Mitsukuni Okabe

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.

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.

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.

Clinicopathological significance of the CRTC3–MAML2 fusion transcript in mucoepidermoid carcinoma

Mucoepidermoid carcinoma is the most common primary malignancy of the salivary gland. We and others showed that CRTC1–MAML2 gene fusion was associated with favorable clinicopathological tumor features. Recently, a novel gene fusion, CRTC3–MAML2, was reported as a rare gene alteration in a case of mucoepidermoid carcinoma. However, its frequency and clinicopathological significance remains unclear. In all, 101 cases of mucoepidermoid carcinoma and 89 cases of non-mucoepidermoid carcinoma of the salivary gland were analyzed, and RNA was extracted from formalin-fixed, paraffin-embedded specimens. In the CRTC family, there have been three genes, CRTC1, CRTC2, and CRTC3. We developed reverse transcription-polymerase chain reaction (RT-PCR) assays for CRTC1–MAML2, CRTC2–MAML2, and CRTC3–MAML2 fusions. Clinicopathological data of the patients were obtained from their clinical records. Of 101 cases of mucoepidermoid carcinoma, 34 (34%) and 6 (6%) were positive for CRTC1–MAML2 and CRTC3–MAML2 fusion transcripts. However, in the 89 cases of non-mucoepidermoid carcinoma, neither transcript was noted. In the former cases, CRTC1–MAML2 and CRTC3–MAML2 fusions were mutually exclusive. The other fusion, CRTC2–MAML2, was not detected. We confirmed that the clinicopathological features of CRTC1–MAML2-positive mucoepidermoid carcinomas indicated an indolent course. CRTC3–MAML2-positive mucoepidermoid carcinomas also had clinicopathologically favorable features; all cases showed a less advanced clinical stage, negative nodal metastasis, no high-grade tumor histology, and no recurrence or tumor-related death after surgical resection of the tumor. It is interesting to note that patients with CRTC3–MAML2-positive tumors (mean 36 years of age) were significantly younger that those with the CRTC1–MAML2 fusion (55 years) and those with fusion-negative tumors (58 years). In conclusion, CRTC3–MAML2 fusion, which is mutually exclusive with CRTC1–MAML2 fusion and specific to mucoepidermoid carcinoma, may be detected more frequently than previously expected. Mucoepidermoid carcinomas possessing CRTC3–MAML2 fusion may be associated with favorable clinicopathological features and patients may be younger than those with CRTC1–MAML2 fusion or those with no detectable gene fusion.

Prognostic Significance of p27 and Ki-67 Expression in Mucoepidermoid Carcinoma of the Intraoral Minor Salivary Gland

p27 and Ki-67, a universal cyclin-dependent kinase inhibitor and a proliferative cell marker, respectively, have been useful in predicting clinical aggressiveness in various human tumors. We studied clinicopathologic significance of these molecules in mucoepidermoid carcinoma of the intraoral minor salivary gland. Expression of p27 and Ki-67 was assessed immunohistochemically in primary mucoepidermoid carcinomas from 31 patients without distant metastasis at surgery. Correlation each of p27 and Ki-67 expression was analyzed with various clinicopathologic parameters including age, sex, primary tumor site, tumor size, nodal metastasis, clinical stage, and histologic grade. The latter was evaluated using a point-scoring scheme of Auclair et al. that consists of five histologic factors (intracystic component, neural invasion, necrosis, mitosis, and anaplasia). p27 expression was correlated inversely with histologic grade (P = .007), but with none of other factors. When the correlation of p27 expression was further examined with each of the histologic factors, it was correlated significantly with intracystic component, but not with neural invasion, necrosis, mitosis, or anaplasia. Ki-67 expression was correlated significantly with histologic grade only in the clinicopathologic factors (P < .0001), and in the histologic factors, with necrosis, mitosis, and anaplasia. Multivariate prognostic analyses were performed to identify independent risk factors for both disease-free and overall survivals. Large tumor size (P = .031, relative risk = 5.5) and low p27 expression (P = .012, relative risk = 5.2) were risk factors for worse disease-free survival. Low p27 expression (P = .015, relative risk = 15.2) was selected as a risk factor for worse overall survival. Other factors including age, sex, tumor site, nodal status, clinical stage, histologic grade, and Ki-67 did not emerge as independent risk factors in either prognostic analysis. These data suggest that p27 may be useful in estimating prognosis of the patients who have mucoepidermoid carcinoma of the intraoral minor salivary gland.

Prognostic significance of p27Kip1, Ki-67, and CRTC1-MAML2 fusion transcript in mucoepidermoid carcinoma: a molecular and clinicopathologic study of 101 cases.

PURPOSE Mucoepidermoid carcinoma (MEC) is the most frequently detected primary malignancy of the salivary gland and is characterized by a marked variation in prognosis. In the present study, we investigated the prognostic significance of p27Kip1, Ki-67, and CRTC1 (also called MECT1, TORC1, and WAMTP1)-MAML2 fusion in MEC. MATERIALS AND METHODS MEC cases (n = 101) were examined for p27Kip1 and Ki-67 expression using immunohistochemistry and for CRTC1-MAML2 fusion transcript using reverse transcriptase-polymerase chain reaction. RESULTS p27Kip1, Ki-67, and the CRTC1-MAML2 fusion transcript were expressed in 71, 31, and 34 of the 101 cases, respectively. p27Kip1 and CRTC1-MAML2 fusion were associated with favorable clinicopathologic tumor features and Ki-67 with aggressive clinicopathologic features. Multivariate survival analyses were performed that included the following 10 clinicopathologic factors: age, gender, tumor site, tumor size, nodal metastasis, clinical stage, histologic grade, p27 expression, Ki-67 expression, and CRTC1-MAML2 fusion. For disease-free survival, only p27Kip1 expression was significant as an independent prognostic factor. For overall survival, p27Kip1 expression, CRTC1-MAML2 fusion, and tumor size were significant. In each analysis, p27Kip1 and CRTC1-MAML2 fusion were independent of the clinical stage. Ki-67 expression was not selected in either multivariate analysis. CONCLUSIONS p27Kip1 and CRTC1-MAML2 fusion were associated with favorable clinicopathologic tumor features, and both were useful in predicting the overall survival of patients with MEC. For disease-free survival, p27Kip1 might be the most useful prognostic factor. In contrast, Ki-67 might not be a very powerful prognostic indicator for either survival point.

API2-MALT1 Fusion Gene in Colorectal Lymphoma

The API2-MALT1 fusion gene was originally identified from a t(11;18)(q21;q21) translocation, a specific chromosomal abnormality that is found in mucosa-associated lymphoid tissue (MALT) lymphoma. Gastric MALT lymphomas positive for the API2-MALT1 fusion gene do not respond to Helicobacter pylori–eradication therapy, but otherwise, the incidence and clinicopathological behavior of colorectal MALT lymphoma with this genetic abnormality are unclear. We examined the API2-MALT1 fusion by multiplex RT-PCR method in 47 cases of MALT lymphoma and 13 cases of diffuse large B-cell lymphoma and evaluated the relevance of API2-MALT1 positivity to the clinical and pathological features. The mean ages of MALT lymphoma and diffuse large B-cell lymphoma patients were 65 (range, 37–87 y) and 58 (range, 14–85 y) years, respectively. API2-MALT1 fusion genes were detected in seven cases (15%) of MALT lymphoma and one case (8%) of diffuse large B-cell lymphoma. In MALT lymphomas, the tumor size in API2-MALT1–positive cases was 62 ± 39 mm (mean ± SD), statistically larger than that in API2-MALT1–negative cases (25 ± 19 mm; P < .01). The API2-MALT1–positive cases demonstrated more advanced clinical stages and a male predominance, compared with API2-MALT1–negative cases. Thus, API2-MALT1–positive tumors should be cared for as a more aggressive subgroup and be followed for a longer time.

Different Keratin Profilesin Craniopharyngioma Subtypes and Ameloblastomas

Craniopharyngiomas are generally considered to arise from the remnants of Rathkes pouch or a misplaced enamel organ. We tried to refine these hypotheses, comparing the subtypes of craniopharyngioma with Rathkes cleft cyst, a known Rathkes pouch derivative, and with ameloblastoma, an enamel organ derivative. Nineteen craniopharyngiomas (14 adamantinomatous and 5 papillary type tumors) and 17 ameloblastomas were immunostained for cytokeratin (CK) 7, CK 8, CK 14, and human hair keratin (HHK). All cases of adamantinomatous craniopharyngioma were CK 7+/CK 8+/CK 14+. Two cases (40%) of papillary craniopharyngioma were CK 7+/CK 8+/CK 14+, whereas the remaining three cases (60%) were CK 7+/CK 8-/CK 14+. Fifteen cases (88%) of ameloblastoma were CK 7-/CK 8+/CK 14+. Only the shadow cells present in adamantinomatous craniopharyngiomas were positive for HHK, which may indicate their follicular differentiation. In Rathkes cleft cyst, ciliated cuboidal cells were CK 7+/CK 8+/CK 14- and metaplastic squamous cells were CK 7+/CK 8/CK 14+. These findings suggest that both subtypes of craniopharyngioma may differ from ameloblastoma in histogenesis, although cytokeratin expression patterns may change during tumor development. Adamantinomatous craniopharyngioma may be related to a heterotopic ectodermal tissue which can differentiate into hair follicles, while papillary craniopharyngioma may arise from Rathkes cleft cyst.

Immunoglobulin VH genes in thymic MALT lymphoma are biased toward a restricted repertoire and are frequently unmutated

Thymic MALT lymphoma shows certain distinctive features among MALT lymphomas, such as expression of IgA isotype, consistent lack of API2–MALT1 gene fusion, and very strong association with autoimmune disease, especially Sjogrens syndrome. To help clarify the nature of the clonal lymphoid infiltrates, we analysed the usage and somatic hypermutation of the Ig heavy chain variable region (VH) genes in nine different cases. The VH rearrangement was potentially functional in all cases and was restricted to the VH3 family. VH usage was biased toward VH3‐30 (five cases) and VH3‐23 (three cases) segments, which have both been frequently expressed by autoimmune B cells. Somatic hypermutation was absent in five cases. Fewer than the expected replacement mutations were found in the framework regions in two cases, indicating a negative antigen selection pressure. Ongoing mutation was absent in all cases. D segment usage was varied, whereas JH segment usage was restricted to JH4. The observed patterns of VH usage and mutations suggested that specific antigens may play a pathologically relevant role in the genesis or progression of thymic MALT lymphoma. Copyright