Classical Mantle Cell Lymphoma Research Articles

Bortezomib Synergizes with a Novel Jak2 Inhibitor, WP-1130, To Inhibit Cell Growth and Induce Apoptosis in “Classic” and “Blastoid-Variant” Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma B cells (NHL-B), a lymphoma with increasing incidence over the last few decades. MCLs are classified into at least two subgroups, “classic” type of MCL and “blastoid-variant”- an even more aggressive type of MCL. Both subgroups are incurable, unusually refractory to standard chemotherapy combinations and associated with poor prognosis. New therapeutic agents with greater efficacy and less toxicity are necessary in MCL. We have shown previously that in MCL cells, the key transcription factor NF-kB, is constitutively active and maintains lymphoma cells survival. We also demonstrated that treating the MCL cells with the proteasome inhibitor bortezomib (Velcade) inhibits constitutive NF-kB activation, leading to G1 cell cycle arrest and apoptosis. Recently, a novel small molecular weight compound called WP-1066, a derivative of AG490, was synthesized by screening a synthetic library for agents that block stat3 activation. WP-1066 has shown to have anti-tumor activity in MCL cells through the inhibition of IL-6 mediated stat3 activation and NF-kB inhibition. A more effective compound called WP-1130, a derivative of WP-1066, was synthesized and is shown to be more potent than WP-1066 in MCL cell lines. Single agents are rarely effective in treating a disease like MCL; therefore, we hypothesized that the combination of bortezomib and WP-1130 would likely be more effective in MCL. We showed that MCL cells, both “classic” and “blastoid-variant”, treated in-vitro with bortezomib in conjunction with WP-1130 resulted in a synergistic growth inhibition and apoptosis induction. The drug concentrations use for bortezomib and WP-1130 that produce the synergistic effects were in the low nanomolar (nM) and micromolar (uM) ranges, respectively. Bortezomib at a concentration of 10 nM induces approximately 15% MCL apoptosis after 48 hr treatment when compare to untreated controls, while WP-1130 at a concentration of 1 uM induces 5% apoptosis. The combination of bortezomib and WP-1130 at the same concentrations induces 60% of MCL cell apoptosis. Bortezomib and WP-1130 showed efficacy in both classic and blastoid-variant MCL cell lines. The apoptotic effects in these cells were correlated with the down-regulation of bcl-2 and the up-regulation of bax proteins. The status of constitutive NF-kB was also examined after drug treatments. While a single agent treatment with low drug concentrations had only minimal effect on NF-kB inhibition, the combination of the two drugs dramatically inhibits NF-kB activation. These two drugs also synergize to inhibit cyclin D1 (a molecular signature of MCL), and c-myc (an oncogene commonly over-expressed in lymphoma B cells). Agents such as bortezomib and WP-1130, that can pharmacologically modulate key intracellular targets such as constitutively expressed NF-kB and cyclin D1 in MCL cells, may be more effective therapeutic agents for the treatment of MCL.

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)

Development of a Blastoid Variant, Mantle Cell Lymphoma Model in Transgenic Mice.

Mantle Cell Lymphoma (MCL) is a poorly understood, aggressive histotype of B-cell non-Hodgkin's Lymphomas (NHL-B) that remains the most therapeutically resistant of the NHL-B. Little is known regarding why MCL is so clinically aggressive and therapeutically refractory. Blastoid variant MCL (MCL-BV) is an even more aggressive form of MCL that appears to be increasing in incidence in the US. It may represent progression from classic MCL, often with leukemic involvement and complex lymphoma karyotypes. Interleukin 14 (IL-14) is cytokine that was identified and cloned from a Burkitt lymphoma (BL) cell line that acts as a growth factor for normal B-lymphocytes. The expression of IL-14a protein and mRNA levels are elevated at lease fifty-fold in B-cell non-Hodgkin's Lymphomas (NHL-B), including mantle cell lymphoma (MCL), in contrast to very low levels of IL-14a in quiescent (Go) B cells by both western and northern blot analysis. To evaluate the role of IL-14 in vivo, we have generated transgenic mice expressing IL-14 with pEuSR. The IL-14 TG mice generally live a normal life span, however when autopsies are performed at 18 months of age, splenomegaly is noted, and 50% have evidence of B cell lymphoma. This lymphoma is CD5+, CD19+, sIgM+, CD21− and contains a monoclonal population of B-lymphocytes with rearranged immunoglobulin genes. Morphologically the lymphoma arising in IL-14 transgenic mice resembles the centroblastic/Immunoblastic histotype of DLBCL. Because of the frequent involvement of c-myc in various B cell malignancies, we crossed Eμ-myc (c-myc TG) mice with the IL-14 TG mice. By 3 months of age, 100% of the double transgenic (DTG) mice develop an aggressive B cell malignancy that is characterized by extensive lymphadenopathy and splenomegaly with intermediate to large atypical lymphoid cells, strongly resembling MCL-BV morphologically. This tumor, like that derived from the IL-14 TG mice, is CD5+, CD19+, sIgM+, CD21−. It is also CD23− and over-expresses Cyclin D1 in monoclonal B lymphoid cells with re-arranged IgH immunoglobulin genes, mimicking the MCL phenotype. At the time of autopsy, tumor infiltration of DTG mice is generally found in all organs evaluated, including peripheral blood, lymph nodes, spleen, liver, bone marrow, thymus and kidneys, consistent with the usual findings in MCL-BV. No tumors are observed in IL-14α TG or c-myc TG mice autopsied at this age. This MCL-BV model allows for the molecular and genotypic characterization of the murine B lymphoid cell compartment from birth to lymphoma development (3 mos.), including histogenesis and functional determination of the growth and survival characteristics of these tumors in DTG bone marrow and peripheral B cell populations. Preliminary comparative in vitro and in vivo (SCID Xeno-transplants) studies in DTG/MCL-BV lymphomas have shown additional molecular similarities to the pathophysiology (e.g constitutive NF-kB activation) of MCL-BV cell lines and patient samples, that should provide insights for future potential therapeutic approaches to 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.

Blastoid variant of mantle cell lymphoma: late progression from classical mantle cell lymphoma and quantitation of minimal residual disease

Classical mantle cell lymphoma (MCL) and its blastoid variant (MCL-BV) are characterized by an extremely poor prognosis. Long-time survivors are rare, only very few patients with an overall survival over 10 years have been reported. We present a case of a 41-year-old male with a 12 yr history of MCL stage I to show, that very late relapses in MCL are possible and may present as a transformation into an aggressive blastoid variant and to illustrate the value of quantitative minimal residual disease (MRD) monitoring for treatment guidance. Diagnostic lymph node and bone marrow samples were investigated by immunohistochemistry. Clonality analysis was performed by immunoglobulin heavy chain gene (IGVH) and t(11;14) PCR. The MRD assessment was done by real-time quantitative PCR (RQ-PCR) on available follow-up samples. By histologic review and sequencing of the clonal IGVH and t(11;14) PCR products we demonstrated a common clonal origin of the leucemic MCL-BV and the classical MCL diagnosed 12 yr earlier. Quantitative MRD assessment revealed significant MRD levels after intensive conventional chemotherapy including Rituximab. Therefore, treatment was early intensified by myeloablative radio-chemotherapy and allogeneic peripheral stem cell transplantation from an unrelated HLA-identical donor. This did not translate into a sustained remission as reflected by persisting MRD levels after transplantation and the patient died from rapid progressive disease 3.5 months after transplant. This report presents a rare case of long-term survivor of MCL with a progression of the original MCL cell clone to MCL-BV and demonstrates the clinical value of quantitative MRD assessment for optimized therapeutic management.

Tissue microarray: A valuable method in diagnosis and prognosis of hematological malignances

BACKGROUND: The novel technology of tissue microarray (TMA) allows rapid and cost-effective analysis of hundreds of markers on the same set of specimens. Limited amount of tissue that could be analyzed and problem of tissue heterogeneity, are the major drawbacks of TMA technique for immunohistochemical characterization of lymphomas. METHODS: In this paper 65 cases of lymphomas were analyzed using TMA with following panel of antibodies: BOB1, Oct2, Bcl2, Bcl6, CD20, CD21, CD23, CD3, CD5, CD10, CD43, CD79a, CD138, Cyclin D1, Ki67, MUM1, Pax5, p53. RESULTS: In 14 patients with diffuse large B-cell lymphoma (DLBCL), 5 were classified as germinative center and 3 as non-germinative center cases according to the Bcl6, CD10, and MUM1 positivity. Other 2 patients were identified as T cell rich B cell lymphoma based on morphology and Oct2 and BOB1 positivity of pleomorphic B lymphocytes. DLBCL with Bcl6+ immunophenotype had better overall survival than Bcl6- cases. All cases of classic mantle cell lymphoma had significantly lower Ki-67 proliferation index than blastoid subtypes. There were 14 cases of chronic lymphocytic leukemia/small cell lymphocytic lymphoma, 6 cases with follicular lymphoma, 5 cases of marginal zone lymphoma, and 7 cases of lymphoplazmacitoid lymphoma. In the indolent lymphoma group, survival of patients with p53+/- was poorer comparing to p53- group. CONCLUSION: We conclude that TMA technique is a valuable method in diagnosis and prognosis of lymphomas, which are considered very heterogeneous group of hematological neoplasms.

Correlation of Quantitative Gene Expression by RQ-PCR with Clinical and Biologic Factors in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and the t(11;14) translocation leading to overexpression of cyclin D1 (CCND1). Recently, a relation between the quantitative expression of proliferation associated genes including CCND1 and the survival of MCL patients has been demonstrated using microarray gene expression profiling. In the current study a set of genes (CCND1, CCND3, CDK2, CDK4, p16, p27, Rb, E2F1, MYC, Ki-67, BMI, MCL-1, MDM2, ATM, TP53) involved in cell cycle regulation, related to MCL pathogenesis, or located in critical genomic regions was analyzed by real-time RT-PCR (RQ-PCR) in 74 MCL cases and correlated with clinical and biologic factors. Diagnoses were based on morphology and the detection of the t(11;14) and/or CCND1 overexpression on protein or mRNA level. 62 cases were classical MCL, 12 blastoid variants. To obtain a quantitative estimate of the relation between the expression the CCND1 1.7 kb (short; missing 3′ UTR) and 4.5 kb (long; containing 3′ UTR) mRNA isoform a first amplicon was localized in the CCND1 coding region and a second one in the CCND1 3′ UTR. FISH analysis was performed in a subset of 40 cases for 3q26, 6q27, 9p21, 11q22–q23, 12q13, 13q14, and 17p13. As a marker for proliferation activity the mitotic index (MI) was determined in 51 cases (mitoses/10 high power fields). Survival information was available for all cases, additional clinical information (IPI, B-symptoms, tumor bulk) for 60 cases. In univariable analysis a statistically significant association between gene expression levels and overall survival was identified for MYC (p=0.003), BMI (p=0.027), and CCND1 (coding region; p=0.028). A multivariable analysis including clinical factors of known prognostic impact (IPI score, B-Symptoms, Bulk, blastoid morphology) was carried out for each of the candidate genes individually. In this analysis a significant impact on survival was observed for MYC (p=0.008), BMI (p=0.021), and E2F1 (p=0.042), and a trend for Ki-67 (p=0.054), the ratio CCND1 coding region/CCND1 3′ UTR (p=0.062), and CCND1 coding region (p=0.082). Notably, and in contrast to the other genes of prognostic significance, BMI overexpression was positively correlated with overall survival times. In line with this, there was a negative correlation of BMI levels with proliferation activity as measured by the MI (p<0.001; r=−0.515). Other genes with a negative MI correlation were MCL-1 (p<0.001; r=−0.532) and p27 (p<0.001; r=−0.589), whereas the ratio CCND1 coding region/CCND1 3′ UTR showed a positive correlation (p<0.001; r=0.523). Genomic losses at 9p21 and 17p13 were associated with reduced levels of p16 (p=0.002) and TP53 (p=0.011), respectively, arguing for a pathogenic relevance of these candidate genes in MCL. However, neither p16 nor TP53 expression levels obtained prognostic significance in our MCL series. In conclusion, focused RQ-PCR analysis of individual candidate genes showed a correlation between quantitative gene expression and survival times in MCL. In addition to CCND1 expression levels of MYC, BMI, and E2F1 provided prognostic information also in the context of clinical factors suggesting those factors being valuable survival predictors and important components of MCL pathogenesis.

Variant t(2;11)(p11;q13) associated with the IgK-CCND1 rearrangement is a recurrent translocation in leukemic small-cell B-non-Hodgkin lymphoma.

Classical t(11;14)(q13;q32) involving IGH-CCND1 is typically associated with aggressive CD5-positive mantle cell lymphoma (MCL). Recently, we identified the IGK variant of this translocation, t(2;11)(p11;q13), in three patients with a leukemic small-cell B-non-Hodgkin lymphoma. In all cases, rearrangements of the IGK and CCND1 genes were demonstrated by fluorescence in situ hybridization. Moreover, we mapped the 11q13 breakpoint of this variant translocation in the 3' region of CCND1 which contrasts with the 5' breakpoints in a standard t(11;14)(q13;q32). Expression of cyclin D1 was shown in two cases analyzed either at diagnosis or during disease progression. All three patients were asymptomatic at presentation and no initial therapy was required. One patient died of a progressive disease 58 months from diagnosis, and two patients showed stable disease after 12 months of follow-up. In two analyzed cases, mutated IGVH genes were identified. Our findings indicate that variant t(2;11)(p11;q13) does not typify a classical MCL but possibly a more indolent leukemic lymphoma originating from an antigen experienced (mutated) B cell.

An Unusual Case of Leukemic Mantle Cell Lymphoma With a Blastoid Component Showing Loss of CD5 and Aberrant Expression of CD10

Characteristically, mantle cell lymphoma (MCL) expresses surface immunoglobulin (sIg), CD19, CD20, and CD5 and lacks CD10 and CD23. Rare CD5-MCL variants have been described. This report describes a case of leukemic MCL with morphologically and immunophenotypically distinct classic MCL and blastoid-variant MCL (BV-MCL) components. The classic MCL had typical morphologic features and immunophenotype (kappa sIg light chain-restricted and CD5+; CD10- and CD23-). The BV-MCL had larger nuclei and open chromatin; these cells also were kappa sIg light chain-restricted; however, they were CD10+ and CD5-. Fluorescence in situ hybridization studies demonstrated cyclin D1-immunoglobulin heavy chain gene fusion in both components; the bone marrow biopsy cellularity was replaced by CD10+ and cyclin D1+ and CD5-BV-MCL. This case illustrates the phenotypic heterogeneity of MCL and underscores the need for histopathologic correlation and, in some instances, ancillary genetic studies to accurately classify B-cell lymphomas.

Immunoglobulin heavy chain genes somatic hypermutations and chromosome 11q22‐23 deletion in classic mantle cell lymphoma: a study of the Swiss Group for Clinical Cancer Research

Mantle cell lymphoma (MCL) shares immunophenotypic and karyotypic features with chronic lymphocytic leukaemia. The latter comprises two distinct entities with prognosis dependent upon immunoglobulin heavy chain (IgH) gene mutational status and the presence of 11q deletion. We evaluated the relevance of IgH gene mutational status, IgV gene family usage and presence of 11q deletion in a series of 42 histologically reviewed classical MCL cases to determine the prognostic impact. VH3 was the most common VH family, with VH3-21 being the most frequent individual VH gene. Approximately 30% of the cases had a IgH somatic mutation rate higher than 2%, but was only higher than 4% in <10% of cases. Half of the cases had deletion of chromosome 11q21-telomere (11q21->ter), with two minimal deleted regions, at 11q22.2 and 11q23.2. There was no association between 11q loss and IgH gene somatic mutation rate; the use of VH3-21 gene could be associated with a better prognosis.

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.

The splenic form of mantle cell lymphoma.

To describe the clinical, immunophenotypic and molecular features, as well as the clinical course of patients with unusual presentation of mantle cell lymphoma (MCL) purely located to the spleen. We describe seven patients presented with splenomegaly and a leukemic picture without lymphadenopathy, fulfilling the diagnostic criteria of MCL. In addition to clinical and pathologic features, patients were studied with respect to surface immunophenotype, including adhesion molecule profile, immunohistochemical expression of cyclin-D1 and bcl-1 rearrangement by polymerase chain reaction. Four patients were male and three female. The median palpable spleen size was 15 cm. A preliminary diagnosis of MCL was made, based on blood cell morphology and immunophenotype. All patients underwent splenectomy for therapeutic purposes. Studies done in blood and splenic lymphocytes revealed the following: 7/7 patients were CD19/CD5, CD20 and CD38 positive; CD10 negative and 6/7 CD23 negative. The adhesion molecule expression pattern was consistent in all patients: L-Selectin and CD11c were negative, CD11alpha and CD18 weakly positive and CD54 strongly positive. The median spleen weight was 1775 g. Histology disclosed a cytologic and architectural pattern consistent with MCL. Cyclin-D1 was positive in 6/6 studied patients. Bcl-1 rearrangement was found in 5/7 patients. Splenectomy was applied as the sole treatment and was beneficial in all patients, with median blood values as following: prior to splenectomy, Ht 29.5%, platelets 110 x 10(9)/l, lymphoma cells 5.0 x 10(9)/L, and at 6 months post-splenectomy, Ht 43%, platelets 311 x 10(9)/l and lymphoma cells 3.0 x 10(9)/L. Of the seven patients, two developed progressive disease 11 and 26 months post-splenectomy. The remaining five are in improving clinical and hematological condition without chemotherapy at a median follow up of 20 months. We conclude that this presentation represents a separate form of MCL which requires splenectomy. It remains to be seen whether it carries a better prognosis than classical MCL.

Identification of two subgroups of mantle cell leukemia with distinct clinical and biological features.

Mantle cell leukemia (MCLeu) has been considered as a leukemic form of mantle cell lymphoma (MCL). However, the presence of certain features rarely observed in MCL, such as transformation to prolymphocytic leukemia (PLL) or indolent clinical course, suggests that MCLeu may represent a distinct disorder. Seven cases of MCLeu with t(11;14)(q13;q32) and BCL1-IGH gene rearrangement were ascertained among 140 newly diagnosed chronic B-cell lymphoproliferative disorders with leukemic expression. Comparative genomic hybridization, FISH for specific gene loci, and immunological studies were preformed in them. In comparison with CLL, MCLeu cases had low immunological scores < or =2 with respect to B-CLL (P<0.0001). Expression of CD38 was absent in 43% of MCLeu and in 44% of B-CLL. Comparative genomic hybridization analysis identified genomic imbalances in 86% of MCLeu with a similar pattern than in MCL: gains of 3q, 8q involving MYC gene and 15q, and losses of 6q, 9p, 13q and 17p affecting P53 gene. Differently from MCL and CLL, genomic loss of 8p was frequently detected in MCLeu (83%). Although clinical presentation of MCLeu was indistinguishable from CLL, all patients but one had disease progression within three years. According to the immunologic and genomic profiles, two distinct subgroups of MCLeu were defined: one related to PLL, showing CD38-, deletion of P53, and MYC amplification and another which corresponds to a leukemic form of classical MCL, presenting with CD38+ and normal P53 and MYC status. MCLeu and MCL are closely related disorders, as they show similar genomic and molecular patterns. However, the deletion of the short arm of chromosome 8 may represent a specific marker for MCLeu. Two distinct subgroups of MCLeu may also be distinguished according to the immunologic and genomic cell profiles.

Increased proteasome degradation of cyclin-dependent kinase inhibitor p27 is associated with a decreased overall survival in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive neoplasm characterized by the deregulated expression of cyclin D1 by t(11;14). The molecular mechanisms responsible for MCL's clinical behavior remain unclear. The authors have investigated the expression of p53, E2F-1, and the CDK inhibitors p27 and p21 in 110 MCLs, relating their expression to proliferative activity (Ki-67). For comparison, they have similarly analyzed low-grade (12 MALT, 16 CLL/SLL) and high-grade (19 DLCL) lymphomas. p53 was detected more frequently in large-cell MCL (l-MCL; 5 of 7) than in classical MCL (s-MCL; 13 of 103) and DLCL (8 of 19). In MCL and DLCL, the percentage of E2F-1+ nuclei was high, correlating with high Ki-67 expression. Most MCLs (91 of 112) and DLCLs (12 of 19) showed a loss of p27; MALT and CLL/SLL, however, were p27 positive. Reverse transcription-polymerase chain reaction and in vitro protein degradation assays demonstrated that MCLs have normal p27 mRNA expression but increased p27 protein degradation activity via the proteasome pathway. Correlation of MCL p53 and p27 expression with clinical data showed an association between reduced overall survival rates and the overexpression of p53 (P =.001), the loss of p27 (P =. 002), or both. Loss of p27 identified patients with a worse clinical outcome among p53 negative cases (P =.002). These findings demonstrated that MCL has a distinct cell cycle protein expression similar to that of high-grade lymphoma. The loss of p27 and the overexpression of p53 in MCL are prognostic markers that identify patients at high risk. The demonstration that low levels of p27 in MCL result from enhanced proteasome-mediated degradation should encourage additional clinical trials. (Blood. 2000;95:619-626) (Blood. 2000;95:619-626)

Expression of Cell Cycle Regulating Proteins in an Unusual Transformation of Mantle Cell Lymphoma

We describe here two patients with mantle cell lymphoma (MCL) who after a few years, developed to the diffuse large cell lymphoma (DLCL)(anaplastic centrocytic lymphoma) growing in a diffuse sheets without the classical MCL component. In both the initial and second biopsy specimens, in each case, tumor cells were positive for cyclin D1, sIgM, sIgD, and CD5, but were negative for CDlO and CD23. In a study of immunoglobulin heavy chain (IgH) gene rearrangement, using the polymerase chain reaction (PCR) method, the products obtained from each paired biopsy tissue sample were the same size, and in one case had an identical sequence to the non-mutated VH gene.Immunohistochemistry was used to examine the expression of p53, p27kipl and cyclin E. Interestingly, there was clear overexpression of p53 protein in case 1 but not in case 2, compared with other typical MCL cases. The expression of p27kipi in the second biopsies of each case was decreased compared with those in the initial biopsies. In case 2, however, p27kipi was clearly expressed in the first and second biopsies, in contrast to other typical MCL cases.Thus these 2 cases demonstrate not only that the variant form of MCL may arise de novo, but also that MCL may transform to DLCL at the time of relapse. Although the mechanism of tumor rogressiodtransforrnation is still poorly understood, the overexpression of p53 or p27kipi may be linked to a cellular mechanism involved in the development of the variant form of MCL.

Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas.

Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.

Blastic variant of mantle cell lymphoma shows a heterogenous pattern of somatic mutations of the rearranged immunoglobulin heavy chain variable genes.

Mantle cell lymphoma is a distinct clinicopathological entity associated with t(11;14) and cyclin D1 overexpression. The majority of cases show uniform morphological and phenotypic features characterized by a monotonous proliferation of small-to-medium-sized irregular B cells that express CD5 and bright surface immunoglobulin IgM and IgD. By sequence analysis of the rearranged immunoglobulin heavy chain variable genes (VH), it has been shown that these lymphoma cells carry little if no somatic mutations, as described for the fetal CD5+ cells or B1 cells. Besides mantle cell lymphoma with classic histological features, a morphological variant of mantle cell lymphoma with blastic features and a more aggressive clinical course has been described. To investigate whether this variant is closely related, by the cell of origin, to typical cases, we analysed the presence and the pattern of somatic mutations of the VH genes in a series of nine cases diagnosed as such. Our cases of blastic mantle cell lymphomas rearrange most frequently VH4 and VH3 family genes. In three cases there was a complete homology to published germline genes, and a near complete homology was documented in another three. In contrast, the remaining three cases showed somatic mutations in their rearranged VH genes. Mutation analysis revealed evidence for antigen selection in one of these three cases. Taken together, these data are similar to those of normal adult-type B1 cells and those described for chronic lymphocytic leukaemia (CLL) but slightly different to those reported for classic mantle cell lymphoma. It is likely that blastic mantle cell lymphoma as well as CLL originates from adult-type B1 cells. More cases will need to be studied to determine whether classic mantle cell lymphoma is different from the blastic subtype and if it arises from fetal-type B1 cells.

Large-cell variants of mantle cell lymphoma: cytologic characteristics and p53 anomalies may predict poor outcome.

Large-cell variants are uncommon in mantle cell lymphoma (MCL). Here we describe the pathologic and clinical findings in five patients with large-cell lymphoma related to MCL (L-MCL), and compare them to a group of classic small-cell MCL (s-MCL) cases. Histologically, the MC origin of the large cells was evinced by their association with a small mantle cell component in the same tissue, or their distribution in a classic mantle zone pattern, or their development in a patient with previous s-MCL. The large cells were either pleomorphic mantle cells (case 1) or transformed blast-like cells (case 2-5). The median nuclear diameter, median nuclear area and proliferation index of L-MCLs and s-MCLs, were statistically different. Immunophenotypic characterization of four specimens of L-MCL and 10 of s-MCLs with a large panel of antibodies showed the classic findings of MCL, i.e. the IgM+ D+/-, CD5+, CD10-, CD23- phenotype in all cases except two (one CD5- and one CD23+), and the association with a loose follicular dendritic cell network. Two of four L-MCLs and 5/10 s-MCLs demonstrated rearrangements of the bcl-1 gene by Southern blot or by polymerase chain reaction (PCR); 2/4 L-MCLs and 1/9 s-MCLs had p53 mutations on single-strand conformation polymorphism analysis; none of the 14 specimens showed rearrangement of bcl-2 by PCR or bcl-6 and c-myc by Southern blot. All patients with 'transformed' histology (versus 37% of all others) died of lymphoma; their survival (15-18 months; median 17) was much shorter than that of all the others (28-117+ months; median 43) (P=0.0035). All three patients with p53 anomalies, two of whom had tumours with transformed histology, died of their disease in a short time (15, 18 and 28 months). In contrast, the presence of bcl-1 rearrangements did not have prognostic implications. This study documents the existence of large-cell variants of MCL and the poor prognosis associated with the 'transformed' cytologic type and/or p53 abnormalities.