Satoru Miyabe

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.

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 factors for keratocystic odontogenic tumor (odontogenic keratocyst): analysis of clinico-pathologic and immunohistochemical findings in cysts treated by enucleation

BACKGROUND The purpose of this study was to determine prognostic factors for the recurrence of keratocystic odontogenic tumors (KCOTs) following simple enucleation by examining clinico-pathologic and immunohistochemical findings. METHODS Following enucleation, the frequency of recurrence among 32 subjects diagnosed with KCOT was analyzed for tumor site, radiographic and histologic features, and immunopositivity for Ki-67 and p53. RESULTS Keratocystic odontogenic tumors in four out of 32 subjects (12.5%) recurred during the follow-up period (median: 33 months, range: 7-114 months). Three out of four subjects (75.0%) among recurrent group showed high expression of Ki-67 (LI >10%) in basal layer and four (4/28; 14.3%) among non-recurrence group (P = 0.025). Expression of p53 among non-recurrent group was observed in 11 subjects (11/28; 39.3%), and in three subjects (3/4; 75.0%) among the recurrent group (P = 0.295). Hazard risk for the recurrence of KCOT was 4.02 (95% CI 1.42-18.14) for high Ki-67 expression in the basal layer by the Cox proportional hazard model (P = 0.009). In our study, none of the other clinico-pathologic variables were associated with the recurrence of KCOT. CONCLUSION The results suggested that the evaluation of Ki-67 expression in KCOT at the time of pathological diagnosis might be helpful for consideration of appropriate adjunctive surgical procedures to avoid a recurrence and may serve as a prognostic marker.

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.

Characterization of mammary analogue secretory carcinoma of the salivary gland: discrimination from its mimics by the presence of the ETV6-NTRK3 translocation and novel surrogate markers

Mammary analogue secretory carcinoma (MASC) is a recently recognized salivary gland tumor harboring an ETV6-NTRK3 translocation similar to secretory carcinoma of the breast. Histologically, MASC mimics papillary-cystic, microcystic, and follicular-type acinic cell carcinoma (AciCC) and low-grade cribriform cystadenocarcinoma (LGCCC) of the salivary gland. Using histology, immunohistochemistry (IHC), and molecular genetic techniques, we reevaluated 18 cases originally diagnosed as AciCC between 1993 and 2012. The last of these methods was used to detect the ETV6-NTRK3 translocation. The results reconfirmed 6 cases as AciCC (3 men; average age, 63 years) and helped us reclassify 10 cases as MASC (6 men; mean age, 46 years) and 2 as LGCCC (2 women; mean age, 48 years). Using IHC, we identified the 3 histologic types according to the expression patterns of vimentin, high-molecular-weight cytokeratin, cytokeratin 19, S-100, mammaglobin, MUC1, GATA-binding protein 3, adipophilin, α-amylase, DOG-1, SOX-10, and p63. The number of tumors diagnosed as MASC indicates that AciCC includes bona fide MASC cases. Because differential diagnosis among zymogen granule-poor AciCC, MASC, and LGCCC tumors is challenging, we recommend using molecular genetic tests for ETV6-NTRK3 for accurate diagnosis. Furthermore, detailed analyses of hematoxylin and eosin-stained tissues and IHC studies using the markers described here should be incorporated into routine practices.

Mammary analogue secretory carcinoma of salivary glands: a clinicopathologic and molecular study including 2 cases harboring ETV6-X fusion.

Mammary analogue secretory carcinoma (MASC) is a recently described low-grade carcinoma with morphologic and genetic similarity, including ETV6-NTRK3 fusion, to secretory carcinoma of the breast. ETV6 is frequently involved in other epithelial and nonepithelial tumors, and many fusion partners of ETV6 have been reported. In the present study, 14 Japanese MASC cases were clinicopathologically and molecularly analyzed. The median age of the patients was 39 years, and the male:female ratio was 6:8. All cases showed histopathologic findings compatible with those previously described for MASC and harbored an ETV6 split as visualized by fluorescence in situ hybridization. Two cases showed thick fibrous septa and invasive features including vascular or perineural tumor involvement, findings that are rare in MASC. In addition, in these 2 cases, non-NTRK3 genes appeared to fuse with ETV6 (ETV6-X fusion). NTRK1 and NTRK2, both members of the NTRK family, were not involved. Of the 14 MASC cases, the ETV6-NTRK3 fusion transcript was positive in 6 cases, and the relative expression level of the ETV6-NTRK3 fusion transcript was variable, ranging from 1 to 5.8. Results of the present study of MASC suggest that (1) ETV6 occasionally fuses with unknown non-NTRK3 genes, (2) ETV6-X cases might have an invasive histology, (3) for molecular diagnosis of MASC, fluorescence in situ hybridization to detect ETV6 splits is the method of choice, and (4) the expression level of the ETV6-NTRK3 fusion transcript is considerably variable. These findings provide a novel insight into the oncogenesis, histopathology, diagnosis, treatment, and prognosis of this newly recognized carcinoma.

Impact of CRTC1/3–MAML2 fusions on histological classification and prognosis of mucoepidermoid carcinoma

Okumura Y, Miyabe S, Nakayama T, Fujiyoshi Y, Hattori H, Shimozato K & Inagaki H
(2011) Histopathology59, 90–97

Clinicopathological significance of MAML2 gene split in mucoepidermoid carcinoma.

CRTC1–MAML2 and CRTC3–MAML2 fusions have been associated with favorable clinicopathological features of mucoepidermoid carcinomas. However, the significance of the MAML2 gene split has not been fully clarified. In the present study, 95 mucoepidermoid carcinomas (paraffin‐embedded materials) were analyzed for CRTC1–MAML2 and CRTC3–MAML2 fusions by RT‐PCR and for the MAML2 gene split by FISH. Quantitative RT‐PCR for the CRTC1–MAML2 transcript was performed in selected cases. MLL gene involvement, which has been reported in some leukemia cases, was examined by FISH in fusion partner‐unknown cases. CRTC1–MAML2 and CRTC3–MAML2 fusions were detected in 37 and 6 cases, respectively. The MAML2 gene split was detected in 62 cases, which included all CRTC1/3–MAML2 fusion‐positive cases. The level of CRTC1–MAML2 transcript expression was highly variable, and its clinicopathological impact was unclear. The MLL gene split was not detected. Mucoepidermoid carcinomas negative for CRTC1/3–MAML2 and positive for the MAML2 gene split (n = 19) showed favorable clinicopathological tumor features similar to those positive for CRTC1/3–MAML2 fusions. Compared with negative cases (n = 33), mucoepidermoid carcinomas positive for the MAML2 split (n = 62) were associated with lower patient age, a mild female predilection, a smaller tumor size, less frequent nodal metastasis, a lower clinical stage, a lower histological grade, and longer overall and disease‐free survival. The MAML2 gene split emerged as an independent prognostic factor for both overall and disease‐free survival in multivariate prognostic analysis. The presence of the MAML2 gene split defines a distinct mucoepidermoid carcinoma subset that is associated clinicopathologically with favorable tumor features. (Cancer Sci 2013; 104: 85–92)

Analysis of VH Gene Rearrangement and Somatic Hypermutation in Sjogren’s Syndrome and IgG4-Related Sclerosing Sialadenitis

IgG4‐related sclerosing sialadenitis is currently considered as an autoimmune disease distinct from Sjogren’s syndrome (SS) and responds extremely well to steroid therapy. To further elucidate the characteristics of IgG4‐related sclerosing sialadenitis, we analysed VH fragments of IgH genes and their somatic hypermutation in SS (n = 3) and IgG4‐related sclerosing sialadenitis (n = 3), using sialolithiasis (n = 3) as a non‐autoimmune control. DNA was extracted from the affected inflammatory lesions. After PCR amplification of rearranged IgH genes, at least 50 clones per case (more than 500 clones in total) were sequenced for VH fragments. Monoclonal IgH rearrangement was not detected in any cases examined. When compared with sialolithiasis, there was no VH family or VH fragment specific to SS or IgG4‐related sclerosing sialadenitis. However, rates of unmutated VH fragments in SS (30%) and IgG4‐related sclerosing sialadenitis (39%) were higher than that in sialolithiasis (14%) with statistical significance (P = 0.0005 and P < 0.0001, respectively). This finding suggests that some autoantibodies encoded by germline or less mutated VH genes may fail to be eliminated and could play a role in the development of SS and IgG4‐related sclerosing sialadenitis.

p53 and ki67 as biomarkers in determining response to chemoprevention for oral leukoplakia

BACKGROUND We performed a randomized controlled chemoprevention trial of oral leukoplakia by administrating a low dose of beta-carotene and vitamin C supplements. 17% of subjects in the experimental arm (4/23) demonstrated clinical remission (complete or partial response) at completion of the trial. The objective of this study was to determine whether baseline expression of p53 and ki67 demonstrated any differences between those responding or not responding to our intervention. A secondary objective was to elucidate any relationship between dietary factors and clinical responses. METHODS For this biomarker study, we included all subjects in the experimental group (n = 23) who were non-smokers. Among 16 who completed the trial for 1 year of supplementation, there were four responders and 12 non-responders at 1-year follow-up. Following immuno-staining for p53 and ki67, the percentage of positive cell nuclei were analyzed as labeling index (LI). RESULTS Expression of p53 was greater in basal layers than in para-basal layers. Mean para-basal LI of p53 was higher in non-responding (26.0) than in responding subjects (11.2) (P = 0.028). ki67 LIs were not significantly different in the two groups. CONCLUSIONS Expression of p53 was inversely related to clinical response to the supplements. Other biomarkers that may recognize subjects responsiveness to chemoprevention require further study.