Hiroshi Inagaki

MECT1-MAML2 Fusion Transcript Defines a Favorable Subset of Mucoepidermoid Carcinoma

Purpose: Mucoepidermoid carcinoma is the most common primary malignancy of the salivary gland. Mucoepidermoid carcinoma translocated gene 1-mastermind-like gene family (MECT1-MAML2) gene fusion was identified from a recurring t(11;19)(q21;p13) translocation, which is often the sole cytogenetic alteration in this disease. This fusion transcript has been frequently detected in mucoepidermoid carcinoma and shown to be involved in the transformation of epithelial cells. However, its clinicopathologic significance remains unclear. Experimental Design: Seventy-one cases of mucoepidermoid carcinoma and 51 cases of nonmucoepidermoid carcinoma salivary gland tumors (including 26 Warthin tumor cases) were retrospectively analyzed. RNA was extracted from archival materials: histologic paraffin specimens in all cases and cytologic specimens in 10 mucoepidermoid carcinoma cases. The MECT1-MAML2 fusion transcript was detected by a reverse transcription-PCR assay, which can be applied to both histologic and cytologic specimens. The presence of the fusion transcript was correlated with relevant clinicopathologic and survival data of the mucoepidermoid carcinoma patients. Results: The MECT1-MAML2 fusion transcript was detected in 27 of the 71 (38%) mucoepidermoid carcinoma cases but not in any case of nonmucoepidermoid carcinoma tumors. The reverse transcription-PCR results showed no difference between histologic and cytologic specimens. Detection of the MECT1-MAML2 fusion transcript was associated with a less advanced clinical stage and a low-grade tumor histology. The presence of the transcript was associated with longer disease-free and overall survivals on univariate analysis and emerged as an independent prognostic factor for longer overall survival on multivariate analysis. Conclusions: The MECT1-MAML2 fusion transcript may be specific to mucoepidermoid carcinoma and associated with a distinct mucoepidermoid carcinoma subset that exhibits favorable clinicopathologic features and an indolent clinical course.

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

PurposeThere are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials.Experimental designNOD/Shi-scid, IL-2Rγnull (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors.ResultsHumanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice.ConclusionsAnti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.

API2-MALT1 Fusion Transcripts Involved in Mucosa-Associated Lymphoid Tissue Lymphoma: Multiplex RT-PCR Detection Using Formalin-Fixed Paraffin-Embedded Specimens

Malignant lymphoma of mucosa-associated lymphoid tissue (MALT) type is a distinct clinicopathological disease entity in the category of extranodal marginal zone B-cell lymphoma. Recently, we and others have shown that the API2 gene on chromosome 11 and the MALT1 gene on chromosome 18 are fused as a result of t(11;18)(q21;q21) in MALT lymphomas. Here we report a detection assay that can be used for formalin-fixed, paraffin-embedded specimens. It consists of a multiplex one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) followed by three parallel multiplex nested polymerase chain reactions. Eight variants of the fusion transcripts have been reported to date. When these variants were used as positive controls, all were successfully detected. The subsequent direct sequencing confirmed the results. Using this rapid and simple method, we could detect API2-MALT1 fusion transcripts in 5 of 15 (33%) archival cases of MALT lymphoma for a frequency comparable with those of RT-PCR assays using frozen materials. The lung was the preferential anatomical site of origin of MALT lymphomas harboring API2-MALT1 fusion. No fusion transcript was detected in any of 20 high-grade B-cell lymphomas. Our multiplex RT-PCR assay, which can be used for routinely-processed paraffin samples, should serve as a useful molecular tool for clarifying the clinicopathological significance of API2-MALT1 fusion in MALT lymphoma.

Primary cutaneous marginal zone B-cell lymphoma: A molecular and clinicopathologic study of 24 Asian cases

Cutaneous marginal zone B-cell lymphoma is a recently proposed entity and constitutes a cutaneous counterpart of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT). Borrelia burgdorferi infection has been suggested as a possible causative agent in European cutaneous cases of marginal zone B-cell lymphoma, whereas API2-MALT1 fusion and BCL10 mutation are highly associated with MALT lymphoma. Aberrant nuclear BCL10 expression may be closely correlated with API2-MALT1 fusion in gastric and pulmonary MALT lymphomas. We examined 24 Asian cases of cutaneous marginal zone B-cell lymphoma for B. burgdorferi involvement, API2-MALT1 fusion, BCL10 cellular expression, and BCL10 mutation. Neither Borrelia DNA nor API2-MALT1 fusion transcript was detected. Nuclear BCL10 expression was evident in tumor cells of 11 of 24 cases, although BCL10 mutation was found in one case only. Clinicopathologically, nuclear BCL10 was more frequently expressed in macroscopically nodular lesions than in plaques or papules (p = 0.0031). These data suggest that 1) B. burgdorferi infection may not play an important role in developing cutaneous marginal zone B-cell lymphoma in Asian cases, 2) neither API2-MALT1 fusion nor BCL10 mutation is closely associated with the pathogenesis, 3) aberrant nuclear BCL10 may frequently be expressed in the absence of these genetic abnormalities, and 4) nuclear BCL10 expression may be clinically important because it was observed in locally aggressive tumors.

Gastric MALT lymphomas are divided into three groups based on responsiveness to Helicobacter Pylori eradication and detection of API2-MALT1 fusion.

Gastric MALT lymphoma shows unique features including regression by Helicobacter pylori eradication and API2-MALT1 fusion. We performed a molecular and clinicopathologic study for 115 cases. All eradication-responsive cases were devoid of API2-MALT1 fusion. All tumors positive for the fusion and all negative for H. pylori infection were nonresponsive to the eradication. Consequently, gastric MALT lymphomas were divided into three groups: Eradication-responsive and fusion-negative (group A, n = 72), eradication-nonresponsive and fusion-negative (group B, n = 22), and eradication-nonresponsive and fusion-positive (group C, n = 21). Group A tumors were characterized by low clinical stage and superficial gastric wall involvement, and group C tumors by low H. pylori infection rate, advanced clinical stage, and nuclear BCL10 expression. All group C tumors showed exclusively low-grade histology. Group B tumors, which have not been well recognized, frequently showed nodal involvement, deep gastric wall involvement, and advanced clinical stage, and sometimes an increased large cell component. A multivariate discriminant analysis revealed that responsiveness to the eradication could be predicted accurately by negative API2-MALT1 fusion, positive H. pylori infection, low clinical stage, and superficial gastric wall invasion, the former being the most important factor for the prediction. This 3-group categorization may be helpful for a comprehensive understanding of gastric MALT lymphoma.

Primary Thymic Extranodal Marginal-Zone B-Cell Lymphoma of Mucosa-Associated Lymphoid Tissue Type Exhibits Distinctive Clinicopathological and Molecular Features

Extranodal marginal-zone B-cell lymphoma (MZBL) of mucosa-associated lymphoid tissue (MALT) arising in the thymus is rare, with the largest series in the literature including only three cases. In the present study, we investigated 15 cases of thymic MALT lymphoma to systematically characterize its clinical, histopathological, and molecular features. There was a marked female predilection (male:female = 1:4), with a mean age of 55 years at diagnosis. There was a strong association with autoimmune disease, especially Sjögrens syndrome. Histologically, the thymic lymphoma showed the characteristic morphological features of extranodal MZBL of MALT type. Cysts were common. Prominent lymphoepithelial lesions were formed by centrocyte-like cells infiltrating and expanding the Hassalls corpuscles and epithelium lining the cysts. Plasmacytic differentiation was apparent in all cases. Notably, 13 of 15 cases expressed immunoglobulin (Ig) A phenotype; IgA expression in thymic MALT lymphoma was in striking contrast with the IgM phenotype observed in most of the Sjögrens syndrome-associated MZBLs and MALT lymphomas at other sites. Epstein-Barr virus was absent, and API2-MALT1 gene fusion, a recently reported MALT lymphoma-specific gene abnormality, was not detected in any case. Although one patient died of disease 85 months after the diagnosis, other patients were alive with overall 3-year and 5-year survival rates being 89% and 83%, respectively. Among the 22 patients reported previously and in the present series, at least 17 patients (77%) were Asians. These data indicate that thymic MALT lymphoma may represent a distinct subgroup of MALT lymphoma characterized by apparent predilection for Asians, a strong association with autoimmune disease, frequent presence of cysts, consistent plasma cell differentiation, tumor cells expressing IgA phenotype, and consistent lack of API2-MALT1 gene fusion.

API2-MALT1 Fusion Defines a Distinctive Clinicopathologic Subtype in Pulmonary Extranodal Marginal Zone B-Cell Lymphoma of Mucosa-Associated Lymphoid Tissue

t(11;18)(q21;q21) is associated with mucosa-associated lymphoid tissue (MALT)-type lymphoma and results in API2-MALT1 fusion. However, its clinicopathologic significance remains unclarified. API2-MALT1 fusion is detected most frequently in MALT lymphomas primarily involving the lung. We therefore screened 51 cases of pulmonary MALT lymphoma for API2-MALT1 fusion, and studied its relationship with clinicopathologic factors including the immunohistochemical expression of BCL10, another MALT lymphoma-associated molecule. The API2-MALT1 fusion transcript was detected in 21 of 51 (41%) cases, and was correlated with the absence of any underlying autoimmune disease, and with a normal serum lactate dehydrogenase, a typical histology without marked plasmacytic differentiation or an increased number of large cells, and aberrant nuclear BCL10 expression. However, its prognostic impact was not identified in the limited follow-up (6 to 187 months, median 27). These data suggest that the API2-MALT1 fusion may be a causative gene abnormality unrelated to autoimmune disease. In addition, this alteration may define a homogeneous MALT lymphoma subtype that is clinically more indolent and histologically more typical. Aberrant nuclear BCL10 expression may have a possible role as a tool to screen for this API2-MALT1 fusion. A large-scale study with a long follow-up is necessary to establish the prognostic significance of API2-MALT1 fusion.

Primary cutaneous marginal zone B-cell lymphoma: a molecular and clinicopathological study of cases from Asia, Germany, and the United States

Primary cutaneous marginal zone B-cell lymphoma is considered the cutaneous counterpart of extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Although its molecular pathogenesis is currently unknown, an etiological link with Borrelia burgdorferi infection has been identified in European, but not in American or Asian cases. To better understand the pathogenesis and the geographical differences of cutaneous marginal zone B-cell lymphoma, 60 cases from the East Asia, Germany, and the United States at their initial presentation were subjected to the following analyses; (1) clinicopathological comparison between the geographical regions, (2) detection of B. burgdorferi DNA, (3) detection of the API2–MALT1 fusion transcript, a gene alteration specific to mucosa-associated lymphoid tissue lymphoma, and (4) inactivation of tumor suppressor genes (death-associated protein kinase (DAPK), p16INK4a, p14ARF, MGMT, TIMP3, CDH1, and RARB) by hypermethylation of the CpG islands. Cases from the three geographical regions showed similar clinicopathological features. However, moderate/marked tissue eosinophilia was found in 9/25 Asian cases, but only 1/23 German cases (P=0.011) and 0/12 American cases (P=0.015). All 60 cases were negative for either Borrelia DNA or API2–MALT1 fusion. Tumors from the three regions were highly methylated for DAPK (38–50% of the cases, mean 43%) and p16INK4a (42–70%, mean 49%), and the positivities were significantly higher than those of nonneoplastic skin (8%, P=0.0010 and 14%, P=0.0032, respectively). Methylation of these genes had no significant association with progressive features of the tumor. Primary cutaneous marginal zone B-cell lymphomas from the three geographical regions have common clinicopathological features, however, moderate/marked tissue eosinophilia is a feature found almost exclusively in Asian cases. Borrelia infection and API2–MALT1 fusion are not significant in this tumor. Methylation of DAPK and p16INK4a genes is a frequent event in this lymphoma at its initial presentation, but may not be associated with tumor progression.

Mucosa-associated lymphoid tissue lymphoma: Molecular pathogenesis and clinicopathological significance

Mucosa‐associated lymphoid tissue (MALT) lymphoma is a low‐grade tumor closely associated with chronic inflammation such as that of Helicobacter pylori gastritis, Sjogrens syndrome, and Hashimotos thyroiditis. Tumor regression by H.u2003pylori eradication alone is well known in gastric MALT lymphoma, but some tumors occur in the absence of pre‐existing chronic inflammation. The understanding of MALT lymphoma biology has significantly improved, and recurrent cytogenetic alterations have been detected. These include the trisomies 3 and 18, and the translocations t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), and t(3;14)(p14.1;q32). At least some of these alterations result in the constitutive activation of the nuclear factor (NF)‐κB pathway, and may exert anti‐apoptotic action. Apoptosis inhibitor 2–MALT lymphoma‐associated translocation 1 (API12‐MALT1) fusion, resulting from t(11;18)(q21;q21), is specific to, and is the most common in, MALT lymphomas, and its clinicopathological significance has been studied extensively. The focus of the present review is on the recent progress made in elucidating MALT lymphomagenesis and its clinicopathological impact, especially in terms of the effect of API2‐MALT1 fusion on this unique tumor.

Defucosylated Anti -CC Chemokine Receptor 4 Monoclonal Antibody Combined with Immunomodulatory Cytokines : A Novel Immunotherapy for Aggressive/Refractory Mycosis Fungoides and Sézary Syndrome

Purpose: Sézary syndrome (SS) and Mycosis fungoides (MF) in the advanced stage have dismal prognoses. Because CC chemokine receptor 4 (CCR4) has an important role in the skin-homing capacity of MF/SS cells, we postulated that anti-CCR4 monoclonal antibody (mAb) could represent a novel therapeutic agent against aggressive/refractory MF/SS. Experimental Design: The defucosylated next-generation therapeutic mAb KM2760 induces enhanced antibody-dependent cellular cytotoxicity (ADCC). Here, we assessed the therapeutic potential of this antibody against aggressive MF/SS tumor cells in vitro and in animal models in vivo. Results: KM2760 induced robust ADCC by peripheral blood mononuclear cell (PBMC) from healthy controls against a MF/SS cell line as well as against primary tumor cells from patients with aggressive MF/SS. KM2760 also showed significant antitumor activity in disseminated and nondisseminated MF/SS mouse models. In addition, ∼30% of autologous MF/SS tumor cells were killed in in vitro assays of KM2760-induced ADCC mediated by patients PBMC after only 4 h, despite the low numbers of natural killer cells present in these PBMCs. It is also shown that ADCC induced by defucosylated therapeutic mAb can be greatly augmented by the immunomodulatory cytokines interleukin-12, IFN-α-2b, and IFN-γ. Conclusions: The present study has encouraged us in the conducting of a phase I clinical trial of a completely defucosylated anti-CCR4 mAb in patients with CCR4-positive T-cell lymphomas, including aggressive MF/SS (ClinicalTrials.gov identifier: NCT00355472). In the near future, the efficacy not only of defucosylated anti-CCR4 mAb single-agent treatment but also of combination therapy with immunomodulatory cytokines will be clinically established to target aggressive/refractory MF/SS.