Kei Ijichi

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 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.

Warthin-like Mucoepidermoid Carcinoma: A Combined Study of Fluorescence In Situ Hybridization and Whole-slide Imaging.

There has been some debate as to whether a subset of metaplastic Warthin tumors (mWTs) harbor the mucoepidermoid carcinoma (MEC)-associated CRTC1-MAML2 fusion. We analyzed 15 tumors originally diagnosed as mWT (mWT-like tumors), 2 of which had concurrent MECs. We looked for the CRTC1/3-MAML2 fusion transcripts and performed immunohistochemistry for p63 and fluorescence in situ hybridization (FISH) for the MAML2 split. To localize MAML2 split–positive cells at the cellular level, whole tumor tissue sections were digitalized (whole-slide imaging [WSI]). The CRTC1-MAML2, but not CRTC3-MAML2 was detected in 5/15 mWT-like tumors. FISH-WSI results showed that all epithelial cells harbored the MAML2 split in fusion-positive mWT-like tumors and were totally negative in fusion-negative mWT-like tumors. A review of the hematoxylin and eosin–stained slides showed that morphology of the “metaplastic” epithelium was virtually indistinguishable between fusion-positive and fusion-negative tumors. However, oncocytic bilayered tumor epithelium, characteristic to typical WT, was always found somewhere in the fusion-negative tumors but not in the fusion-positive tumors. This distinguishing histologic finding enabled 5 pathologists to easily differentiate the 2 tumor groups with 100% accuracy. The age and sex distribution of fusion-positive mWT-like tumor cases was similar to that of fusion-positive MEC cases and significantly different from those of fusion-negative mWT-like tumor and typical WT cases. In addition, only fusion-positive mWT-like tumors possessed concurrent low-grade MECs. In conclusion, a subset of mWT-like tumors were positive for the CRTC1-MAML2 fusion and had many features that are more in accord with MEC than with WT. The term Warthin-like MEC should be considered for fusion-positive mWT-like tumors.

Prediction of Chemosensitivity Using Multigene Analysis in Head and Neck Squamous Cell Carcinoma

Aims: The main purpose of the current study was to find predictive biomarkers that can be routinely used for the response to chemotherapy in head and neck squamous cell carcinoma (HNSCC). Methods: From this standpoint, we selected the histoculture drug response assay (HDRA) to assess in vitro chemosensitivity, and real-time reverse transcription polymerase chain reaction to investigate the gene expression profile of individual tumors as available predictive biomarkers. Using both surgery and biopsy specimens, we analyzed their gene expression profiles using the 18 markers that we thought were likely predictors of the response to anti-cancer agents. Results: Statistically significant associations were found between 5-fluorouracil (5-FU) sensitivity in HDRA and HER-2 mRNA expression level (p = 0.0030). Moreover, HER-2 expression was significantly associated with cisplatin sensitivity (p = 0.0089). Cisplatin sensitivity in HDRA was also demonstrated to have a significant association with epidermal growth factor receptor (EGFR) expression in which the group with cisplatin resistance tended to have a higher expression level than the sensitive group (p = 0.0385). Conclusion: HER-2 and EGFR may be possible reliable predictive biomarkers for anti-cancer therapy, and might help in the decision-making process for individual patients with HNSCC.

Efficacy of gemcitabine and cetuximab combination treatment in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) may be curable with surgery, radiation and chemotherapy in its early stages. However, recurrence and metastasis often prevail following primary treatment in advanced stage cases and are associated with significant morbidity and mortality. In this study we investigated the combination therapy of gemcitabine and cetuximab for HNSCC. The UM-SCC-6 and UM-SCC-23 HNSCC cell lines were analyzed following treatment with gemcitabine and cetuximab. To determine the mechanism of action of this combination treatment, the cell cycle distributions following gemcitabine and/or cetuximab treatment were analyzed by flow cytometry and apoptosis assay. Gemcitabine and cetuximab combination treatment exerted an enhanced cytotoxic effect. The cell cycle analysis demonstrated that cells accumulated in the S phase following gemcitabine treatment and G1 arrest occurred following cetuximab treatment. An increase in sub-G1 phase cells was also observed following treatment with the two drugs. In an apoptosis assay, caspase 3/7 activity was found to be higher when administering a combination of gemcitabine and cetuximab compared to each agent administered alone. Gemcitabine and cetuximab are individually effective against HNSCC and an enhanced growth inhibitory effect may be expected when these agents are used in combination.

MYB, MYBL1, MYBL2 and NFIB gene alterations and MYC overexpression in salivary gland adenoid cystic carcinoma

Adenoid cystic carcinoma (AdCC) is one of the most common salivary gland malignancies and the long‐term prognosis is poor. In this study, we examined alterations of AdCC‐associated genes, MYB, MYBL1, MYBL2 and NFIB, and their target molecules, including MYC. The results were correlated to clinicopathological profile of the patients.

Hypersensitivity to cisplatin after hRev3 mRNA knockdown in head and neck squamous cell carcinoma cells

Resistance to platinum-based chemotherapy frequently poses a significant problem in the treatment of head and neck squamous cell carcinomas (HNSCCs). In this study, we isolated cisplatin-resistant UM-SCC-23 CDDP/R cells from a UM-SCC-23 head and neck squamous cell carcinoma cell line. The UM-SCC-23 CDDP/R cells were approximately 3.5-fold more resistant to cisplatin than the UM-SCC-23 cells. Translesion DNA synthesis (TLS) is one of the major pathways involved in post-replication repair. To ascertain whether TLS is involved in cisplatin resistance in human cancer cells, we analyzed expression of the Rev3 gene, which encodes the catalytic subunit of DNA polymerase ζ (Polζ), using quantitative PCR. Expression of hRev3 mRNA in the UM-SCC-23 CDDP/R cells was approximately 1.4-fold higher than in the UM-SCC-23 cells. In both types of cells, expression of hRev3 mRNA was suppressed using siRNAs. Cisplatin sensitivity after hRev3 mRNA knockdown increased approximately 2-fold in UM-SCC-23 cells and approximately 3-fold in UM-SCC-23 CDDP/R cells. This study suggests that hRev3 knockdown with siRNAs increases sensitivity to cisplatin. Inhibition of the hRev3 gene may therefore prove to be a novel clinical strategy for reducing resistance to platinum-based chemotherapy in HNSCC.

Surgical treatment of parapharyngeal space tumors: A report of 29 cases

The present study aimed to establish a strategy for parapharyngeal space (PPS) tumor surgery based on preoperative symptoms, clinical signs, imaging and histological examination. A retrospective cohort study was conducted with 29 adult patients who underwent surgery for primary PPS tumors from 2008-2015. The following data was obtained: Preoperative symptoms of the patient, histological type of the tumor, surgical approach and complications. Of the 29 patients who underwent surgery to remove a PPS tumor, 16 presented with neurogenic tumors and 13 with salivary gland tumors. The most common symptom was the presence of a neck mass. Preoperative computed tomography and magnetic resonance imaging were performed to evaluate the size and location of the tumors. The majority of the salivary gland tumors were located in the prestyloid space, whereas all neurogenic tumor cases were poststyloid. A total of 21 of the 29 patients (72.4%) underwent a preoperative fine needle aspiration (FNAC) examination. FNAC contributed to preoperative diagnosis in 9 of these cases (42.9%). In parotid tumors of the prestyloid space, facial nerve palsy was a common postoperative complication. In the case of neurogenic tumors, a common complication was the postoperative palsy of the nerve of tumor origin. The strategy for PPS surgery, from the preoperative diagnosis to the operative method, remains controversial. The data on pre- and postoperative symptoms, imaging and histological diagnosis, and the selection of surgical method depending on a neurogenic or salivary tumor origin in the present study indicated that improving the method of examination, carefully selecting the method of approach and accurately managing surgery leads to complete tumor removal, and that the use of nerve-preserving techniques may reduce the likelihood of complications.

Selection of Therapeutic Treatment with Alternating Chemoradiotherapy for Larynx Preservation in Laryngeal Carcinoma Patients

OBJECTIVE We analyzed the efficacy of treatments that included alternating chemoradiotherapy in laryngeal cancer patients. METHODS Alternating chemoradiotherapy consisted of chemotherapy with 5-fluorouracil (600 mg/m(2)/day) on Days 1-6 and cisplatin (80 mg/m(2)) on Day 7 followed by radiotherapy with 30 Gy. Additional chemoradiotherapy was administered to responders, and laryngectomy was performed in non-responders. The contribution of alternating chemoradiotherapy to laryngeal preservation was compared with that of radiotherapy in patients with T2 disease and with that of laryngectomy in patients with T3/T4 disease. RESULTS Analysis of 87 patients was conducted. The 5-year overall survival rate of T2 patients (n = 46) was 88.9% for definitive radiotherapy and 82.5% for alternating chemoradiotherapy. The laryngectomy-free rate in T2 patients was 90.5% for definitive radiotherapy and 80.0% for alternating chemoradiotherapy. In patients with T3/T4 disease (n = 41), the 5-year overall survival rate was 86.9% for alternating chemoradiotherapy and 67.4% for laryngectomy. The laryngectomy-free rate in T3/T4 patients was 91.7% for alternating chemoradiotherapy and 0.0% for laryngectomy. CONCLUSIONS In advanced carcinoma of the larynx, alternating chemoradiotherapy treatment might enable larynx preservation.

Expression of cancer/testis antigens in salivary gland carcinomas with reference to MAGE ‐A and NY ‐ ESO ‐1 expression in adenoid cystic carcinoma

Cancer/testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. CTAs are highly immunogenic proteins, and thus represent ideal targets for cytotoxic T‐lymphocyte‐mediated specific immune therapy. The aim of this study was to screen CTA expression in various types of salivary gland carcinoma and to clarify clinicopathological significance of MAGE‐A and NY‐ESO‐1 expression in adenoid cystic carcinomas (AdCCs) of the salivary gland, which is one of the most common salivary gland carcinomas, and usually has a fatal outcome.