An Assessment Rubric for a Resident Training Program in Surgery: A Single-Institution Experience.

Using a Collaborative Action Research model, our research team established a one-month clinical resident training program for first- and second-year clinical residents. We created and implemented an assessment rubric to assess the residents' progress toward independent practice in surgery, and thereby, to evaluate the program itself. The program included training in three areas: basic techniques and procedures in the operating room, surgical ward management, and academic activities. The rubric measured the residents' performance according to three achievement levels: Level 1 (demonstration), Level 2 (active help) and Level 3 (passive help). The program and rubric implementation began in June 2019 and continued until March 2020, when the program outcomes and shortcomings were analyzed. Among nineteen clinical residents, a total of nine clinical residents participated in the study. Most participants reached achievement Level 3 for their performance of basic techniques in the operating room. Finally, we discussed ideas for improvement and drafted plans for an improved rubric to complete the action research cycle. Our research team found the rubric to be a useful tool in evaluating the status of the new clinical resident training program.

Expanding on the professional role of dental hygienists as key managers of medical-dental and hospital-dental clinic collaboration in a local Japanese hospital without a dentistry department: From a questionnaire survey after a web seminar.

INTRODUCTION: Perioperative oral management (POM) was introduced into the Japanese universal health insurance system in 2012. Collaboration with dental clinics is important for hospitals without a dentistry department. A dental hygienist newly assigned as a member of the patient flow management centre led a seminar to promote collaboration via the web. This study represents the first step to explore the possible role of the hospital-based dental hygienist in the field of regional medical-dental cooperation of POM by assessing their willingness to participate in providing this type of care by a survey. METHODS: The rate of attendees' satisfaction and the current problems of the collaboration for POM were evaluated through a questionnaire survey after the web seminar. RESULTS: All respondents reported satisfaction with the web seminar although it was the first experience of an online seminar for half of the respondents. All hospital dentists, but only 47.8% of dentists working at clinics, had participated in POM. Dental hygienist tended to show greater desire to participate in POM than dentists. All respondents appreciated the role played by the dental hygienist as a key manager of medical-dental collaboration between the hospital and local clinics. CONCLUSION: The hospital-based dental hygienist can play a role in planning and management of web seminars for POM, to raise awareness and promote regional medical-dental cooperation of POM.

Application of amplicon-based targeted sequencing with the molecular barcoding system to detect uncommon minor EGFR mutations in patients with treatment-naïve lung adenocarcinoma.

BACKGROUND: In lung cancer, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitizing mutations co-existing with rare minor EGFR mutations are known as compound mutations. These minor EGFR mutations can lead to acquired resistance after EGFR tyrosine kinase inhibitor treatment, so determining the mutation status of patients is important. However, using amplicon-based targeted deep sequencing based on next-generation sequencing to characterize mutations is prone to sequencing error. We therefore assessed the benefit of incorporating molecular barcoding with high-throughput sequencing to investigate genomic heterogeneity in treatment-naïve patients who have undergone resection of their non-small cell lung cancer (NSCLC) EGFR mutations. METHODS: We performed amplicon-based targeted sequencing with the molecular barcoding system (MBS) to detect major common EGFR mutations and uncommon minor mutations at a 0.5% allele frequency in fresh-frozen lung cancer samples. RESULTS: Profiles of the common mutations of EGFR identified by MBS corresponded with the results of clinical testing in 63 (98.4%) out of 64 cases. Uncommon mutations of EGFR were detected in seven cases (10.9%). Among the three types of major EGFR mutations, patients with the G719X mutation had a significantly higher incidence of compound mutations than those with the L858R mutation or exon 19 deletion (p = 0.0052). This was validated in an independent cohort from the Cancer Genome Atlas dataset (p = 0.018). CONCLUSIONS: Our findings demonstrate the feasibility of using the MBS to establish an accurate NSCLC patient genotype. This work will help understand the molecular basis of EGFR compound mutations in NSCLC, and could aid the development of new treatment modalities.

Tumor-suppressive effect of LRIG1, a negative regulator of ErbB, in non-small cell lung cancer harboring mutant EGFR.

Epidermal growth factor receptor (EGFR) is a member of the ErbB (HER) family that is known to play important roles in the pathogenesis of various human cancers. Mutations of the EGFR gene are commonly found as oncogenic driver mutations and have been targeted for treatment of non-small cell lung cancer (NSCLC). Leucine-rich repeat and immunoglobulin-like domain protein-1 (LRIG1) is a cell-surface protein that is known as a negative regulator of the ErbB (HER) family. In this study, we first confirmed that the expression levels of LRIG1 were much lower in NSCLC than in non-malignant cells or tissues. Next, we focused on the effect of LRIG1 in NSCLC. For this purpose, we established clones stably overexpressing LRIG1, using EGFR-mutant (HCC827, HCC4011 and NCI-H1975) and wild-type (A549) cells. Transfection of LRIG1 was associated with a decrease in the expression and phosphorylation levels of EGFR in the HCC827, HCC4011 and NCI-H1975 cells. It was also associated with strong suppression of the cell proliferative, invasive, migratory and tumorigenic potential of the HCC827 cells. On the other hand, no such effects were observed in the A549 cells. In addition, LRIG1 also downregulated the expression and phosphorylation levels of other tyrosine kinase receptors, such as HER2, HER3, MET and IGF-1R, and prevented the epithelial-to-mesenchymal transition induced by TGF-ß in the HCC827 cells. These findings suggest that LRIG1 exerts important tumor-suppressive effects in EGFR-mutant NSCLC and has the potential to become a novel therapeutic target for EGFR-mutant NSCLC.

Therapeutic strategies for afatinib-resistant lung cancer harboring HER2 alterations.

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non-small-cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan-HER inhibitor afatinib could be a useful therapeutic agent as HER2-targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib-resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib-resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial-to-mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)-like features. The afatinib-resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC-like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.

Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer.

Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-human epidermal growth factor receptor 2 (HER2) drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. In this study, we propose novel treatment options for patients with HER2-positive gastric cancer. First, we determined the molecular profiles of 12 gastric cancer cell lines, and examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines. Additionally, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. In the drug sensitivity analysis, both afatinib and neratinib produced an antitumor effect in most of the HER2-amplified cell lines. However, some cells were not sensitive to the drugs. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene that inhibits the activation of insulin-like growth factor-1 receptor (IGF-1R), were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, showed a notable synergistic effect. Among 123 clinical samples, we found 19 cases of HER2 amplification and three cases of oncogenic mutations. In conclusion, afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer.

Antitumor effect of afatinib, as a human epidermal growth factor receptor 2-targeted therapy, in lung cancers harboring HER2 oncogene alterations.

Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.

Human collagen XV is a prominent histopathological component of sinusoidal capillarization in hepatocellular carcinogenesis.

BACKGROUND: Increased expression of collagen XV has been reported in hepatocellular carcinogenesis in mice. The aim of this study was to confirm the previous murine findings in human hepatocellular carcinoma (HCC) specimens, along with the histopathological distribution of collagen XV in tumoral tissues. METHODS: Sixty-three primary HCC specimens were examined. Immunostaining of collagen XV and quantitative reverse transcriptional PCR of COL15A1, which encodes collagen XV, were performed. RESULTS: Positive staining of collagen XV was observed in all tumoral regions, regardless of differentiation level or pathological type of HCC, along the sinusoid-like endothelium, whereas collagen XV was not expressed in any non-tumoral region. The intensity score of collagen XV immunostaining and the mRNA value of COL15A1 were significantly correlated. COL15A1 expression in tumors was 3.24-fold higher than in non-tumoral regions. Multivariate analysis showed that COL15A1 expression was significantly higher in the absence of hepatitis virus and moderately differentiated HCC. CONCLUSIONS: COL15A1 mRNA was up-regulated in HCC and collagen XV was expressed along the sinusoid-like endothelium of HCC but not in non-tumoral regions, which implies that collagen XV contributes to the capillarization of HCC.

Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib.

Afatinib is an irreversible epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR-TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non-small cell lung cancer. In this study, we established various afatinib-resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib-resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827-ACR, was established from one of the MET amplified-cell lines. Several afatinib-resistant cell lines including HCC827-ACR displayed epithelial-to-mesenchymal transition (EMT) features and epigenetic silencing of miR-200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib-resistant cells and found that MET amplification, EMT, and stem cell-like features are observed in cells with acquired resistance to EGFR-TKIs. This finding may provide clues to overcoming resistance to EGFR-TKIs.

Preclinical evaluation of microRNA-34b/c delivery for malignant pleural mesothelioma.

The microRNA-34s (miR-34s) have p53 response elements in their 5'-flanking regions and demonstrate tumor-suppressive functions. In malignant pleural mesothelioma (MPM), we previously reported that expression of miR-34b and miR-34c (miR-34b/c) was frequently downregulated by methylation in MPM cell lines and primary tumors. The forced overexpression of miR-34b/c showed significant antitumor effects with the induction of apoptosis in MPM cells. In this study, we examined the in vivo antitumor effects of miR-34b/c using adenovirus vector on MPM. We subcutaneously transplanted NCI-H290, a human MPM cell line, into BALB/C mice and injected adenovirus vector expressing miR-34b/c, luciferase driven by the cytomegalovirus promoter (Ad-miR-34b/c or Ad-Luc), or PBS control into tumors over 5mm in diameter. A statistically significant growth inhibition of the tumor volume was observed in the Ad-miR-34b/c group from day 6 onward compared to the Ad-Luc group. The inhibition rate of Ad-miR-34b/c, compared to the tumor volume treated with Ad-Luc, was 58.6% on day 10 and 54.7% on day13. Our results indicate that adenovirus-mediated miR-34b/c gene therapy could be useful for the clinical treatment of MPM.

Silenced expression of NFKBIA in lung adenocarcinoma patients with a never-smoking history.

Nuclear factor of κ-light polypeptide gene enhancer in B cells inhibitor α (NFKBIA), which is a tumor suppressor gene, was found to be silenced in lung adenocarcinomas. We examined NFKBIA expression, mutations in the EGFR and K-ras genes, and EML4-ALK fusion in 101 resected lung adenocarcinoma samples from never-smokers. NFKBIA expression was evaluated using immunohistochemistry. NFKBIA expression was negative in 16 of the 101 samples (15.8%). EGFR and K-ras mutations and EML4-ALK fusion were detected in 61 (60.5%), 1 (1.0%), and 2 (2.0%) of the 101 samples, respectively, in a completely mutually exclusive manner. Negative NFKBIA expression was observed significantly more frequently among the tumors with none of the three genetic alterations compared to those with such alterations (p = 0.009). In addition, negative NFKBIA expression was significantly more frequent among the EGFR-wild type samples compared to the EGFR-mutant samples (p = 0.013). In conclusion, NFKBIA expression was silenced in adenocarcinomas without EGFR/K-ras mutations or EML4-ALK fusion, suggesting that the silencing of NFKBIA may play an important role in the carcinogenesis of adenocarcinomas independent of EGFR/K-ras mutations or EML4-ALK fusion.

Duodenal carcinoma from a duodenal diverticulum mimicking pancreatic carcinoma.

An 81-year-old man was found to have a pancreatic head tumor on abdominal computed tomography (CT) performed during a follow-up visit for sigmoid colon cancer. The tumor had a diameter of 35mm on the CT scan and was diagnosed as pancreatic head carcinoma T3N0M0. The patient was treated with pylorus-preserving pancreaticoduodenectomy. Histopathological examination showed that the tumor had grown within a hollow structure, was contiguous with a duodenal diverticulum, and had partially invaded the pancreas. Immunohistochemistry results were as follows:CK7 negative, CK20 positive, CD10 negative, CDX2 positive, MUC1 negative, MUC2 positive, MUC5AC negative, and MUC6 negative. The tumor was diagnosed as duodenal carcinoma from the duodenal diverticulum. Preoperative imaging showed that the tumor was located in the head of the pancreas and was compressing the common bile duct, thus making it appear like pancreatic cancer. To the best of our knowledge, this is the second report of a case of duodenal carcinoma from a duodenal diverticulum mimicking pancreatic carcinoma.

Totally laparoscopic colectomy with intracorporeal anastomosis for colonic liposarcoma: A case report.

Liposarcoma is a type of soft tissue sarcoma. Primary colonic liposarcomas are extremely rare. An 86-year-old man with diarrhea and anorexia visited our outpatient clinic at Okayama City Hospital. Colonoscopy and computed tomography imaging revealed a large submucosal tumor in the descending colon with a maximum diameter of approximately 10 cm. He underwent totally laparoscopic left hemicolectomy with intracorporeal anastomosis. The histopathological diagnosis was a well-differentiated liposarcoma without lymph node metastases. The patient's postoperative course was uneventful. We herein report a rare case of totally laparoscopic colectomy with intracorporeal anastomosis in an elderly patient with colonic liposarcoma.

An experience of total laparoscopic partial colectomy with intracorporeal triangulating anastomosis in an obese patient with descending colon cancer.

A 68-year-old woman was transferred to the emergency room of Okayama City Hospital because of worsening epigastric pain. After the examination, she was diagnosed with descending colon cancer, and laparoscopic colectomy was planned. However, exteriorization of the bowels to produce anastomosis was difficult because the rich adipose tissue of the mesocolon hardly stretched, and the abdominal wall was thick as the patient was obese. Therefore, an intracorporeal triangulating end-to-end anastomosis was performed. The colon was divided at 10 cm either side from the tumor using an endoscopic linear stapler. After the resection of the stumps with staples, the posterior walls were tied with stay sutures and then stapled with an endoscopic linear stapler. The anterior wall was stapled twice in the same manner, and the intracorporeal anastomosis was completed. The patient's postoperative course was uneventful. This technique provided an alternative intracorporeal anastomotic technique as troubleshooting in laparoscopic colectomy.

Ileal perforation induced by acute radiation injury under gefitinib treatment.

Enteritis is one of the side effects of radiotherapy to the abdominal cavity. Radiation enteritis involves damage to mucous membranes in the acute phase and to stromal tissues in the late phase. Perforation of the intestine tends to occur in the late phase, and rarely in the acute phase. However, we describe here a case of intestinal perforation occurring in the acute phase after irradiation in a patient who received gefitinib treatment. Gefitinib, one of the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), is widely used to treat non-small cell lung cancer (NSCLC) patients, but is simultaneously known to inhibit wound healing. We suspect that gefitinib may affect regeneration of the small intestinal mucosa injured by irradiation. A 76-year-old woman had NSCLC with metastases to the 5th lumbar, sacral, and right iliac bones. To control the pain from bone metastasis, anterior-posterior opposing portal irradiation (total 35 Gy) was started, and was completed over 22 days. On day 25 after starting radiotherapy, the patient began to take gefitinib. On day 35, she presented with acute peritonitis, and an emergency laparotomy was performed. The terminal ileum was affected by radiation enteritis and there were two pin-hole perforations. In the surgical specimen, no cancerous lesions were detected, and immunohistochemical staining of phosphorylated EGFR (pEGFR) was negative. pEGFR has an important role in mucous membrane repair after irradiation. Intestinal perforation in the acute phase of radiation enteritis may be associated with impaired mucosal repair mechanisms due to the use of an EGFR-TKI such as gefitinib, as evidenced by the absence of pEGFR.

Aberrant methylation of p21 gene in lung cancer and malignant pleural mesothelioma.

Suppression of p21 has been implicated in the genesis and progression of many human malignancies. DNA methylation is an important mechanism of gene silencing in human malignancies. In this study, we examined the expression status and aberrant methylaion of p21 in lung cancers and malignant pleural mesotheliomas (MPM). We used 12 small cell lung cancer (SCLC) cell lines, 13 non-small cell lung cancer (NSCLC) cell lines, 50 primary NSCLCs, 6 MPM cell lines and 10 primary MPMs. The expression and methylation of p21 was examined by reverse transcription-PCR (RT-PCR), Western blotting and methylation-specific PCR (MSP) assay. Loss of p21 protein expression was observed in 7 SCLC cell lines (58.3%), 5 NSCLC cell lines (38.5%) and 3 MPM cell lines (50%) while mRNA expression was lost in 2 SCLC cell lines (16.7%), 2 NSCLC cell lines (15.4%) and none of the MPM cell lines. Aberrant methylation of p21 was found in 8.3% of SCLC cell lines, 30.2% of NSCLCs and 6.3% of MPMs. Among primary NSCLCs, methylation in adenocarcinomas was significantly more frequent than in squamous cell carcinomas. Loss of p21 expression was frequently observed in lung cancers and MPMs and aberrant methylation was one of the mechanisms of suppression of p21, especially in NSCLCs.

Impact of endoscopic and histological evaluations of two different types of mesh plug for a groin hernia model.

PURPOSE: The biological responses to mesh in vivo have been evaluated in some papers, but the in vivo condition of mesh and plugs have not been sufficiently evaluated. This study evaluated the endoscopic observations and histological assessments of mesh plugs using swine models. METHODS: An artificial abdominal hernia was established in the porcine abdomen, and repaired using three different sizes of two types of plug, Proloop (ATRIUM Medical Corporation, Hudson, NH, USA) or Perfix (BARD Medical Division, Covington, GA, USA). The in vivo conditions of each plug were periodically observed using a laparoscope. Moreover, a histological evaluation of the plugs was performed 3 months after implantation. RESULTS: The laparoscopic observation revealed that inversion of the plugs occurred in 10 out of 18 cases repaired with Perfix, while no case repaired with Proloop inverted. The large and medium sizes of Perfix plugs were inclined by an average of more than 30°. In addition, the triangular shape of Perfix plugs was broken and the vertical/horizontal ratio was enlarged during the observation period, while Proloop plugs shrank both vertically and horizontally. The inflammatory cell count was significantly lower within the Proloop plugs than within Perfix plugs. CONCLUSION: Proloop plugs are apparently superior because they are stable even 3 months after implantation.

Usefulness of cumulative smoking dose for identifying the EGFR mutation and patients with non-small-cell lung cancer for gefitinib treatment.

We examined the diagnostic accuracy of the cumulative smoking dose for identifying the epidermal growth factor receptor (EGFR) exon 19 deletion and L858R mutation among Japanese patients with non-small-cell lung cancer (NSCLC). EGFR mutations in exon 19 and exon 21 were determined in 1001 NSCLC patients. A receiver-operating characteristic (ROC) curve methodology was applied to estimate the diagnostic accuracy. EGFR mutations were detected in 314 patients (31.4%). A cumulative smoking dose of less than 13 pack-years (PY) was the optimal cut-off point for predicting a positive EGFR mutation status, producing a balance between the sensitivity (73.5%) and the specificity (77%). The area under the ROC curve was 0.77, indicating that the smoking dose had a moderate diagnostic accuracy. The median survival time or the median progression-free survival time of patients who had smoked less than 13 pack-years (PY) were 18.6 and 6.3 months, respectively, while those of patients with equal to or more than 13 PY were 9.6 and 2.4 months, respectively. The overall survival (OS) and progression-free survival (PFS) rates were significantly different between the two groups (OS: hazard ratio [HR] = 0.64, 95% confidence interval [CI] = 0.51-0.80, P = 0.0001) (PFS: HR = 0.58, 95% CI = 0.47-0.71, P < 0.0001). Our study indicated that the smoking dose predicted EGFR mutations with a moderate diagnostic accuracy. Thus, patients who have smoked less than 13 PY might be candidates for gefitinib treatment when EGFR mutation status cannot be determined.

Breast cancer with cartilaginous and/or osseous metaplasia diagnosed by lymph nodal metastasis: a case report.

Breast cancer with cartilaginous and/or osseous metaplasia is a type of metaplastic carcinomas and is a rare disease. We report the case of a 49 year-old female who underwent right mastectomy for a large breast tumor. Histological examinations revealed a mixed tumor with both stromal and epithelial elements;the stroma showed poor differentiated spindle-shape and multiform cells with a massive osseous matrix, and atypical epithelial cells, which mainly existed on the surface of the cysts, showed nucleic atypia. The tumor was diagnosed as a malignant phyllodes tumor with osteosarcomatous differentiation;it was not identified as a metaplastic carcinoma because of the lack of proof of a cancerous component. Two years after a mastectomy, swelling of the axillary lymph nodes was found and a biopsy was performed. Histological findings for the lymph node indicated a metastasis of the invasive ductal carcinoma. The primary tumor was re-examined and was considered to be the origin of the lymph nodal metastasis. Lymph nodal metastasis of cancer proved that the primary tumor had cancerous potential, and the pathological diagnosis was altered to a breast cancer with cartilaginous and/or osseous metaplasia.

The aberrant promoter methylation of BMP3b and BMP6 in malignant pleural mesotheliomas.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta superfamily. Recent studies have showed that aberrant methylation of BMP genes is present in several types of human cancer. We examined the expression and methylation status of BMP3b and BMP6 in malignant pleural mesotheliomas (MPMs). The expression status of BMP3b, and BMP6 mRNAs were examined in seven MPM cell lines by RT-PCR assay. The expression of BMP3b was completely suppressed in 2 and partially suppressed in 2 of 7 cell lines and expression of BMP6 was partially suppressed in 2 cell lines. Methylation status of BMP3b in cell lines was determined by methylation-specific assay to find aberrant methylation in 6 cell lines which include 4 cell lines with suppressed BMP3b expression. Partial methylation of BMP6 was found in 2 cell lines whose expression was partially suppressed. Treatment with 5-Aza-dC restored BMP3b expression in methylated cell lines. Next, we examined the methylation status in 57 surgically resected MPM cases and found aberrant methylation of BMP3b in 9 (53%) out of 17 cases from Japan and 3 (8%) of 40 cases from USA and that of BMP6 in 4 (24%) cases from Japan and 12 (30%) cases from USA, showing significant difference in frequency of BMP3b methylation between MPMs of the two countries (P=0.0004). Our study indicated that BMP3b and BMP6 genes were suppressed by DNA methylation and methylation of BMP3b is significantly frequent in Japanese MPMs, suggesting its pathogenic role and the ethnic difference in MPMs.