Patrick A. Lennon

B-cell lymphomas with MYC/8q24 rearrangements and IGH@BCL2 /t(14;18)(q32;q21): an aggressive disease with heterogeneous histology, germinal center B-cell immunophenotype and poor outcome

B-cell lymphomas with MYC/8q24 rearrangement and IGH@BCL2/t(14;18)(q32;q21), also known as double-hit or MYC/BCL2 B-cell lymphomas, are uncommon neoplasms. We report our experience with 60 cases: 52 MYC/BCL2 B-cell lymphomas and 8 tumors with extra MYC signals plus IGH@BCL2 or MYC rearrangement plus extra BCL2 signals/copies. There were 38 men and 22 women with a median age of 55 years. In all, 10 patients had antecedent/concurrent follicular lymphoma. Using the 2008 World Health Organization classification, there were 33 B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (henceforth referred to as unclassifiable, aggressive B-cell lymphoma), 23 diffuse large B-cell lymphoma, 1 follicular lymphoma grade 3B, 1 follicular lymphoma plus diffuse large B-cell lymphoma, 1 B-lymphoblastic lymphoma, and 1 composite diffuse large B-cell lymphoma with B-lymphoblastic lymphoma. Using older classification systems, the 33 unclassifiable, aggressive B-cell lymphomas most closely resembled Burkitt-like lymphoma (n=24) or atypical Burkitt lymphoma with BCL2 expression (n=9). Of 48 cases assessed, 47 (98%) had a germinal center B-cell immunophenotype. Patients were treated with standard (n=23) or more aggressive chemotherapy regimens (n=34). Adequate follow-up was available for 57 patients: 26 died and 31 were alive. For the 52 patients with MYC/BCL2 lymphoma, the median overall survival was 18.6 months. Patients with antecedent/concurrent follicular lymphoma had median overall survival of 7.8 months. Elevated serum lactate dehydrogenase level, ≥2 extranodal sites, bone marrow or central nervous system involvement, and International Prognostic Index >2 were associated with worse overall survival (P<0.05). Morphological features did not correlate with prognosis. Patients with neoplasms characterized by extra MYC signals plus IGH@BCL2 (n=6) or MYC rearrangement with extra BCL2 signals (n=2) had overall survival ranging from 1.7 to 49 months, similar to patients with MYC/BCL2 lymphomas. We conclude that MYC/BCL2 lymphomas are clinically aggressive, irrespective of their morphological appearance, with a germinal center B-cell immunophenotype. Tumors with extra MYC signals plus IGH@BCL2 or MYC rearrangement plus extra BCL2 signals, respectively, appear to behave as poorly as MYC/BCL2 lymphomas, possibly expanding the disease spectrum.

Sonic Hedgehog Signaling Pathway Is Activated in ALK-Positive Anaplastic Large Cell Lymphoma

Deregulation of the sonic hedgehog (SHH) signaling pathway has been implicated in several cancers but has not been explored in T-cell lymphomas. Here, we report that the SHH/GLI1 signaling pathway is activated in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL). We show that SHH, but not its transcriptional effector GLI1, is amplified in ALK+ ALCL tumors and cell lines, and that SHH and GLI1 proteins are highly expressed in ALK+ ALCL tumors and cell lines. We also show that inhibition of SHH/GLI1 signaling with cyclopamine-KAAD, as well as silencing GLI1 gene expression by small interfering (si)RNA, decreased cell viability and clonogenicity of ALK+ ALCL cells. Transfection of wild-type or mutant NPM-ALK into 293T cells showed that only wild-type NPM-ALK increased GLI1 protein levels and activated SHH/GLI1 signaling as shown by increase of CCND2 mRNA levels. Inhibition of ALK tyrosine kinase and phosphatidylinositol 3-kinase (PI3K)/AKT or forced expression of pAKT down-regulated or up-regulated SHH/GLI1 signaling, respectively. Inhibition of GSK3beta in 293T cells also increased protein levels of GLI1. In conclusion, the SHH/GLI1 signaling pathway is activated in ALK+ ALCL. SHH/GLI1 activation is the result of SHH gene amplification and is further mediated by NPM-ALK through activation of PI3K/AKT and stabilization of GLI1 protein. There is a positive synergistic effect between the SHH/GLI1 and PI3K/AKT pathways that contributes to the lymphomagenic effect of NPM-ALK.

Prognostic value of MYC rearrangement in cases of B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma

B‐cell lymphoma, Unclassifiable with features intermediate between diffuse large B‐cell lymphoma (DLBCL) and Burkitt lymphoma, for convenience referred to here as unclassifiable B‐cell lymphoma, is a category in the 2008 World Health Organization system used for a group of histologically aggressive neoplasms that are difficult to classify definitively. Currently, there is no established standard therapy for these neoplasms.

Splenic marginal zone lymphomas are characterized by loss of interstitial regions of chromosome 7q, 7q31.32 and 7q36.2 that include the protection of telomere 1 (POT1) and sonic hedgehog (SHH) genes

To further characterize the genotypic features of splenic (S) and nodal (N) marginal zone lymphomas (MZL) we compared eight SMZL and five NMZL by array‐based comparative genomic hybridization (aCGH). Arbitrarily, aberrations were divided into major imbalances, defined as gains or losses involving five or more contiguous genetic loci, and minor imbalances, defined as those involving four or fewer loci. SMZL, but not NMZL, demonstrated major imbalances. These included deletions involving various lengths of 7q (three cases), and 14q23q24 (one case) and gains of 9p13p21 (one case), 13q21q33 (one case) and 16p13.1 (one case). Common minor imbalances in SMZL were: loss of sonic hedgehog gene (SHH) at 7q36.2 (four cases), loss of protection of telomere 1 gene (POT1) at 7q31.32 (three cases), and gain of glioma associated oncogene 1 (GLI1) at 12q13.2 (three cases). Common minor alterations in NMZL were: loss of the fas‐associated via death domain gene (FADD) at 11q13.2 (three cases) and gain of GLI1 (five cases). In conclusion, SMZL, but not NMZL, demonstrates large genomic imbalances and frequent loss of the 7q31.32 and 7q36.2 regions involving POT1 and SHH, respectively. In NMZL, loss of FADD and gain of GLI1 are frequent events.

Distinct patterns of cytogenetic and clinical progression in chronic myeloproliferative neoplasms with or without JAK2 or MPL mutations

Chronic myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET) and primary myelofibrosis (PMF), result from interactions between initiating growth factor mutations and secondary genomic changes. Codon 617 mutation of the JAK2 kinase is found in 40-50% of ET/PMF, whereas the mutation of codon 515 in the JAK2-linked thrombopoietin receptor MPL is found in approximately 20% of JAK2-unmutated cases of ET and PMF. Using quantitative mutation assays, we compared patterns of clinical and cytogenetic progression in MPL-mutated MPN (n=21) to those with JAK2 V617F mutation (n=383) or neither mutation (n=109). Among patients with MPL mutations, ET was seen in 9 and PMF in 12. Median mutation levels in pretreatment ET samples were significantly higher for MPL-mutated cases (60%) than for JAK2-mutated cases (24%; P=0.01), as was presentation with anemia. Differential genomic changes included +9 in JAK2-mutated cases and chromosome 1 alterations in MPL-mutated ones, implicating dosage effects related to gene copy number. Decreases in the levels of MPL mutation were seen in sequential marrow samples from some patients under treatment with biologic therapies, but not in those treated with kinase inhibitors, consistent with selective response of the MPL-mutated clone similar to the responses seen in JAK2-mutated MPN.

Therapy may unmask hypoplastic myelodysplastic syndrome that mimics aplastic anemia

Rarely, patients who present with pancytopenia and are diagnosed initially with aplastic anemia (AA) subsequently develop a myelodysplastic syndrome (MDS). There has been controversy regarding whether the initial diagnosis of AA is correct or whether these patients have hypocellular MDS at the onset of pancytopenia.

Translocation (18;22)(q21;q11) in B-cell lymphomas: a report of 4 cases and review of the literature

Follicular lymphomas characteristically carry t(14;18)(q32;q21) which results in IGH-BCL-2 fusion. Variant translocations that juxtapose the BCL-2 gene with the immunoglobulin kappa (2p11) and lambda (22q11) light chain genes are rare. We report 4 cases of B-cell lymphoma/leukemia associated with t(18;22)(q21;q11). The t(18;22)(q21;q11) was the sole aberration identified by conventional cytogenetics in 2 cases. Three cases were classified as chronic lymphocytic leukemia, and one as follicular lymphoma based on morphology and immunophenotype. Fluorescence in situ hybridization analysis was performed on all 4 cases using a BCL-2 breakapart probe. The BCL-2 gene was rearranged in all cases. Immunoglobulin lambda light chain gene rearrangement was shown in 3 cases using bacterial artificial chromosome probes spanning the variable and constant clusters of the IGlambda gene. Each case was negative for MALT-1 rearrangement using a MALT-1 breakapart probe. These cases illustrate that t(18;22)(q21;q11) is more commonly observed in chronic lymphocytic leukemia and may represent either an initial or secondary genetic event.

Chronic myeloid leukemia in blast phase associated with t(3;8)(q26;q24)

We previously reported a recurrent t(3;8)(q26;q24) translocation involving EVI1 in five patients with myelodysplastic syndrome or acute myeloid leukemia. Here we report the same structural abnormality in a case of chronic myeloid leukemia in blast phase. The t(3;8)(q26;q24) occurred several months after the initial diagnosis of chronic myeloid leukemia, while the patient was being treated with a tyrosine kinase inhibitor. We confirmed rearrangement of EVI1 by fluorescence in situ hybridization assay using a dual-color break-apart probe set that spans the EVI1 region. Our findings demonstrate that, similar to other recurrent translocations involving 3q26, such as t(3;3) and t(3;21), the t(3;8)(q26;q24) is implicated not only in myelodysplastic syndrome and acute myeloid leukemia, but also in progression of chronic myeloid leukemia. These findings extend the known disease spectrum associated with this cytogenetic aberration.

Defining causative factors contributing in the activation of hedgehog signaling in diffuse large B-cell lymphoma

Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-κB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling.

Bacterial artificial chromosome array-based comparative genomic hybridization using paired formalin-fixed, paraffin-embedded and fresh frozen tissue specimens in multiple myeloma

Multiple myeloma (MM) is a neoplasm of malignant plasma cells that often harbors many chromosomal aberrations. Currently, fresh frozen tissues (FT) are considered the most reliable for molecular genetic analysis; however, formalin‐fixed, paraffin‐embedded (FFPE) tissues are easily retrievable. Compared with conventional cytogenetics, bacterial artificial chromosome (BAC) array‐comparative genomic hybridization (CGH) allows more sensitive detection of chromosomal abnormalities.