Search

Supplementary Table 1 from Activation of DNA Methyltransferase 1 by EBV Latent Membrane Protein 2A Leads to Promoter Hypermethylation of PTEN Gene in Gastric Carcinoma

CpG island promoter methylation of tumor suppressor genes is one of the most characteristic abnormalities in EBV-associated gastric carcinoma (GC). Aberrant promoter methylation and expression loss of PTEN were evaluated in cancer tissues of GC by methylation-specific PCR and immunohistochemistry, respectively, showing that both abnormalities occurred concurrently in EBV-associated GC. PTEN abnormalities were reiterated in GC cell lines MKN-1 and MKN-7 infected with recombinant EBV, and DNA methyltransferase 1 (DNMT1) was commonly overexpressed in both cell lines. Stable and transient transfection systems in MKN-1 similarly showed that viral latent membrane protein 2A (LMP2A) up-regulated DNMT1, leading to an increase in methylation of the PTEN promoter. Importantly, the level of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) increased in the nuclei of LMP2A-expressing GC cells, and knockdown of STAT3 counteracted LMP2A-mediated DNMT1 overexpression. Immunohistochemistry for both pSTAT3 and DNMT1 showed diffuse labeling in the nuclei of the cancer cells in GC tissues, especially in EBV-associated GC. Taken together, LMP2A induces the phosphorylation of STAT3, which activates DNMT1 transcription and causes PTEN expression loss through CpG island methylation of the PTEN promoter in EBV-associated GC. LMP2A plays an essential role in the epigenetic abnormalities in host stomach cells and in the development and maintenance of EBV-associated cancer. [Cancer Res 2009;69(7):2766–74]

An 85-year-old man with a history of aortic dissection suddenly fainted, underwent cardiac heart arrest, and died. An autopsy was performed, but the cause of death was not grossly identified. Congo red staining detected amyloid deposits in systemic organs, including the heart, lungs, liver, and kidneys. Immunohistochemical (IHC) analysis revealed immunoglobulin (Ig) λ light chain (-λ) in systemic blood vessels and transthyretin (TTR) in the heart and lungs. Ig-λ was predominantly positive in the blood vessels of the lungs, while TTR was detected in the alveolar septum. In the heart, Ig-λ was positive in the endocardium and blood vessels, and TTR was positive in nodular deposits between cardiomyocytes. The concurrent deposition of Ig-λ and TTR in the heart was further substantiated by laser microdissection (LMD)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) at each deposition site. Despite systemic deposition of Ig-λ, bone marrow biopsy findings were not diagnostic for multiple myeloma. In summary, we present an autopsy case of concurrent Ig-λ and TTR deposition as revealed by IHC and LC-MS/MS. When Congo red staining and IHC results are indeterminate due to the deposition of multiple amyloid proteins, LMD-LC-MS/MS is useful for determining the precursor protein.

International audience; Abstract Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error ( p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections ( p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.; v2.5, 25 March 2021.

Histiocytic sarcoma is a relatively new disease category and the gastrointestinal origin is sporadic. We report a case of a 74-year-old woman who underwent chemotherapy and proximal gastrectomy for extremely rare, advanced gastric histiocytic sarcoma. The resected specimen was subjected to numerous immunostainings to meet the diagnostic criteria of histiocytic sarcoma and was positive for the histiocyte markers' cluster of differentiation 68 and lysozyme. The markers of Langerhans cells, follicular dendritic cells, and myelocyte were all negative. Six reports of surgical resection of histiocytic sarcoma originating in the stomach exist, including our case. We reviewed the clinical course and the histological and immunohistochemical diagnostic features of surgically resected gastric histiocytic sarcoma.

Background: Non-palpable breast lesions represent about 25-35% of all breast cancers diagnosed in developed countries, according to findings based on the evolution of imaging modalities such as MRI, and the uses reliable biopsy techniques such as ultrasound-guided or stereo-guided vacuum assisted biopsy. Wire -guided localization (WGL), radio-guided localization (RGL), and SAVI SCOUT localization (SSL) have been presented as a surgical procedure for non-palpable breast lesion. The reported disadvantages of WGL are related to mechanical stimulation of wire plucking, kinking, and patient discomfort. RGL has issues regarding radioactive licensing, handling, and waste management, and the SSL system requires high start-up costs. To eliminate these problems, we verified the magnetically guided localization (MGL) method for breast lesion localization by means of the combinations of the magnetic probe TAKUMI (ISO13485, Matrix cell Research Institute Inc., Tokyo, Japan) and the guiding-marker system® (Hakko, Tokyo, JAPAN). The TAKUMI is a novel handheld magnetic probe with a permanent magnet and a Hall magnetic sensor for detecting magnetic substances. It was newly developed at the University of Tokyo under a grant from the Japan Agency for Medical Research and Development (AMED). The aim of this study was to evaluate the feasibility and safety of our MGL system. Method: Patients were enrolled in this multi-center, open-label trial from January 2019 to March 2019 at two University Hospitals. The inclusion criteria were age 20 or older female patients who had non-palpable breast lesions, for which breast-conserving surgery or tumor resection was performed. Patients who had a metal allergy, pregnancy, or inflammatory breast cancer were excluded. The guiding-marker system® consists of a stainless-steel hook (φ 0.28 × 10 mm) connected with a nylon thread and an a 21-gauge 10 cm long steel needle.The marker was inserted into the center of the target lesions using ultrasound guidance or stereo guidance within 4 days before surgery. The TAKUMI was used to determine whether the guiding marker was detectable or not before, during, and after the surgical resection of the specimen. The resection rate of the guiding marker, surgical margin status, and re-operation rates were evaluated as the primary outcomes, and the volume and weight of the excised specimen were evaluated as secondary outcomes. The study protocol of the evaluation of magnetic probe system for detecting of non-palpable lesions of the breast was approved by the Institutional Review Board of Nippon Medical School Foundation (CRB3180001) and was registered at https://jrct.niph.go.jp/re (protocol record jRCTs032180422). Written informed consent was obtained from all participants. Results: Forty-one patients were recruited into this study. Thirty-eight patients (92.6%) underwent breast-conserving surgery for breast cancer treatment, and 3 (7.3%) underwent tumor resection for biopsy purposes. All guiding markers were resected during the initial surgical operation. Three out of 38 breast cancer patients (7.9%) were diagnosed as margin positive in frozen section analysis; 2 (5.2%) underwent additional resection during the initial surgery due to the positive margin, and 1 (2.6%) underwent subsequent surgery due to the positive margin on final pathology. Eight patients (21%) underwent boost radiation therapy due to the close margin ( Citation Format: Tomoko Kurita, Kanae Taruno, Seigo Nakamura, Hiroyuki Takei, Katsutoshi Enokido, Takashi Kuwayama, Yoko Kanada, Sadako Akashi, Maki Nakai, Meishi Hankyo, Keiko Yanagihara, Takashi Sakatani, Kentaro Sakamaki, Akihiko Kuwahata, Masaki Sekino, Moriaki Kusakabe. Magnetic guided localization for non-palpable breast lesion: A multi-center open-label trial in Japan [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-20-17.

Background Non-functioning parathyroid carcinoma is an extremely rare malignancy among endocrine tumors. We report a case in which non-functional oxyphilic parathyroid carcinoma was diagnosed from clinical symptoms and pathological diagnosis. Case presentation The patient was a 42-year-old man with no medical or family history of note. He had presented to a local hospital with a neck mass 2 months earlier. Medullary thyroid carcinoma was diagnosed and he was referred to our department. A 3.5-cm mass was observed in the left thyroid lobe. Laboratory data for thyroid functions, thyroglobulin, anti-thyroglobulin antibodies, anti-thyroid peroxidase antibodies, serum calcium, and parathyroid hormone (PTH) were all within normal ranges. Ultrasonography revealed a 40-mm irregular, hypoechoic mass throughout the left thyroid lobe. Follicular thyroid tumor was suspected from fine-needle aspiration cytology. Left lobectomy was performed. Pathological features revealed a thick fibrous capsule around the tumor, and a thick fibrous band was observed inside the tumor. Both capsular invasions and vascular invasions were observed. Tumor cells were eosinophilic and displayed solid growth. Immunohistochemically, tumor cells were negative for thyroid transcription factor-1, negative for thyroglobulin, negative for chromogranin A (positive for normal parathyroid tissue within the nodule), positive for PTH, and positive for parafibromin. Ki-67 labeling index was 10%. Based on these findings, non-functional oxyphilic parathyroid carcinoma was diagnosed. One and a half years postoperatively, calcium and PTH were within normal ranges, and he has shown no evidence of recurrence or metastasis. Conclusions Non-functioning oxyphilic parathyroid carcinoma is an extremely rare malignancy, and definitive diagnosis is difficult to obtain preoperatively. Few reports have been made worldwide, and information on the long-term prognosis is scarce. Long-term surveillance by imaging is mandatory, since no indices that can be used as a marker for postoperative recurrence and metastasis have been identified.

Current immunohistochemistry methods for diagnosing abnormal cells, such as cancer cells, require multiple steps and can be relatively slow compared with intraoperative frozen hematoxylin and eosin staining, and are therefore rarely used for intraoperative examination. Thus, there is a need for novel rapid detection methods. We previously demonstrated that functionalized fluorescent ferrite beads (FF beads) magnetically promoted rapid immunoreactions. The aim of this study was to improve the magnetically promoted rapid immunoreaction method using antibody-coated FF beads and a magnet subjected to a magnetic field. Using frozen sections of xenograft samples of A431 human epidermoid cancer cells that express high levels of epidermal growth factor receptor (EGFR) and anti-EGFR antibody-coated FF beads, we reduced the magnetically promoted immunohistochemistry procedure to a 1-min reaction and 1-min wash. We also determined the optimum magnetic force for the antibody reaction (from 7.79 × 10−15 N to 3.35 × 10−15 N) and washing (4.78 × 10−16 N), which are important steps in this technique. Furthermore, we stained paraffin-embedded tissue arrays and frozen sections of metastatic breast cancer lymph nodes with anti-pan-cytokeratin antibody-coated FF beads to validate the utility of this system in clinical specimens. Under optimal conditions, this ultra-rapid immunostaining method may provide an ancillary method for pathological diagnosis during surgery. (J Histochem Cytochem 58:XXX–XXX, 2010)

Aims: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. Methods and results: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot-spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799–0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot-spot method (0.83; 95% CI = 0.74–0.90) did not. Visually, interobserver concordance in location of selected hot-spots varies between cases. The median times for scoring were 9 and 6 min for global and hot-spot methods, respectively. Conclusions: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further.

Adding taxane to an anthracycline-based regimen improves survival in node-positive breast cancer patients, as shown by clinical trials and meta-analyses. However, no studies have analyzed the number of metastatic lymph nodes in patients with estrogen receptor (ER)-positive cancer. This study investigated whether adding a taxane to an anthracycline-based regimen improved prognosis in node-positive, ER-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer patients in a real-world setting. Using Japanese Breast Cancer Society registry data, we compared disease-free survival (DFS) of patients with ER-positive, HER2-negative breast cancer, excluding those receiving neoadjuvant chemotherapy, between those who received an anthracycline-based regimen followed by a taxane-based regimen (A + T) and those who received only an anthracycline-based regimen (A w/o T), stratified by lymph node status. A Cox proportional hazards model was used to evaluate DFS in both groups. There were 4566 eligible patients with ER-positive, HER2-negative breast cancer. During the median follow-up period of 60 months, there were 481 recurrences and 149 deaths. There was no significant difference in DFS between the A + T and A w/o T groups among patients with 1–3 positive nodes, while there was a significant difference among patients with ≥ 4 positive nodes. In patients with ER-positive, HER2-negative breast cancer, adding taxane to an anthracycline regimen did not improve DFS in patients with metastasis in 1–3 lymph nodes. We considered that the group without the addition of taxane might be present in patients with ER-positive, HER2-negative lymph node metastases.

BACKGROUND A retrospective study of the real-world use of neoadjuvant endocrine therapy (NET) is important for standardizing the role of NET in breast cancer care. METHODS In a consecutive series of women with operable breast cancer who received NET for ≥28 days, associations of NET objectives, NET outcomes, adjuvant chemotherapy use after NET, and survival with clinicopathological factors were examined. RESULTS NET objectives were reduction in surgical extent in 49 patients, avoidance of surgery in 31, and treatment until scheduled surgery in 8. The mean duration of NET was 349.5 (range, 34-1,923), 869.8 (range, 36-4,859), and 55.8 (range, 39-113) days, respectively, in these cohorts (success rate: 79.6%, 64.5%, and 100%, respectively), and the differences were significant. Among patients in the former two cohorts, progression-free survival was significantly better in patients with stage 0 or I disease, ductal carcinoma in situ or invasive ductal carcinoma, ≥71% estrogen receptor (ER) positivity, and the surgical extent reduction cohort than the other counterparts. Postoperative chemotherapy use was significantly associated with lymph node metastasis, a high Ki67 labeling index, lymphovascular invasion, and a high preoperative endocrine prognostic index at the time of surgery after NET. Better recurrence-free survival after surgery was significantly associated with high ER expression after NET or high progesterone receptor expression before or after NET. CONCLUSIONS NET can help reduce surgical extent or avoid surgery in women with early breast cancer, ductal carcinoma, or high ER expression. NET may also aid in decisions related to postoperative systemic therapy to improve survival.

Background The caudal type homeobox 2 transcription factor (CDX2) is a specific and sensitive marker for intestinal carcinoma, but usually not expressed in breast cancer. In CDX2-positive metastatic cancer of occult primary, the origin is highly suspicious of an enteric carcinoma. Case presentation A 50-year-old woman complained of enlarged lymph nodes (LNs) in the right axilla. Mammography and ultrasonography scans showed no abnormal findings in her breasts. Core needle biopsy (CNB) revealed metastatic adenocarcinoma. Immunohistochemical staining was positive for CDX2 intensely. The primary tumor was suspicious of intestinal adenocarcinoma. A dynamic contrast-enhanced magnetic resonance imaging scan revealed an accentuated lesion which was detected using a second-look ultrasound, and diagnosed invasive ductal carcinoma by CNB. A partial mastectomy of the right breast with level I and II axillary LN dissection was performed. A few cells of primary cancer were expressed CDX2 and estrogen receptor. The final pathological diagnosis was T1bN3aM0 stage IIIC. The fluorescent double staining showed that CDX2 simultaneously expressed on the Ki67 positive cells of metastatic tumors. The adjuvant treatment included chemotherapy and radiation, followed by tamoxifen administration. The patient survived without any recurrences over the following 36 months. Conclusions We report a rare case of CDX2-positive metastatic breast cancer in the axillary LNs. As some literatures reported vitamin D pathways induced cancer cell apoptosis and inhibition, these metastatic cells of our case might play the effort of autoregulation of inhibiting progression.

Objective The Japan Clinical Oncology Group 1505 trial is a single-arm multicentre prospective study that examined the possibility of non-surgical follow-up with endocrine therapy for patients with low-grade ductal carcinoma in situ. In that study, the eligible criteria included histopathological findings comprising low to intermediate nuclear grade and absence of comedo necrosis, and cases were entered according to the local histopathological diagnosis. Nuclear grade is largely based on the Consensus Conference criteria (1997), whereas comedo necrosis is judged according to the Rosen’s criteria (2017). The purpose of this study was to standardize and examine the interobserver agreement levels of these histopathological criteria amongst the participating pathologists. Methods We held slide conferences, where photomicrographs of haematoxylin–eosin-stained slides from 68 patients with ductal carcinoma in situ were presented using PowerPoint. The nuclear grade and comedo necrosis statuses individually judged by the pathologists were analysed using κ statistics. Results In the first and second sessions, where 22 cases each were presented, the interobserver agreement levels of nuclear grade whether low/intermediate grade or high grade were moderate amongst 29 and 24 participating pathologists, respectively (κ = 0.595 and 0.519, respectively). In the third session where 24 cases were presented, interobserver agreement levels of comedo necrosis or non-comedo necrosis were substantial amongst 25 participating pathologists (κ = 0.753). Conclusion Although the concordance rates in nuclear grade or comedo necrosis were not high in a few of the cases, we believe that these results could provide a rationale for employing the present criteria of nuclear grade and comedo necrosis in the clinical study of ductal carcinoma in situ.

Breast cancer survival outcomes vary across different ethnic groups. We clarified the differences in clinicopathological and survival characteristics of breast cancer among Japanese, US residents with Japanese origin (USJ), and US residents with other origins (USO). Using Surveillance, Epidemiology, and End Results (SEER) 18 dataset and Japanese Breast Cancer Society (JBCS) registry, we included patients first diagnosed with breast cancer between 2004 and 2015. We categorized the patients into three groups based on the database and the recorded ethnicity: Japanese (all those from the JBCS registry), USJ (those from SEER with ethnicity: Japanese), and USO (those from SEER with ethnicity other than Japanese). Excluding patients diagnosed after 2012, stage 0, and 4 patients, we examined the overall survival (OS) and breast cancer-specific survival (BCSS) using the Kaplan–Meier method and Cox proportional hazards models, adjusting for age, sex, cancer stage, and hormone receptor (HR) status. We identified 7362 USJ, 701,751 USO, and 503,013 Japanese breast cancer patients. The proportion of HR-positive breast cancer was the highest among USJ (71%). OS was significantly longer among Japanese and USJ than USO (Hazard ratio 0.46; 95% Confidence Interval [CI] 0.45–0.47 for Japanese and 0.66 [95% CI 0.59–0.74] for USJ) after adjusting for baseline covariates. BCSS was also significantly higher in the two groups (HR 0.53 [95% CI 0.51–0.55] for Japanese and 0.53 [95% CI 0.52–0.74] for USJ). In stage I–III breast cancer, Japanese and US residents with Japanese origin experienced significantly longer survival than US residents with non-Japanese origins.

Aim Infants with fetal growth restriction (FGR) are at an increased risk of perinatal morbidity and mortality. The long noncoding RNA H19 gene is expressed abundantly in placental villi and recent studies suggest that it regulates FGR. However, the role of H19 in the FGR placenta remains unclear. This study aimed to clarify the relationship between H19 expression and FGR using normotensive placentas after 34 weeks of gestation. Methods Formalin-fixed paraffin-embedded tissues from human placentas collected from pregnancies resulting in small for gestational age (SGA) and appropriate for gestational age (AGA) newborns were used. The histopathological features of placenta tissues, such as villous stromal fibrosis, the numbers of terminal villi, villous vessels and cytotrophoblasts were analyzed using hematoxylin and eosin, Masson's trichrome staining and immunostaining. The localization and expression of H19 in the placentas were demonstrated by in situ hybridization and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. Moreover, the expression levels of H19-regulated molecules such as IGF2 and decorin (DCN) were measured by RT-qPCR. Results Histopathological features of the placental villous were not different between placentas associated with SGA and AGA. H19 localized to the villous stroma, endothelial cells and cytotrophoblasts. Moreover, the expression level of H19 in SGA placentas was significantly lower than that in AGA placentas. The expression levels of IGF2 and DCN in SGA placentas tended to be lower than those in AGA placentas similarly to H19. Conclusion This study highlights the potential importance of regulatory events mediated by H19 in SGA placentas without histopathological abnormalities in late pregnancy.

Programmed death-ligand 1 (PD-L1) expression on immune cells (ICs) is a predictive marker for PD-L1 checkpoint blockade in patients with triple-negative breast cancer (TNBC). However, the level of PD-L1 expression and the percentage of cells that are PD-L1+ are continuous variables not dichotomous variables for tumor-infiltrating lymphocytes (TILs) and other cells. Multiplexed immunohistochemistry was applied to 31 archived surgical specimens from untreated TNBC patients. TIL levels were visually scored, and CD8+ T cells and PD-L1+ ICs were quantified using an automated multispectral imaging system. PD-L1 expression was assessed within a multiplexed context (CD8 combined spectral composite). The mean value of stromal TILs (i.e., the percentage of the stromal area with a dese mononuclear infiltrate) was 20%. The frequency of patients with PD-L1-positive tumor cells (TC) and ICs was 38.7% and 32.2%, respectively, with a significant association between them. TIL levels were correlated with CD8+ T cell infiltration in the stroma (Spearman r = 0.795, p

Platelet-rich plasma (PRP) contains numerous growth factors and promotes bone fracture healing. The aim of this study was to evaluate the effectiveness of the controlled release of PRP from biodegradable gelatin hydrogel for promoting healing in a rabbit ischemic sternal model. PRP was prepared from the whole blood of a Japanese white rabbit. Sixteen rabbits were randomized into four groups (each n = 4) and all underwent median sternotomy and bilateral internal thoracic artery removal. Before the sternum was closed, the following solutions were applied between the sternum incisions in three of the groups: 30 mg of gelatin hydrogel incorporating 300 μL of phosphate-buffered saline, 300 μL of a solution form of PRP, or 30 mg of gelatin hydrogel incorporating 300 μL of PRP (PRP + Gel). The fourth group acted as a control. Sternal healing was evaluated by histology and microcomputed tomography 7 days after the intervention. The PRP + Gel group showed a significantly higher proportion of fibrosis within the fracture area (an indicator of sternal healing) than the other groups and a significantly higher mean intensity of osteocalcin. These results indicate that the controlled release of PRP from locally applied gelatin hydrogel was markedly effective in enhancing sternal healing in the early postoperative period. This novel therapy could potentially help prevent complications, such as deep sternal wound infection and could result in early postoperative ambulation after median sternotomy.

Breast carcinoma with osteoclast-like giant cells (OGCs) is a rare tumor; however, their clinicopathological aspects remain unclear. We described the clinicopathological characteristics of 8 patients with breast carcinoma with OGCs. Immuno-phenotypes of the OGCs were comparatively examined with that of foreign body giant cells (FBGCs) in 4 cases of granulomatous reaction (GR) without cancerous elements. In most cancers, tumors displayed cribriform and tubular growth patterns. Three cases showed moderate to high nuclear grade, while all the other tumors had low nuclear grade. Six patients were estrogen receptor (ER) positive, while triple negative phenotype was identified in 2 patients. During the follow-up period, 1 patient had local recurrence of the tumor, and all the patients remained alive. All OGCs and FBGCs expressed CD68, a pan-macrophage marker. OGCs in all the breast cancers showed moderate to high expression of CD163 - a marker of M2-macrophage with pro-tumoral function - whereas its expression in FBGCs was low to moderate (p=0.04). CD86 - a marker of M1-macrophage with a tumoricidal activity - was positive in the OGCs of 3 breast cancers, and in the FBGCs of 3 GR cases (p=0.15). The expression of CD163 was significantly higher than that of CD86 in the OGCs of breast cancer (p

Accurate pre-operative localization of nonpalpable lesions plays a pivotal role in guiding breast-conserving surgery (BCS). In this multicenter feasibility study, nonpalpable breast lesions were localized using a handheld magnetic probe (TAKUMI) and a magnetic marker (Guiding-Marker System®). The magnetic marker was preoperatively placed within the target lesion under ultrasound or stereo-guidance. Additionally, a dye was injected subcutaneously to indicate the extent of the tumor excision. Surgeons checked for the marker within the lesion using a magnetic probe. The magnetic probe could detect the guiding marker and accurately localize the target lesion intraoperatively. All patients with breast cancer underwent wide excision with a safety margin of ≥5 mm. The presence of the guiding-marker within the resected specimen was the primary outcome and the pathological margin status and re-excision rate were the secondary outcomes. Eighty-seven patients with nonpalpable lesions who underwent BCS, from January to March of 2019 and from January to July of 2020, were recruited. The magnetic marker was detected in all resected specimens. The surgical margin was positive only in 5/82 (6.1%) patients, these patients underwent re-excision. This feasibility study demonstrated that the magnetic guiding localization system is useful for the detection and excision of nonpalpable breast lesions.

An 82-year-old woman who had been suffering from repeated obstructive jaundice for 7 years was referred to our hospital. Although endoscopic aspiration of the mucin in the common bile duct had been temporally effective, origin of the mucin production had not been detectable. The patient thus had been forced to be on long-term follow-up without curative resection. Endoscopic retrograde cholangioscopy on admission revealed massive mucin in the common bile duct. In addition, an anomalous bile duct located proximal to the gallbladder was identified. Since the lumen of the anomalous duct was irregular and the rest of biliary tree was completely free of suspicious lesions, the anomalous duct was judged to be the primary site. Surgical resection of the segment 4 and 5 of the liver combined with the extrahepatic biliary tract was performed. Pathological diagnosis was compatible to intraductal papillary neoplasm with high-grade intraepithelial dysplasia of the anomalous bile duct. The patient has been free from the disease for 6.5 years after resection. This is the first case of intraductal papillary neoplasm derived from an anomalous bile duct, which was resected after long-term conservative treatment. The present case suggested the slow growing character of natural history of the neoplasm.

Farnesoid X receptor (FXR) exhibits critical anti-cancer functions in several types of cancer, including colorectal cancer, in vitro and in vivo. However, the underlying mechanism remains unclear. We evaluated pharmacological activation of FXR with the synthetic agonist GW4064 using comprehensive proteomic analysis in colorectal cancer cell lines (HCT116, SW480, and DLD1). Among the commonly detected proteins in all three cell lines, death receptor 5 (DR5) was the most up-regulated protein, and key autophagy-related proteins, such as microtubule-associated protein 1 light chain 3 alpha/beta (MLP3A/3B) and p62 sequestosome-1 (SQSTM), were also differentially expressed. Western blot analysis showed that GW4064 stimulation induced activation of the extrinsic death signaling pathway in all cell lines and induced activation of the intrinsic death signaling pathway in DLD1 cells. Western blotting showed that DR5 up-regulation was associated with inhibition of autophagic activity. These results suggest that FXR activation induced DR5 up-regulation through inhibition of autophagic activity and the DR5-related death signaling pathway. In addition, DR5 selective ligand, also known as TRAIL, has been widely used for anti-cancer treatment in several clinical trials. Co-treatment of TRAIL with GW4064 synergistically inhibited colorectal cancer cell proliferation as compared with single treatments. To the best of our knowledge, our results provide novel insights into FXR function in cancer cell lines. These findings may contribute to a new therapeutic strategy for colorectal cancer.

Toll‑like receptor 4 (TLR4), a key regulator of the innate immune system, is expressed not only in immune cells, but also in a number of cancer cells. A biological role for TLR4 in cutaneous squamous cell carcinoma (SCC), however, is unclear. In this study, we first examined TLR4 expression and localization in cases of SCC, actinic keratosis (AK) and Bowen's disease (BD) by immunohistochemistry. TLR4 expression was significantly higher in the SCC than in the AK or BD tissues. We then determined the TLR4 expression level in vivo, in 3 histological subtypes of SCC. TLR4 expression in poorly differentiated SCC was significantly lower compared with that of the moderately and well‑differentiated type. In addition, the CD44 immunoreactivity tended to be high in the cell membrane of poorly differentiated SCC. Of note, poorly differentiated SCC is a risk factor of unfavorable outcomes in affected patients. We then assessed the biological role of TLR4 in HSC‑1 and HSC‑5 SCC cells and HaCaT human keratinocytes. TLR4 knockdown by transfection with siRNA accelerated HSC‑1 and HaCaT cell migration and invasion compared to the control siRNA‑transfected cells. TLR4 knockdown resulted in an increased CD44 expression and in an enhanced filopodia protrusion formation, particularly in HSC‑1. On the whole, these results suggest that a reduced TLR4 expression enhances the malignant features in SCC cases and cultured SCC cell lines. TLR4 may thus play an anti‑tumor role in cutaneous SCC.

Decreased cell adhesion has been reported as a significant negative prognostic factor of lung cancer. However, the molecular mechanisms responsible for the cell incohesiveness in lung cancer have not yet been elucidated in detail. We herein describe a rare histological variant of lung adenocarcinoma consisting almost entirely of individual cancer cells spreading in alveolar spaces in an incohesive pattern. A whole exome analysis of this case showed no genomic abnormalities in CDH1 or other genes encoding cell adhesion molecules. However, whole mRNA sequencing revealed that this case had an extremely high expression level of mucin 21 (MUC21), a mucin molecule that was previously shown to inhibit cell‐cell and cell‐matrix adhesion. The strong membranous expression of MUC21 was found on cancer cells using mAbs recognizing different O‐glycosylated forms of MUC21. An immunohistochemical analysis of an unselected series of lung adenocarcinoma confirmed that the strong membranous expression of MUC21 correlated with incohesiveness. Thus, MUC21 could be a promising biomarker with potential diagnostic and therapeutic applications for lung adenocarcinoma showing cell incohesiveness.

BACKGROUND AND OBJECTIVES Emerging evidence suggests that the presence of atypical mitoses is associated with poor prognosis in some types of cancer, but its clinical significance remains uncertain. Here, we investigated the occurrence of atypical mitoses in breast cancers. METHODS Mitotic figures, including normal and atypical mitoses, were assessed in resected histological sections from 109 patients with invasive carcinoma of no special type (ICNST). Comparisons with clinicopathological features and biomarkers such as Ki67 and phosphohistone H3 (PHH3) were performed. RESULTS The total number of mitotic figures, including atypical mitoses, was higher in situ and invasive ductal carcinoma components than in normal ducts. Morphological characteristics of atypical mitoses included multipolar, lagged, ring, asymmetrical mitoses, and anaphase bridge. Patients with higher total mitoses and PHH3, and the presence of atypical mitoses showed reduced overall survival (OS), compared to those with lower total mitoses and PHH3, and without atypical mitoses (P = 0.03, 0.02, and

Objectives: Evaluation of Intraductal Components (ICs) of breast cancer, such as ductal extension or segmental distribution, is essential before surgery. Evaluation is mainly performed using contrast-enhanced MRI as its sensitivity and detectability are superior to those of contrast-enhanced CT. Dual Energy CT (DECT) has been shown to have a high contrast enhancement effect with high resolution in soft tissues such as breast tissue. Thus, we assessed the utility of DECT for evaluating an IC of breast cancer. Methods: Fifty-five patients with a biopsy-proven breast cancer have undergone contrast-enhanced DECT and MRI for preoperative investigation. We compared the imaging findings of both modalities and correlated them with the pathological outcome. Results: Pathologically, an IC of breast cancer was identified in 26 patients. Existence of an IC was identified in 32 patients on DECT and 35 patients on MRI. The sensitivity, specificity and accuracy for detecting an IC were 76.9%, 58.6% and 67.3% with DECT and were 73.1%, 44.8%, and 58.2% with MRI, respectively. Conclusions: The appearance of ICs on DECT was highly correlated with the pathological outcome. The advantages of DECT include high detectability of an IC, clinically acceptable image quality, imaging in the face-up position (consistent with the surgical position) and simultaneous assessment of distant metastasis without radiation dose penalty. Thus, DECT is a useful supporting modality for the detection of an IC of breast cancer.

Breast carcinoma with osteoclast-like giant cells (OGCs) is a rare disease characterized by the infiltration of OGCs in the tumor; however, cytological aspects of this tumor type remain elusive. We examined the cytological features in fine needle aspiration (FNA) biopsy smears obtained from 5 patients who were histologically diagnosed with breast carcinoma with OGCs. We compared FNA and clinicopathological findings with results from the published literature. Histological assessment of the resected samples showed that all tumors exhibited a histological grade 1 phenotype with a predominant cribriform architecture. Four patients were estrogen receptor positive, and 1 patient showed a triple negative phenotype. All patients survived without tumor recurrence. In the FNA smears, tumor cells were arranged in loosely cohesive clusters, characterized by varying degrees of OGCs infiltration and rare formation of solid tumor nests. Occasionally, 2- or 3-dimensional clusters of tumor cells were found, accompanied by OGCs at the peripheral regions. In all patients, tumor cells were small without severe nuclear atypia. None of the patients showed significant background necrosis. In summary, cytological features of breast carcinoma with OGCs are characterized by loose aggregates of low grade tumor cells, the presence of OGCs, and the absence of necrosis, all of which were consistent with features reported previously. This peculiar form of breast tumors should be included in the differential diagnosis, when physicians encounter FNA findings including low grade ductal carcinoma with the admixture of multinucleated giant cells or OGCs.

Rosai-Dorfman disease (RDD) is a benign histiocytic proliferative disorder characterized by the accumulation of histiocytes in lymph nodes and various other organs. RDD seldom involves the central nervous system, and cases of purely intracranial RDD are particularly rare. We report a case of purely intracranial RDD involving the brainstem that was diagnosed at autopsy. A 68-year-old woman visited our hospital because of visual disturbances and loss of energy. Magnetic resonance imaging revealed an obscure mass in the brainstem. Despite exhaustive work-ups, the etiology of the intracranial mass remained unclear. The patient died of respiratory depression, and an autopsy was performed for pathological investigation. Macroscopically, a pink pale mass 2.5 cm in diameter was found in the brainstem, with no attachment to the dura. Histologically, it was composed of histiocytic cells with incorporation of small lymphocytes (emperipolesis). Immunohistochemical staining revealed that the cells were positive for CD68 and S100 and negative for CD1a, consistent with a diagnosis of RDD. Purely intracranial RDD is extremely rare and considered benign. To date, nine cases (including ours) have been reported. To our knowledge, this is the first case of intracranial RDD with autopsy. Although generally considered benign, RDD involving the brainstem might be lethal.

We hypothesized that a reduction in the size of the lymph nodes after neoadjuvant therapy for locally advanced rectal carcinoma would be associated with decreased lymph node metastases and/or a better prognosis. Between March 2006 and April 2012, 71 patients with primary rectal cancer received neoadjuvant chemoradiation therapy (CRT). For all lymph nodes 5 mm or larger in size, the major and minor axes were measured on CT scan images, and the product was calculated. The lymph node size was determined before and after CRT. The patients were divided into three groups based on the lymph node size before and after treatment. Group A exhibited a reduction in size of 60 % or more, Group B a reduction of less than 60 % and Group C had no lymph node enlargement before treatment. The incidence of lymph node metastases on pathological examination was 15 % in Group A and 50 % in Group B (p = 0.006). The five-year disease-free survival in Group A was 84 % compared with 78 % in Group B (log rank p = 0.34). The five-year overall survival in Group A was 92 % compared with 74 % in Group B (log rank p = 0.088). A reduction in the size of enlarged lymph nodes after neoadjuvant therapy may be a useful prognostic factor for recurrence and survival.

A lack of consistent methods to evaluate Ki67 expression is problematic in terms of accurately predicting prognosis in breast cancer. Accordingly, this study aimed to identify the causes of discrepancies in Ki67 labeling index measurements by different observers under different conditions using breast cancer samples.This Japanese study group compared and assessed immunohistochemical (IHC) analysis of the Ki67 labeling index when measured by different pathologists. Six pathologists (pathologists A-F) in Japan participated in this ring study. One hundred and ten surgical cases of estrogen receptor-positive and human epidermal growth factor receptor 2-negative invasive breast cancer treated in 2007 were identified from the breast cancer database of Tokai University Hospital and were included in this study.For all 6 pathologists, the Ki67 labeling index were significantly different between grade 3 and grade 1 cases and between grade 3 and grade 2 cases, whereas the index tended to be different between grade 1 and grade 2 cases. Further, the Ki67 labeling indexes measured by the 6 pathologists were strongly correlated (ρ: 0.73-0.88). The IHC scores recorded by pathologist A were in moderate to good agreement with those recorded by the others in patients with a Ki67 labeling index of13.25 % and in those with a Ki67 labeling index of13.25 % (κ = 0.429-0.660). The Ki67 low and high concordance rates between pathologist A and the others were 0.452-0.778 and 0.862-0.979, respectively. The most pertinent reason for discrepancy in scores seemed to be the selection of the area for counting and the quality of nuclear staining.The Ki67 labeling index measured by 6 pathologists without method standardization was in fair to good agreement. We plan to undertake a second ring study, pending recommendations by the international Ki67 panel.

Aims: (i) Determine whether between-observer reproducibility for Ki67 when assessed on whole sections according to a standardized scoring protocol is adequate for clinical application. (ii) Compare between-observer reproducibility of Ki67 scores assessed on hot-spots to scores using a global method that averages across a tissue section. Background: The nuclear proliferation biomarker Ki67 has multiple potential roles in breast cancer, including aiding decisions based on prognosis, but unacceptable levels of between-laboratory variability have been observed. The International Ki67 in Breast Cancer Working Group has undertaken a systematic program to determine whether Ki67 measurement can be analytically validated and standardized across labs. In phase 1, variability in visual interpretation was identified as an important source of variability. Phases 2 and 3a showed that adherence to defined scoring methods substantially improved reproducibility in scoring tissue microarrays and core-cut biopsies. We now assess whether acceptable reproducibility can be achieved on whole sections. Methods: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 to assemble 4 sets of 30 stained tumor sections, circulated around 23 labs in 12 countries. Ki67 was scored by 2 methods by all labs: (a) global: 4 fields of 100 tumor cells each were selected to reflect observed heterogeneity in nuclear staining (b) hot-spot: the field with highest Ki67 percentage of tumor cells with nuclear staining was selected and up to 500 cells scored. Ki67 scores were log2-transformed for statistical analyses and back-transformed for presentation. The primary objective was to assess whether either method could achieve an intraclass correlation coefficient (ICC) significantly greater than 0.8, considered substantial to almost-perfect reproducibility. Secondary objectives were to assess which method had highest observed ICC and to assess whether observers identified the same “hot-spots”. Results: ICC for the global method was 0.87 (95%CI: 0.799-0.93), marginally meeting the prespecified success criterion. The ICC for the hot-spot method was 0.83 (95%CI: 0.74-0.90) and had a CI extending below the success criterion. Across the 23 labs, geometric mean value of the 30 scores ranged from 8.5 to 19.6 for the global method and from 12.8 to 30.3 for the hot-spot method. The overall mean (95% CI) of these values was 12.9 (11.9-14.0) and 20.9 (19.1-22.8), respectively. Visually, between-laboratory agreement in location of selected hot-spot varies between cases. The median times for scoring were 9 and 6 minutes for global and hot-spot methods respectively. Conclusions: The global method marginally met the prespecified criterion of success; it should now be evaluated for clinical validity in appropriate cohorts of cases. The hot-spot method was observed to have slightly less reproducibility between labs. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between labs further. (Supported by a grant from the Breast Cancer Research Foundation) Citation Format: Nielsen TO, Leung SCY, Zabaglo LA, Arun I, Badve SS, Bane AL, Bartlet JMS, Borgquist S, Chang MC, Dodson A, Ehinger A, Fineberg S, Focke CM, Gao D, Gown AM, Gutierrez C, Hugh JC, Kos Z, Lænkholm A-V, Mastropasqua MG, Moriya T, Nofech-Mozes S, Osborne CK, Penault-Llorca FM, Piper T, Sakatani T, Salgado R, Starczynski J, Sugie T, van der Vegt B, Viale G, Hayes DF, McShane LM, Dowsett M. Analytical validation of a standardized scoring protocol for Ki67 assessed on breast excision whole sections: An international multicenter collaboration [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-01.

BRG1 and BRM, two core catalytic subunits in SWI/SNF chromatin remodeling complexes, have been suggested as tumor suppressors, yet their roles in carcinogenesis are unclear. Here, we present evidence that loss of BRG1 and BRM is involved in the progression of lung adenocarcinomas. Analysis of 15 lung cancer cell lines indicated that BRG1 mutations correlated with loss of BRG1 expression and that loss of BRG1 and BRM expression was frequent in E‐cadherin‐low and vimentin‐high cell lines. Immunohistochemical analysis of 93 primary lung adenocarcinomas showed loss of BRG1 and BRM in 11 (12%) and 16 (17%) cases, respectively. Loss of expression of BRG1 and BRM was frequent in solid predominant adenocarcinomas and tumors with low thyroid transcription factor‐1 (TTF‐1, master regulator of lung) and low cytokeratin7 and E‐cadherin (two markers for bronchial epithelial differentiation). Loss of BRG1 was correlated with the absence of lepidic growth patterns and was mutually exclusive of epidermal growth factor receptor (EGFR) mutations. In contrast, loss of BRM was found concomitant with lepidic growth patterns and EGFR mutations. Finally, we analyzed the publicly available dataset of 442 cases and found that loss of BRG1 and BRM was frequent in E‐cadherin‐low, TTF‐1‐low, and vimentin‐high cases and correlated with poor prognosis. We conclude that loss of either or both BRG1 and BRM is involved in the progression of lung adenocarcinoma into solid predominant tumors with features of epithelial mesenchymal transition and loss of the bronchial epithelial phenotype. BRG1 loss was specifically involved in the progression of EGFR wild‐type, but not EGFR‐mutant tumors.

Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non–small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion–positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.

The effectiveness of neoadjuvant chemotherapy is evaluated on the basis of pathological responses and survival outcome, because achievement of a pathological complete response (pCR) is a good predictor of long-term survival. However, few studies have assessed the survival of breast cancer patients who received neoadjuvant chemotherapy including trastuzumab. The records of 161 breast cancer patients who received neoadjuvant chemotherapy between January 2006 and December 2011 were retrospectively reviewed. The patients were categorized into 4 subgroups on the basis of the status of the estrogen receptor (ER), the progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). HER2-positive patients received trastuzumab-based regimens. Pathological responses and survival were analyzed on the basis of breast cancer subtypes. The pCR results obtained were: luminal A and B (ER and/or PR-positive, HER2-negative), 6.3 % (5/79 cases); luminal–HER2 hybrid (ER and/or PR-positive, HER2-positive), 25.0 % (5/20 cases); HER2-enriched (ER and PR-negative, HER2-positive), 63.0 % (17/27 cases); and triple-negative (ER and PR-negative, HER2-negative), 25.7 % (9/35 cases). Achievement of pCR was a good predictor of disease-free survival in the HER2-enriched group. Overall survival of patients with pCR was slightly, but not significantly, better in the HER2-enriched and triple-negative subgroups. Responses and survival after neoadjuvant chemotherapy including trastuzumab of patients with HER2-positive tumors differed among disease subtypes. Our findings suggest that disease subtype is an important determinant of the efficacy of neoadjuvant chemotherapy.

e24123Background: Standardized evaluation of TIL is currently based on the extent of lymphocytic infiltration into the tumor stroma for breast cancer. However, the distributions of subpopulations a...

Epstein-Barr virus (EBV) associated lymphoproliferative disease (LPD) comprises a wide spectrum of clinical and pathological features [1]. This variety is most systematically categorized in post-transplant lymphoproliferative disorders (PTLD) [2], in which subtypes are ordered according to disease progression from reactive polyclonal proliferation to large cell lymphoma. However, whether this categorization is applicable to LPDs other than PTLD is not well explored. Here, we present a nontransplant case of EBV-LPD that initially presented as a polyclonal self-limiting proliferation and later transited to large cell lymphoma. This transition was associated with the progression of lung cancer and its therapy. Our case demonstrates that stepwise progression of LPD is a feature that can be observed in non-PTLD cases of EBV-LPD.

Epstein-Barr virus (EBV)-associated gastric carcinoma (GC) is a distinct subtype with characteristic clinicopathological features. To better characterize its cellular characteristics, 43 cases of EBV-associated GC, 68 cases of EBV-negative GC, and non-neoplastic gastric mucosa in adults and fetuses were examined immunohistochemically. We quantified the expression of the major tight-junction protein claudin (CLDN) -1, -3, -4, -7, and -18 together with gastric mucins (MUC5AC and MUC6), intestinal mucin (MUC2), and CD10. EBV-associated GC showed a high frequency of CLDN18 expression (84%) and a low frequency of CLDN3 expression (5%). This expression profile corresponded to that of normal gastric epithelium in adults and fetuses. Almost half of the EBV-associated GC cases demonstrated gastric mucin expression, whereas the other half lacked mucin or CD10 expression. In contrast, as demonstrated by the expression profiles of CLDN3 and CLDN18, EBV-negative GC comprised a heterogeneous group of four different CLDN phenotypes: gastric, intestinal, mixed, and an undifferentiated type with variable expression patterns of mucins. These results indicate that EBV-associated GC is considerably homogenous with regard to cellular differentiation and that it preserves well the nature of the cells of origin. EBV-associated GC may undergo distinct carcinogenic processes, which differ from those of EBV-negative GC. (J Histochem Cytochem 57:775–785, 2009)

Extranodal non-Hodgkin's lymphoma (NHL) is a rare breast disease. Here we report three cases of primary NHL of the breast. The first patient was a 29-year-old woman with a firm mass in her right breast with ipsilateral axillary lymphadenopathy. An excisional biopsy revealed NHLs. Clinical stage was IIAE. The tumor and enlarged lymph nodes had successfully been treated following the combination therapy. The second patient was a 70-year-old women with an elastic hard mass in her left breast. An excisional biopsy revealed NHLs and clinical stage was 1AE. The tumor disappeared following the combination therapy. The third patient was a 67-year-old women with a hard mass in her left breast. Core needle biopsy revealed NHLs and clinical stage was 1AE. The tumor disappeared following chemotherapy. All patients are alive with no evidence of recurrence 4-8 years after the initial treatment. Although a standard treatment has yet to be established, an initial treatment with combination therapy without surgical intervention including axillary dissection appears to be appropriate for this rare disease.

CpG island promoter methylation of tumor suppressor genes is one of the most characteristic abnormalities in EBV-associated gastric carcinoma (GC). Aberrant promoter methylation and expression loss of PTEN were evaluated in cancer tissues of GC by methylation-specific PCR and immunohistochemistry, respectively, showing that both abnormalities occurred concurrently in EBV-associated GC. PTEN abnormalities were reiterated in GC cell lines MKN-1 and MKN-7 infected with recombinant EBV, and DNA methyltransferase 1 (DNMT1) was commonly overexpressed in both cell lines. Stable and transient transfection systems in MKN-1 similarly showed that viral latent membrane protein 2A (LMP2A) up-regulated DNMT1, leading to an increase in methylation of the PTEN promoter. Importantly, the level of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) increased in the nuclei of LMP2A-expressing GC cells, and knockdown of STAT3 counteracted LMP2A-mediated DNMT1 overexpression. Immunohistochemistry for both pSTAT3 and DNMT1 showed diffuse labeling in the nuclei of the cancer cells in GC tissues, especially in EBV-associated GC. Taken together, LMP2A induces the phosphorylation of STAT3, which activates DNMT1 transcription and causes PTEN expression loss through CpG island methylation of the PTEN promoter in EBV-associated GC. LMP2A plays an essential role in the epigenetic abnormalities in host stomach cells and in the development and maintenance of EBV-associated cancer. [Cancer Res 2009;69(7):2766–74]

EBV-associated gastric carcinoma is a distinct subset of gastric carcinoma infected with EBV, which shows latency I type expression of EBV latent genes (EBNA1, EBER, BARF0, and LMP2A). To clarify the role of EBV in this type of gastric carcinoma, the cell biological characteristics (growth, apoptosis, and migration) were evaluated in gastric carcinoma cell lines (MKN-1, TMK1, MKN-74 and MKN-7) with and without infection of recombinant EBV harboring the neomycin resistance gene. The infection reiterated the latency I type infection, and the only difference observed in EBV-infected gastric carcinoma cell lines was the resistance to serum deprivation–induced apoptosis. Comparative analyses of transcripts of apoptosis-associated genes in MKN-1 and EBV–MKN-1 and subsequent quantitative reverse transcription-PCR analysis showed up-regulation of the cellular survivin gene in EBV-infected gastric carcinoma cell lines. Small interfering RNA–mediated knockdown of survivin increased apoptosis in EBV–MKN-1 to the level of the original MKN-1 cells. Transfection of EBV-latent genes into MKN-1 showed that LMP2A, but not EBNA1, EBER, or BARF0, up-regulated survivin gene expression. LMP2A-mediated survivin up-regulation in gastric carcinoma cells was inhibited with a nuclear factor-κB (NF-κB) inhibitor, Bay 11-7082. In parallel with these findings in vitro, survivin expression was frequent in carcinoma tissues of gastric carcinoma by immunohistochemistry, and significantly more in EBV-associated gastric carcinoma (12 of 13) than in EBV-negative gastric carcinoma in the advanced stage (P = 0.0307). Thus, EBV uses its latent protein, LMP2A, to activate the NF-κB–survivin pathway to rescue EBV-infected epithelial cells from serum deprivation, and up-regulation of survivin may play a role in the progression of this specific type of gastric carcinoma infected with EBV. [Cancer Res 2008;68(5):1427–35]

Objective Mucinous carcinoma (MCA) may show neuroendocrine differentiation (ND), but the cytological features characteristic of ND remains elusive. We compared fine needle aspiration (FNA) findings of MCA between cases with high and low degrees of ND. Methods Histological sections of 37 MCA cases were immunohistochemically evaluated for expression of chromogranin A and synaptophysin, and were graded as 0 to 3+ degrees of ND. They were divided into low ND (grade 0 and 1+) and high ND (grade 2+ and 3+) groups. Pre-operative FNA samples of each group were assessed for cytological features. Results The mean age of the high ND group (n = 18) was higher than the low ND group (n = 19, P = 0.01). In FNA samples of the high ND group, 17 cases showed moderate to severe degrees of discohesiveness, but low ND cases mainly showed no or only mild discohesiveness (P < 0.001). Nine of the low ND cases displayed overlapped, cohesive cell clusters, whereas, in the high ND cases, the cells were arranged in a loose, flat and monolayered pattern (P = 0.045). Fourteen of the high ND cases had round nuclei, but oval nuclei were predominant in the low ND cases (P = 0.027). The nuclei were eccentrically located in 12 of the high ND cases but were centrally located in 14 of the low ND cases (P = 0.01). Conclusions Mucinous carcinoma with high ND may be diagnosed by the presence of discohesiveness, a flat, monolayered pattern, and round or eccentrically located nuclei. Features of ND in carcinomas in other organs, such as intracytoplasmic granules and coarse chromatin, may not be reliable cytological features of ND in MCA.