Multiple Myeloma Oncogene Research Articles

MTDH is an oncogene in multiple myeloma, which is suppressed by Bortezomib treatment.

Metadherin (MTDH) is identified as an oncogene in multiple cancers including breast cancer, bladder cancer and endometrial cancer. However, the function of MTDH in multiple myeloma (MM) is still unexplored. In this study, we disclose that MTDH is an oncogene in MM. This is characterized by an elevation mRNA level of MTDH and chromosomal gain of MTDH locus in MM cells compared to normal samples. Moreover, MTDH expression significantly increased in MMSET translocation (MS) subgroup, one of the high-risk subgroups in MM, and was significantly correlated with MM patients' poor outcomes in Total Therapy 2 (TT2) cohort. Further knockdown of MTDH expression by shRNA in MM cells induced cell apoptosis, inhibited MM cells growth in vitro and decreased xenograft tumor formation in vivo. Interestingly, opposite to TT2, MM patients with high-MTDH expression showed favorable survival outcomes in the TT3 cohort, while Bortezomib treatment was the major difference between TT2 and TT3 cohort. Furthermore we proved that Bortezomib suppressed pre- and post-transcription levels of MTDH expression of MM cells in vitro and in vivo. Finally, our studies demonstrated that MTDH is a transcriptional gene of MMSET/NFκB /MYC signaling in MM cells, which is inhibited by Bortezomib treatment.

Prognostic impact of B-cell lymphoma 6 in primary CNS lymphoma.

We investigated the prognostic significance of B-cell differentiation status and common B-cell differentiation markers in a post hoc analysis of 119 patients with primary CNS lymphoma (PCNSL) homogeneously receiving high-dose methotrexate (HDMTX)-based chemotherapy within the prospective G-PCNSL-SG1 trial. We evaluated protein expression of B-cell lymphoma 2 (BCL2), BCL6, CD10, and multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) by immunohistochemistry and analyzed the association with survival. The median follow-up of all patients was 67.5 months. Median progression-free survival (PFS) was 10.61 months (95% CI: 4.23-17.00). Median overall survival (OS) was 28.85 months (95% CI: 17.96-39.73). Eighty-nine tumors expressed BCL2 (92.7%), 24 (20.5%) expressed CD10, 60 (54.1%) expressed BCL6, and 87 (79.0%) expressed MUM1/IRF4. On the basis of the Hans algorithm, 80 tumors (73.4%) were classified to the non-germinal center B group, suggesting a post-germinal center origin of PCNSL. Expression of BCL6 (cutoff point 30%), but none of the other markers, was associated with shorter PFS (P = .047) and OS (P = .035). On multivariate analysis, BCL6 expression was associated with shorter PFS (hazard ratio: 1.95, 95% CI: 1.22-3.12, P = .005) but not OS (hazard ratio: 1.85, 95% CI: 0.71-4.80, P = .21). Classification according to Hans algorithm and expression status of the single B-cell markers BCL2, CD10, and MUM1/IRF4 did not correlate with prognosis. The findings are limited by the fact that only 23% of all G-PCNSL-SG1 patients could be included in the analysis. If validated in an independent cohort, BCL6 may assume clinical relevance as an unfavorable prognostic biomarker in PCNSL.

Evaluation of BCL6 and MUM1 Expression in Patients with Diffuse Large B Cell Lymphoma (DLBCL) and Their Correlation with Staging and Prognosis of the Disease

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common type of Non-Hodgkin Lymphoma (NHL) which includes about (25%) of the cases. Patients with DLBCL have a rapidly growing mass that is located in the neck or abdominal region. Multiple Myeloma Oncogene (MUM1) has an important role in lymphoid cell differentiation. MUM1/IRF4 is as oncogene related to myeloma and translocation (p25; q32), t (6; 14) and leads to a change in the heavy chain of Immunoglobulin (IGH). BCL6 exists in approximately (100%) of follicular cell cancer cases, (100%) of Burkitt lymphoma, (80%) of large B-cell lymphoma, and in more than (80%) of nodular lymphocytic Hodgkin lymphoma cases. BCL6 is related to cancers with a high proliferation cell index. In some studies BCL6 is expressed as a prognostic factor in LBCL patients

ALK-positive diffuse large B-cell lymphoma of the duodenum: A case report and review of the literature.

Anaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (ALK+ DLBCL) is characterized by the presence of immunoblastic or plasmablastic cells with a strong ALK protein expression that is frequently associated with t(2;17)(p23;q23). The present study reports a case of ALK+ DLBCL in a 26-year-old male with a duodenal mass. Histologically, the neoplastic cells demonstrated prominent plasmablastic differentiation with abundant amphophilic cytoplasma and central nucleoli. Paraffin immunohistochemistry revealed: an exclusively cytoplasmic granular expression of ALK; CD138, immunoglobulin A (IgA) and CD79α positivity; and focal expression of multiple myeloma oncogene 1 (Mum-1), CD30 and epithelial membrane antigen (EMA). However, the immunohistochemical staining was negative for CD3, CD38 and CD20. Fluorescence in situ hybridization (FISH) analysis using an ALK break-apart probe revealed the presence of ALK gene rearrangements in the patient. To the best of our knowledge, the current case represents the first example of primary extranodal ALK+ DLBCL presenting as a duodenal mass.

A Rare Case of Acute Leukemic Presentation of Blastic Plasmacytoid Dendritic Cell Neoplasm without Cutaneous Lesions

A Rare Case of Acute Leukemic Presentation of Blastic Plasmacytoid Dendritic Cell Neoplasm without Cutaneous Lesions

Epstein-Barr Virus-Positive Primary Cutaneous Diffuse Large B-Cell Lymphoma in an Immunocompetent Patient

Dear Editor: Epstein-Barr virus (EBV) is known to induce a number of malignancies, including diffuse large B-cell lymphoma (DLBCL) in immunosuppressed patients. Recently, however, age-related immune impairment was recognized as a predisposing factor in the development of EBV-driven lymphoproliferative processes in elderly patients1. Herein, we present a case of EBV-positive primary cutaneous DLBCL in an immunocompetent patient. A 66-year-old woman presented with a 6-month history of multiple erythematous macules and patches on her trunk and both extremities (Fig. 1). There was no specific past history except for hypertension. A skin biopsy was performed and histological examination of the specimen revealed multifocal and nodular infiltrates of atypical medium- and large-sized mononuclear cells with enlarged nuclei showing one or more prominent nucleoli (Fig. 2A, B). Immunohistochemical examination revealed that the tumor cells were positive for CD20, bcl-2 and latent membrane protein 1 (LMP1), but negative for CD3, CD5, CD10, CD30, and bcl-6. In situ hybridization for EBV-encoded RNA (EBER) was positive in the majority of tumor cells (Fig. 2C). Bone marrow biopsy revealed no lymphomatous involvement. Based on these findings, the patient was diagnosed with EBV-positive primary cutaneous DLBCL. She was treated with combination chemotherapy (R-CHOP) and reached complete remission. After a year, however, the disease recurred. She was treated with radiotherapy, but the disease progressed. Fig. 1 Multiple erythematous macules and patches were observed on her trunk (A) and both extremities (B). Fig. 2 (A, B) Skin biopsy revealed multifocal and nodular infiltrates of atypical medium- and large-sized mononuclear cells with enlarged nuclei showing one or more prominent nucleoli (HE A: ×40, B: ×400). (C) In situ hybridization ... Primary cutaneous DLBCL is a malignant proliferation of large B cells, presenting primarily in the skin with solitary or clustered erythematous or reddish-brown tumors. Histologically, dense diffuse infiltrates characterized by predominance of large cells with round nuclei are observed. Tumor cells express B-cell markers (CD20, CD79a). Most of the cases are positive for bcl-2 protein and multiple myeloma oncogene-1 (MUM-1), but are negative for CD10. EBV-related primary cutaneous DLBCL in non-immunocompromised patients is extremely rare. So far, only 11 cases have been reported1. All of the cases in the literature were of DLBCL except for 1 case. All of the cases of DLBCL occurred in elderly patients (over 55 years), presenting more frequently with multiple lesions at various locations. Many cases of DLBCL required combination chemotherapy, although one case underwent spontaneous remission1. Immunohistochemistry and in situ hybridization are the most important tests for the diagnosis of EBV-positive DLBCL. The tumor cells are usually positive for CD45 as well as for B-cell markers such as CD20, CD19, CD79a, and PAX-5. The cases are usually negative for germinal center markers, CD10 and bcl-6, and frequently positive for MUM-1. EBV-associated latent antigens such as LMP1 and EBNA-2 are positive in 94% and 28% of cases, respectively. Moreover, up to 50% of cases express CD30 but are negative for CD15. EBER in situ hybridization is the most reliable method for the diagnosis of EBV-positive DLBCL. It has the highest diagnostic sensitivity in the diagnosis of EBV-positive DLBCL2. So far, the prognosis of the newly recognized subtype of DLBCL remains in doubt, and there is no uniformly accepted treatment due to the small number of reported cases. On the basis of several literatures, EBV-positive DLBCLs seem to have worse response to chemotherapy than EBV-negative DLBCLs3-5. However, the response to rituximab-containing regimens has not yet been properly evaluated, and further studies are needed to clarify the role of rituximab and EBV-targeted therapy in EBV-positive DLBCL2. The proportion of EBV-related DLBCL cases in the elderly may be higher than that estimated at present. Therefore, we propose that EBV expression should be assessed in primary cutaneous DLBCLs. More information needs to be obtained regarding this new entity and further investigation is necessary.

Selective Inhibition of Tumor Oncogenes by Disruption of Super-Enhancers

Chromatin regulators have become attractive targets for cancer therapy, but it is unclear why inhibition of these ubiquitous regulators should have gene-specific effects in tumor cells. Here, we investigate how inhibition of the widely expressed transcriptional coactivator BRD4 leads to selective inhibition of the MYC oncogene in multiple myeloma (MM). BRD4 and Mediator were found to co-occupy thousands of enhancers associated with active genes. They also co-occupied a small set of exceptionally large super-enhancers associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impacted genes with super-enhancers, including MYC. Super-enhancers were found at key oncogenic drivers in many other tumor cells. These observations have implications for the discovery of cancer therapeutics directed at components of super-enhancers in diverse tumor types.

MLL-HOXA9 and Calcineurin Are Novel Therapeutic Targets in Multiple Myeloma

Abstract 4007Multiple myeloma is one of incurable hematological malignancies and many novel drugs including histone deacetylase (HDAC) inhibitors are currently undergoing preclinical and clinical evaluation. Treatment of U266 and KMS-11, multiple myeloma cell lines, by LBH589, an HDAC inhibitor, inhibited proliferation and induced apoptosis of these cell lines at low nanomolar concentrations. Here, we discovered that expression of HOXA9, a homeobox protein, was suppressed in the presence of LBH589. Transcription of HOXA9 mRNA immediately decreased in LBH589-treated myeloma cell lines. HOXA9 is a candidate oncogene in multiple myeloma and knockdown of HOXA9 is shown to block proliferation of myeloma cell lines. Our results suggest that HOXA9 is one of the targets of anti-myeloma effects elicited by HDAC inhibitors. MLL (mixed-lineage leukemia), a trithorax group protein, is shown to be essential for persistent expression of HOXA9 in human leukemia cells. We examined the effect of LBH589 on MLL in myeloma cell lines and found that expression of MLL protein was suppressed without decrease of MLL mRNA. These results indicate that LBH589 induces degradation of MLL protein. In the previous studies, it was shown that HDAC inhibitors targeted heat shock protein 90 (HSP90). The molecular chaperone HSP90 is essential for the protein-folding ability of several proteins. We evaluated the expression of MLL in myeloma cell lines treated by HSP90 inhibitor. We found that the expression of MLL proteins was suppressed by the treatment of 17-AAG, an inhibitor of HSP90. These results suggest that HDAC inhibitors induce degradation of MLL proteins via inhibition of chaperone function of HSP90. This inhibition of MLL-HOXA9 by HDAC inhibitors are supposed to block proliferation of myeloma cells.Furthermore, we tried to find a cooperative factor of MLL to investigate the roles of MLL in pathophysiology of multiple myeloma. For this purpose, we picked up PPP3CA, catalytic subunit of calcineurin, as one of the molecules. Those were highly co-expressed with MLL in multiple myeloma patients. We revealed that PPP3CA was degraded by the treatment of LBH 589 or 17-AAG. These results suggest that PPP3CA is protected from protein degradation by HSP90 as in the case of MLL and that LBH589 induces degradation of PPP3CA through inhibition of chaperone function of HSP90. We also found that co-treatment of myeloma cell lines by LBH589 and FK506 showed more anti-proliferative effect than LBH589 alone. FK506 selectively inhibits the function of calcineurin B, regulatory subunit of calcineurin. Expression of PPP3CB, the other isozyme of catalytic subunit of calcineurin, was upregulated when myeloma cell lines were treated with LBH589 and this upregulation of PPP3CB was supposed to be the result of compensation for downregulation of PPP3CA. It is suggested that combination of FK506 with LBH589 should display enhanced anti-myeloma effects by inhibiting the interaction between upregulated PPP3CB and calcineurin B. These results indicate that calcineurin-signaling pathway plays an important role in the persistent proliferation of myeloma cells. Surprisingly, bortezomib also suppressed expression of PPP3CA via inhibition of HDAC6.Our study is the first report to demonstrate that MLL-HOXA9 and calcineurin are the important targets of HDAC inhibitors in the treatment for multiple myeloma. MLL and PPP3CA, catalytic subunit of calcineurin, are highly co-expressed in multiple myeloma patients. These results suggest that HDAC inhibitors including LBH589 display anti-myeloma effects by inhibiting both MLL-HOXA9 and calcineurin pathways. These findings will lead to understanding of a novel mechanism of survival and growth of myeloma cells and be helpful to establish a new strategy of therapy for multiple myeloma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Multicentric Castleman Disease with Complete Duodenal Obstruction in an HIV Patient

Abstract 4834Multicentric Castleman's Disease(MCD) can have an aggressive course making early diagnosis and treatment important. Although rare, peripancreatic involvement is known in Castleman's disease and an unusual presentation should be considered especially in the setting of HIV. An excisional lymph node biopsy is required for diagnosis. High plasma titers of Human Herpes Virus 8 DNA should also raise this possibility. Coexistence of Castleman disease and body cavity lymphoma has been reported but is rare. Case Presentation:A 42 year old African American gentleman with a history of HIV and CD4 count 271 presented with intractable nausea and vomiting of 1 month duration; associated with epigastric pain and weight loss. Past history was significant for presentation with jaundice 6 months earlier when he was found to have a pancreatic head mass with portocaval and retroperitoneal lymphadenopathy raising the suspicion of pancreatic adenocarcinoma. Endoscopic ultrasound guided fine needle aspiration of the mass had revealed a poorly differentiated malignant neoplasm that could not be further characterized by immunohistochemistry. Considering the HIV status of the patient, there was a strong suspicion of lymphoma. A PET-CT was planned to identify an appropriate site for tissue diagnosis but the patient was lost to follow up. Repeat imaging during the current admission revealed increasing size of the pancreatic mass causing complete obstruction of second part of duodenum. The patient subsequently underwent gastro-jejunostomy to bypass the duodenal obstruction and an excisional biopsy of two mesenteric lymph nodes was performed during the procedure. Pathology revealed HHV-8 positive plasma cell variant of Castleman's disease. Patient also had high plasma titers of HHV-8. Furthermore, the patient was also found to have bilateral pleural effusions and a thoracentesis was performed. Pleural cytology revealed large plasmablastic lymphoid cells that were positive for HHV-8, CD30, CD45, CD138, Ebstein-Barr virus (EBV), epithelial membrane antigen, and multiple myeloma oncogene-1. These findings suggested a diagnosis of body cavity lymphoma. Discussion:Although rare, the association of Multicentric Castleman's disease (MCD) with HIV is well recognized. The incidence is independent of CD4 count and the use of HAART. There are isolated case reports of pancreatic involvement with Castleman's disease and only one involving the duodenum. While most patients with peripancreatic involvement presented with abdominal discomfort, nausea or systemic symptoms, our patient was severely symptomatic from complete duodenal obstruction. Our case also highlights that the diagnosis can be elusive and requires excisional lymph node biopsy. Moreover, HIV MCD is fluorodeoxyglucose avid, and a PET-CT may help identify the gland to biopsy next in difficult to diagnose cases. Body cavity lymphoma is also HHV-8 related and this diagnosis should be considered in any HIV patient with effusions. Pleural fluid cytology and immunohistochemistry usually clinch the diagnosis. Disclosures:No relevant conflicts of interest to declare.

A Rare Case of Multicentric Extracavitary Solid Primary Effusion Lymphoma (ECPEL) Diagnosed by Upper Endoscopy and Colonoscopy

Purpose: A 52-year-old male with HIV/AIDS on HAART (non-compliant) was admitted for hypotension, anemia, weight loss and malaise. Laboratory findings: CD4 cells 3% (30% to 61%); CD8+cells 78% (12% to 42%); CD4:CD8 ratio 0.04 (0.86 to 5.0); white blood cell count 9.7 (4.8 to 10.8); hemoglobin 3.4 g (11.0 to 15.6 g). Total bilirubin 6.4 mg (0.2 to 1.2 mg/dl), direct bilirubin 4.9 (0.0 to 0.5 mg/dl), lactate dehydrogenase 960 (125 to 243 U/L). Imaging Studies: CT scan of chest, abdomen and pelvis showed several regions of soft tissue prominence in the pancreatic head, upper retro peritoneum and ascending colon. Bone marrow biopsy: Large granular lymphocytes that could be reactive or neoplastic. Immunohistochemistry showed MUM1 (multiple myeloma oncogene 1) positive and negative for CD20, CD3, CD30, CD15, CD79a, PAX-5, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). EGD and colonoscopy were performed for anemia and abnormal findings on CT scan. EGD showed 3 friable nodules, the largest measuring 2x3 cm in the body of the stomach. The nodules bled on contact and were biopsied (Figure 1). Colonoscopy showed a large friable mass seen in the transverse colon and was biopsied. Histopathology of gastric nodules and colonic mass were consistent with solid Primary Effusion Lymphoma. The neoplastic cells were positive for Epstein Barr (EBV) and Kaposi's Sarcoma Virus (KSHV/HHV8). PEL is seen in body cavities such as the pleural space, pericardium and peritoneum. Extracavitary Solid Primary Effusion Lymphoma (ECPEL) is a rare occurrence and is often associated with Kaposi's sarcoma or multicentric Castleman's disease. It is seen in less than 1% of HIV/AIDS related lymphomas and is also reported rarely after solid organ transplantation. Detection of HHV8 in the neoplastic cells is the only confirmatory diagnostic test in PEL and ECPEL. This case represents an even rarer condition with ECPEL, involving multiple sites, and readily diagnosed by upper endoscopy and colonoscopy.Figure: [1338] Nodule in the body of stomach.

Primary plasmacytoma of the thyroid gland: a case report of a rare neoplasia

Extramedullary plasmacytoma comprises 3%-5% of all plasma cell neoplasms, and approximately 80% of the cases occur in the upper respiratory tract. Primary thyroid plasmacytomas (PTP) are rare tumors. The authors report a case of PTP in a male patient with dyspnea and dysphagia. Physical examination and computerized tomography (CT) scan revealed a solid tumor affecting the thyroid gland, measuring 12 cm in its greatest dimension. Surgical biopsy was performed. Microscopy revealed a hypercellular malignant neoplasm composed of round plasmacytoid cells arranged in solid nests, which showed a positive immunoexpression for CD138, epithelial membrane antigen (AME), kappa light chains and multiple myeloma oncogene 1 (MUM1). The diagnosis of PTP was accordingly established.

Pediatric Anaplastic Lymphoma Kinase–Positive Large B-cell Lymphoma: A Case Report and Review of the Literature

We present a case of pediatric anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK-positive LBCL) with a cervical, mesentery, and pelvis cavity mass. Histologic examination of the cervical mass revealed that the lesion was composed of diffuse large immunoblastic-like or plasmablastic-like tumor cells with a sinusoidal growth pattern. The tumor cells were strongly immunoreactive for ALK; revealed a granular cytoplasmic distribution; and were diffusely positive for CD45, CD4, CD138, epithelial membrane antigen, and multiple myeloma oncogene-1 but negative for CD20 and CD79a. The patient underwent 5 courses of cyclophosphamide, doxorubicin, vincristine, prednisone, and etoposid and obtained a remarkable clinical response with regression of mesentery and pelvis cavity mass. We suggest that this distinct subtype of large B-cell lymphoma should belong to the spectrum of pediatric lymphomas and that radiologic examination should be performed to inspect the progression of disease even if the patients experienced complete remission at initial chemotherapy.

The Irf4 Gene, a Susceptibility Locus for Chronic Lymphocytic Leukemia (CLL), Controls Establishment of Follicular and Marginal Zone B Cell Compartments in Mice

Abstract 285Chronic lymphocytic leukemia (CLL) originates from the malignant transformation of mature B cells. Recently, single nucleotide polymorphisms (SNPs) in the 3'UTR region of the Irf4 gene have been associated with an increased risk of developing CLL in independent patient cohorts. IRF4 is a member of the interferon regulatory factor (IRF) family of transcription factors, and in the B lineage is essential for plasma cell differentiation in T-dependent immune responses. Irf4 has been demonstrated to act as an oncogene in multiple myeloma. Conversely, evidence suggests that the corresponding SNPs in the Irf4 gene lead to a reduction of IRF4 mRNA expression, and it was reported that tumor cells in half of CLL cases show reduced IRF4 protein expression compared to normal B cells. Together, these observations suggest that aberrant downregulation of IRF4 expression in mature B cells may contribute to CLL development. However, the normal function of IRF4 in mature B cells is incompletely understood. In order to investigate how IRF4 deficiency affects the biology of mature B cells, we investigated the consequences of deleting Irf4 specifically in B cells in vivo using a conditional Irf4 knockout mouse line.We and others had previously observed that Irf4−/− mice develop an expansion of B cells with a marginal zone (MZ) phenotype (IgMhiIgDloCD23–CD21+). By demarcating the MZ with the MOMA-1 marker, we here show that B cells in Irf4−/− mice localized preferentially in the MZ area, causing MZ hyperplasia. In contrast, the area where follicular B cells normally home contained few B cells. B cell autonomy of the observed phenotype was ascertained by crossing a “floxed” Irf4 allele with CD19-Cre mice to achieve B cell conditional deletion. We then crossed the floxed Irf4 allele with a transgenic mouse that allows inducible deletion of Irf4 specifically in B cells. Whereas flow-cytometric analysis revealed an unchanged ratio between cells with a follicular (IgMloIgDhiCD23+CD21int) vs. a MZ B cell surface phenotype upon Irf4 deletion, immunohistochemical (IHC) stainings of spleen sections for a marker protein (eGFP) that signals gene deletion demonstrated that the Irf4-deleted cells localized preferentially in the MZ, leading to MZ hyperplasia. Together, these results suggest that deletion of Irf4 in B cells alters chemokine responsiveness and migratory capacity. In agreement, global gene expression profile analysis of B cells purified from Irf4−/− and Irf4+/+ mice identified a set of differentially expressed genes with known functions in cell migration and homing. Notably, PLXND1 and the chemokine receptor CXCR7 showed 4.5 and 6.5-fold upregulation, respectively, while the G protein coupled receptor RGS13 and the adhesion molecule ALCAM (CD166) showed 3.5 and 5-fold downregulation in Irf4−/− vs. Irf4+/+ B cells.Unexpectedly, we observed in the profiling analysis that expression of the known NOTCH target genes Deltex1 and Hes5 was significantly upregulated (3 and 5-fold) in Irf4−/− vs. Irf4+/+ B cells. Despite unchanged NOTCH1 and NOTCH2 mRNA levels, Western blot and immunofluorescence analysis showed that Notch2, a gene known to be indispensible for MZ B cell development, was strongly upregulated in Irf4−/− B cells, suggesting that IRF4 is indirectly involved in NOTCH2 repression at a post-transcriptional level. Together with the altered migratory properties of Irf4−/− vs. Irf4+/+ B cells, these findings indicate that a balanced expression of IRF4 and NOTCH2 in B cells is required for establishing the follicular and MZ B cell compartments in mice, and suggest that IRF4 maintains the cellular identity of follicular B cells.The results may have implications for understanding CLL pathogenesis, as both NOTCH1 and NOTCH2 transmembrane receptors were reported to be expressed and activated in CLL B cells, and since NOTCH1 was recently found to be aberrantly activated in a fraction of CLL cases due to genetic mutations. Alterations in the balance of the transcriptional network established by NOTCH and IRF4 either through mutations, polymorphisms, or microenvironmental factors may disrupt normal B cell physiology and thereby contribute to tumorigenesis by an as yet unknown mechanism. Recently, a small fraction of CLL patients were identified that have a recurrent heterozygous somatic mutation in exon 2 of Irf4, providing additional rationale for determining how alterations in IRF4 function may promote CLL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

2.15 The Irf4 Gene, a Susceptibility Locus for Chronic Lymphocytic Leukemia, Controls Establishment of Follicular and Marginal Zone B-Cell Compartments in Mice

Abstract 285 Chronic lymphocytic leukemia (CLL) originates from the malignant transformation of mature B cells. Recently, single nucleotide polymorphisms (SNPs) in the 39UTR region of the Irf4 gene have been associated with an increased risk of developing CLL in independent patient cohorts. IRF4 is a member of the interferon regulatory factor (IRF) family of transcription factors, and in the B lineage is essential for plasma cell differentiation in T-dependent immune responses. Irf4 has been demonstrated to act as an oncogene in multiple myeloma. Conversely, evidence suggests that the corresponding SNPs in the Irf4 gene lead to a reduction of IRF4 mRNA expression, and it was reported that tumor cells in half of CLL cases show reduced IRF4 protein expression compared to normal B cells. Together, these observations suggest that aberrant downregulation of IRF4 expression in mature B cells may contribute to CLL development. However, the normal function of IRF4 in mature B cells is incompletely understood. In order to investigate how IRF4 deficiency affects the biology of mature B cells, we investigated the consequences of deleting Irf4 specifically in B cells in vivo using a conditional Irf4 knockout mouse line. We and others had previously observed that Irf4 −/− mice develop an expansion of B cells with a marginal zone (MZ) phenotype (IgM hi IgD lo CD23 – CD21 + ). By demarcating the MZ with the MOMA-1 marker, we here show that B cells in Irf4 −/− mice localized preferentially in the MZ area, causing MZ hyperplasia. In contrast, the area where follicular B cells normally home contained few B cells. B cell autonomy of the observed phenotype was ascertained by crossing a “floxed” Irf4 allele with CD19-Cre mice to achieve B cell conditional deletion. We then crossed the floxed Irf4 allele with a transgenic mouse that allows inducible deletion of Irf4 specifically in B cells. Whereas flow-cytometric analysis revealed an unchanged ratio between cells with a follicular (IgM lo IgD hi CD23 + CD21 int ) vs. a MZ B cell surface phenotype upon Irf4 deletion, immunohistochemical (IHC) stainings of spleen sections for a marker protein (eGFP) that signals gene deletion demonstrated that the Irf4 -deleted cells localized preferentially in the MZ, leading to MZ hyperplasia. Together, these results suggest that deletion of Irf4 in B cells alters chemokine responsiveness and migratory capacity. In agreement, global gene expression profile analysis of B cells purified from Irf4 −/− and Irf4 +/+ mice identified a set of differentially expressed genes with known functions in cell migration and homing. Notably, PLXND1 and the chemokine receptor CXCR7 showed 4.5 and 6.5-fold upregulation, respectively, while the G protein coupled receptor RGS13 and the adhesion molecule ALCAM (CD166) showed 3.5 and 5-fold downregulation in Irf4 −/− vs. Irf4 +/+ B cells. Unexpectedly, we observed in the profiling analysis that expression of the known NOTCH target genes Deltex1 and Hes5 was significantly upregulated (3 and 5-fold) in Irf4 −/− vs. Irf4 +/+ B cells. Despite unchanged NOTCH1 and NOTCH2 mRNA levels, Western blot and immunofluorescence analysis showed that Notch2 , a gene known to be indispensible for MZ B cell development, was strongly upregulated in Irf4 −/− B cells, suggesting that IRF4 is indirectly involved in NOTCH2 repression at a post-transcriptional level. Together with the altered migratory properties of Irf4 −/− vs. Irf4 +/+ B cells, these findings indicate that a balanced expression of IRF4 and NOTCH2 in B cells is required for establishing the follicular and MZ B cell compartments in mice, and suggest that IRF4 maintains the cellular identity of follicular B cells. The results may have implications for understanding CLL pathogenesis, as both NOTCH1 and NOTCH2 transmembrane receptors were reported to be expressed and activated in CLL B cells, and since NOTCH1 was recently found to be aberrantly activated in a fraction of CLL cases due to genetic mutations. Alterations in the balance of the transcriptional network established by NOTCH and IRF4 either through mutations, polymorphisms, or microenvironmental factors may disrupt normal B cell physiology and thereby contribute to tumorigenesis by an as yet unknown mechanism. Recently, a small fraction of CLL patients were identified that have a recurrent heterozygous somatic mutation in exon 2 of Irf4 , providing additional rationale for determining how alterations in IRF4 function may promote CLL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma within a cavernous hemangioma of the cavernous sinus

Cavernous angiomas or hemangiomas and malignant lymphomas rarely involve the cavernous sinus. We report the case of a 72-year-old man with right circumorbital pain and right oculomotor nerve dysfunction because of a mass in the right cavernous sinus. It was removed via a transsphenoidal approach and histological examination revealed the mass was a cavernous hemangioma containing atypical large B cells in some sinusoidal vessels; no other evidence of lymphoma was detected on (18)F-2-fluoro-2-deoxy-D-glucose positron-emission tomography or bone marrow biopsy. The patient underwent gamma knife radiosurgery (GKS) of the right cavernous sinus, but no systemic chemotherapy was administered. Although good local control was achieved, the patient developed a systemic diffuse large B-cell lymphoma (DLBCL) 1.5 years after the GKS. The atypical lymphocytes of the cavernous hemangioma and biopsied lymph nodes expressed multiple myeloma oncogene-1 protein. This is a rare case of DLBCL occurring within a cavernous sinus that was diagnosed as hemangioma of the cavernous sinus by neuroimaging, surgical findings, and rapid-freezing histological diagnosis. The case indicates a importance of surgical sampling and detailed histopathological analysis.

Expression of Interferon Regulatory Factor-4 (IRF4/MUM1) Is Associated with Inferior Overall Survival In Peripheral T-Cell Lymphoma

AbstractAbstract 140 Background:Peripheral T-cell lymphomas (PTCLs) have poor outcomes, and there is a lack of prognostic biomarkers and therapeutic targets to guide treatment. We recently proposed that the transcription factor, interferon regulatory factor-4 (IRF4, also called multiple myeloma oncogene-1 [MUM1]), might be oncogenic in PTCLs based on its expression in association with translocations between IRF4 and the T-cell receptor gene, TRA@. IRF4 is a therapeutic target in multiple myeloma, where high expression is a poor prognostic factor. In addition, at least two germline IRF4 single nucleotide polymorphisms (SNPs), rs12203592 and rs872071, are associated with IRF4 expression and disease risk and progression in various lymphoid neoplasms. However, the prognostic effects of IRF4/MUM1 expression and IRF4 SNPs in PTCLs are unknown. Methods:Forty seven newly diagnosed PTCL patients with available tissue were identified from the University of Iowa/Mayo Clinic Lymphoma SPORE Molecular Epidemiology Resource. There were 5 anaplastic large cell lymphomas, 14 cutaneous T-cell lymphomas (CTCLs), 6 cytotoxic T-cell lymphomas (cytTCLs: 4 extranodal NK/T-cell and 2 enteropathy type), and 22 PTCLs, not otherwise specified (NOS). Patients were diagnosed between September 2002 and February 2008 and systematically followed through March 2010 for overall survival (OS). Tumor cell IRF4/MUM1 expression was examined by immunohistochemistry on paraffin tissue sections (MUM1p clone; Dako). Positivity was defined as >30% of tumor cells with nuclear staining, as in previous studies. rs12203592 and rs872071 were genotyped in peripheral blood DNA. Cox regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for both IRF4/MUM1 expression and SNPs with OS. Chi-squared tests were used to assess the relationship between IRF4/MUM1 expression and IRF4 SNPs. Results:The median age at diagnosis was 60 years (range, 24–88). At a median follow-up of 62 months (range, 29–89), 22 (47%) of the patients had died. Fifteen (32%) of the 47 T-cell patients had IRF4/MUM1 positive tumors. IRF4/MUM1 positivity was associated with poorer overall survival (HR=4.3; 95% CI: 1.8–10.2; p=0.0008). This association was seen across PTCL subtypes, including PTCL, NOS (HR= 6.5; 95% CI: 1.5–27.7; p=0.01), CTCL (HR=13.4; 95% CI: 1.2–150.1; p=0.03), and cytTCL (HR=5.8; CI: 0.5–65.9; p=0.15). The minor allele (T) in SNP rs12203592 was positively associated with IRF4/MUM1 positivity in a dominant model, with IRF4/MUM1 expression in 60% of patients with CT or TT genotypes compared to 19% of patients with CC genotype (p=0.01). Patients with the CT/TT genotype at rs12203592 also had inferior overall survival (HR=3.7; 95% CI: 1.4–9.5; p=0.007). The rs872071 SNP showed no significant association with either IRF4/MUM1 expression (p=0.38) or overall survival (p=0.71). Conclusions:This study is the first to demonstrate that IRF4/MUM1 expression is poor prognostic factor in PTCLs. This association was observed across PTCL subtypes, including the most common subtype, PTCL, NOS. IRF4/MUM1 expression and poor survival in our patients also were associated with the minor allele (T) in the IRF4 SNP rs12203592; these findings are consistent with previous in vitro data showing the major allele (C) represses IRF4 promoter activity. Interestingly, rs872071 was not associated with IRF4/MUM1 expression or prognosis, similar to findings in multiple myeloma. In contrast, rs872071 is a risk and prognostic factor in chronic lymphocytic leukemia and classical Hodgkin lymphoma, diseases in which IRF4/MUM1 expression has been associated with favorable prognosis. IRF4/MUM1 expression is a poor prognostic factor in PTCLs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Giant Primary Cutaneous Spindle Cell B-Cell Lymphoma of Follicle Center Cell Origin

Spindle cell B-cell lymphoma is a rare morphologic variant of B-cell lymphoma that is generally associated with follicle center cell origin. It is typically found on the skin and presents as single nodule or plaque with a diameter of several centimeters. It is also characterized by abnormal spindle cells with elongated or spindle-shaped nuclei, and usually stained positive for Bcl-6 and negative for multiple myeloma oncogene 1 (MUM-1). In this report, we describe a giant primary cutaneous spindle cell B-cell lymphoma measured 20 cm × 25 cm, substantially larger than all the previously reported cases. Histologic examination revealed that the neoplasm was mainly located in the dermis and subcutaneous fat, and had infiltrated into striated muscles of the patient's back. The neoplasm cells contained elongated or spindle-shaped nuclei. Immunohistochemistry results demonstrated that the neoplasm cells were stained positive for CD20, CD79, and Bcl-6, negative for Bcl-2 and MUM-1, and focally positive for CD5, CD10, CD31, and CD43. These results collectively indicated that the neoplasm was of follicle center cell origin. The neoplasm was excised and the patient was still alive without systemic involvement after 4 years of follow-up.

Bcl-2 protein frequency in patients with high-risk diffuse large B-cell lymphoma.

Gene expression and immunohistochemical profiling of diffuse large B-cell lymphoma (DLBCL) have revealed important prognostic subgroups: germinal center B-cell-like (GCB-like) DLBCL and activated B cell-like (ABC-like) DLBCL. Although few reports on high-risk DLBCL are available, the prognosis for the GCB-like subgroup has been shown to be better than that of the ABC-like subgroup. The role of Bcl-2 as a predictor of survival in DLBCL cases is unclear and its expression varies between the two subgroups of DLBCL. In this study, we analyzed the frequency and prognostic impact of Bcl-2 protein expression in high-risk DLBCL cases. Retrospective cohort study among DLBCL patients treated at Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP). The prognostic impact of the expression of the proteins CD10, Bcl-6, MUM1 (multiple myeloma oncogene-1) and Bcl-2 on high-risk DLBCL cases was evaluated by means of immunohistochemistry. Seventy-three patients aged 18-60 years were evaluated for all these markers. Twenty-four cases (32.9%) were GCB-like and 49 (67.1%) were ABC-like, with no difference regarding complete remission, disease-free survival or overall survival rates. Twenty-seven patients (37%) showed Bcl-2 expression, which was the only independent factor predicting a worse prognosis for overall survival according to multivariate analysis. Bcl-2 protein was expressed in 37% of the high-risk DLBCL patients, without any difference between the ABC-like DLBCL and GCB-like DLBCL cases.

A Common Genetic Variant in the 3′UTR of IRF4/MUM1 Associates with Risk of Disease and Poor Prognosis in Chronic Lymphocytic Leukaemia.

High frequency low penetrance risk alleles for chronic lymphocytic leukaemia (CLL) have been identified at 6p25.3 (rs872071, IRF4/MUM1), 11q24.1 (rs735665, GRAMD1B), 15q23 (rs7176508), 2q37.1 (rs13397985, SP140), 2q13 (rs17483466, ACOXL) and 19q13.32 (rs11083846, PRKD2). Given a role in determining risk of disease it is plausible that these allelic variants also play a role in disease progression and overall survival. In order to test this hypothesis, polymorphic status was determined in a case series of 403 patients diagnosed with CLL recruited via 5 clinical centres in the United Kingdom. Mean follow-up time was 4447 days and 169 patients were deceased at the time of last follow-up. Kaplan-Meier survival analysis and the log-rank test were used to investigate the prognostic significance of constitutional genetic markers. Polymorphic variation at rs735665, rs7176508, rs13397985, rs17483466 and rs11083846 was not significantly association with time from diagnosis to first treatment (p>0.05, log-rank test). However, a genetic variant in the 3′UTR of the interferon regulatory factor 4 (IRF4)/multiple myeloma oncogene 1 (MUM1) gene was significantly associated with poor prognosis. Specifically, carriers of the disease-associated variant at rs872071 had a significantly shorter mean time from diagnosis to first treatment (3983 days), compared to non-carriers (7340 days)(p=0.001), although there was no significant difference in overall survival (p=0.29). The association with time to first treatment remained significant in multivariate Cox regression analysis that included age, gender, immunoglobulin heavy chain variable region mutational status and stage of disease at diagnosis in the model (p=0.021). IRF4 is expressed in germinal centre (GC) and post-GC B-cells, and is important for B-cell maturation and homeostasis. Taken together with the aetiological evidence, these data suggest that common allelic variation in IRF4 not only affects risk of CLL, but also predicts poor prognosis.

Recurrent translocations involving the IRF4 oncogene locus in peripheral T-cell lymphomas

Oncogenes involved in recurrent chromosomal translocations serve as diagnostic markers and therapeutic targets in hematopoietic tumors. In contrast to myeloid and B-cell neoplasms, translocations in peripheral T-cell lymphomas (PTCLs) are poorly understood. Here, we identified recurrent translocations involving the multiple myeloma oncogene-1/interferon regulatory factor-4 (IRF4) locus in PTCLs. IRF4 translocations exist in myeloma and some B-cell lymphomas, but have not been reported previously in PTCLs. We studied 169 PTCLs using fluorescence in situ hybridization and identified 12 cases with IRF4 translocations. Two cases with t(6;14)(p25;q11.2) had translocations between IRF4 and the T-cell receptor-alpha (TCRA) locus. Both were cytotoxic PTCLs, unspecified (PTCL-Us) involving bone marrow and skin. Eight of the remaining ten cases were cutaneous ALCLs without TCRA rearrangements (57% of cutaneous ALCLs tested). These findings identified IRF4 translocations as a novel recurrent genetic abnormality in PTCLs. Cytotoxic PTCL-Us involving bone marrow and skin and containing IRF4/TCRA translocations might represent a distinct clinicopathologic entity. Translocations involving IRF4 but not TCRA appear to occur predominantly in cutaneous ALCLs. Detecting these translocations may be useful in lymphoma diagnosis. Further, due to its involvement in translocations, MUM1/IRF4 protein may play an important biologic role in some PTCLs, and might represent a possible therapeutic target.