromosome 17 abnormalities ( P = 0.03). We conclude that blastoid MCL tumors often show complex cytogenetic aberrations. Some abnormalities correlate with morphologic features, suggesting that morphologic variants of blastoid MCL may arise via different molecular pathways.