A case of hyperprogressive disease following atezolizumab therapy for pulmonary pleomorphic carcinoma with epidermal growth factor receptor mutation.

A 66-year old man with non-smoking history was diagnosed with pulmonary pleomorphic carcinoma of the right lower lobe. The carcinoma metastasized to the brain, lungs, pleura, and mediastinal lymph nodes. It was positive for epidermal growth factor receptor (EGFR) L858R mutation, and tumor cells highly expressed programmed death-ligand 1(PD-L1). Atezolizumab was initiated as the fourth treatment. After three days, he developed cardiac tamponade and immediately underwent pericardial drainage. Computed tomography showed bilateral ground-glass opacity (GGO), significant worsening of multiple lung metastases, and increased size of metastatic lesions. Newly developed metastasis was noted in the lung, and the patient's respiratory condition rapidly deteriorated. He died of respiratory failure on day 13 after atezolizumab administration. The autopsy showed widespread metastasis in all lobes of the bilateral lungs, cardiac tamponade due to carcinomatous pericarditis, carcinomatous lymphangiopathy, and multiple lung metastases, which were thought to be comprehensively the cause of death. These symptoms suggested hyperprogressive disease (HPD). Hence, we report the first case of HPD following atezolizumab therapy for pulmonary pleomorphic carcinoma with EGFR mutation.

Prognostic Prediction of Oral Squamous Cell Carcinoma by E-Cadherin and N-Cadherin Expression in Overall Cells in Tumor Nests or Tumor Cells at the Invasive Front.

Epithelial-mesenchymal transition (EMT) is a significant process in the invasion and metastasis of cancers including oral squamous cell carcinoma (OSCC), and the cadherin switch has been identified as one of the hallmarks of EMT. The aim of the present study was to evaluate the significance of the cadherin switch in the prognosis of OSCC and generate a model for prognostic predictions. Seventy-six biopsy and/or initial surgical specimens from OSCC patients were immunohistochemically analyzed for the expression of E-cadherin and N-cadherin in either overall OSCC cells in tumor nests or in OSCC cells at the invasive front. Among 76 OSCC cases, overall OSCC cells in tumor nests were negative for the expression of E-cadherin in 10 cases and positive for that of N-cadherin in 53 cases. Among 10 cases negative for the expression of E-cadherin, 4 cases were positive for that of N-cadherin. In OSCC cells at the invasive front, the expression of E-cadherin was negative in 62 cases, while that of N-cadherin was positive in 39 cases. Among 62 cases negative for the expression of E-cadherin, 33 cases were positive for that of N-cadherin. A logistic regression analysis showed that a model using the evaluation of N-cadherin expression in overall OSCC cells in tumor nests with a cut-off point of 70 years old was the best fit model. These results suggest that N-cadherin has significant value in prognostic predictions for OSCC patients.

Low-risk population among patients with tumor-node-metastasis stage III/IV oral squamous cell carcinoma.

A novel system auxiliary to the Union for International Cancer Control classification may allow the prognosis of patients with malignant tumors at similar stages to be predicted, as currently this is challenging. The present study generated a novel system to predict populations at low risk among patients with stage III/IV oral squamous cell carcinoma (OSCC). A total of 41 patients who were diagnosed at stages III/IV OSCC and underwent surgical tumor resection were analyzed. Band-like or follicular lymphocyte infiltration, intraepithelial micro-abscess formation and natural killer (NK) cell infiltration were histopathologically evaluated. Cox's proportional hazards regression model was used to identify prognostic factors, and a set of factors was selected from a combination of those prognostic factors to create a logic covariate model. A logic regression analysis for 41 patients with OSCC revealed that the presence of intraepithelial micro-abscesses and a lower density of NK cells were significantly associated with a favorable prognosis among patients with stage III/IV OSCC. These results suggested that the host innate immune responses, including neutrophil and NK cell infiltrations, are useful for prognostic prediction in patients with advanced malignant tumors.

FOXC2 expression is associated with tumor proliferation and invasion potential in oral tongue squamous cell carcinoma.

Forkhead box protein C2 (FOXC2) is a gene encoding a transcription factor that controls the generation of mesodermal tissue including vascular and lymphatic tissues. FOXC2 has previously been associated with EMT and tumor angiogenesis in various cancers. Moreover, a relationship between the expression of FOXC2 and poor prognosis has been reported in various cancers. We herein examined the clinicopathological significance of FOXC2 in oral tongue squamous cell carcinoma (OTSCC) and attempted to clarify the function of FOXC2 in OTSCC cell lines in vitro. The overexpression of FOXC2 was more frequent in cancers with higher grades according to the pattern of invasion (grade 4 vs. 1-3; p < 0.05). A correlation was observed between the expression of FOXC2 and that of VEGF-A and -C (VEGF-A; p < 0.05, VEGF-C; p < 0.001). The high-FOXC2 expression group had a significantly poorer prognosis than that of the low-expression group (p < 0.001). Multivariate analysis indicated that the overexpression of FOXC2 may also be an independent prognostic factor, similar to N classification (N0 vs 1/2; p < 0.05), stage classification (stage I/II vs III/IV; p < 0.05), pattern of invasion (grade 1-3vs 4; p < 0.05), local recurrence (local recurrence (+) vs (-); p < 0.01), and the overexpression of FOXC2 (FOXC2 overexpression (-) vs.(+); p < 0.05). In the OTSCC cell line analysis, the expression of FOXC2 was also associated with proliferation and invasion potential. These results strongly suggest that the overexpression of FOXC2 may be a potent predictor of survival in OTSCC patients.

Expression of the cancer stem cell markers CD44v6 and ABCG2 in tongue cancer: effect of neoadjuvant chemotherapy on local recurrence.

The efficacy of neoadjuvant chemotherapy (NAC) is controversial, and no report supports NAC with a high evidence level. Recently, we showed that a deep surgical margin was resected very close to the tumor site in many NAC-treated oral squamous cell carcinoma patients, suggesting that NAC may lead to local recurrence and poor outcomes. The purpose of this study was to evaluate the effect of NAC on tumor local recurrence using cancer stem cell marker immunohistochemistry. We retrospectively analyzed 89 patients who underwent radical surgery for tongue cancer, and examined the effect of NAC on tumor local recurrence. Cancer stem cell marker (CD44v6 and ABCG2) expression was detected by immunohistochemistry. In our study, the local recurrence rate was 12.4%. CD44v6 and ABCG2 expression was significantly associated with regional lymph node metastasis, pattern of invasion, depth of invasion, perineural invasion and local recurrence, respectively. Tumor local recurrence was a significant independent predictive factor of the 5-year disease specific survival. CD44v6 or ABCG2 positivity in NAC-treated patients was significantly associated with tumor local recurrence. It was suggested that local recurrence in NAC-treated cases is associated with cancer stem-like cells. We propose that NAC leads to the selection and/or residue of more aggressive cancer stem-like cells.

Prognostic value of podoplanin expression in oral squamous cell carcinoma--a regression model auxiliary to UICC classification.

Podoplanin, a type I transmembrane glycoprotein with an effect of platelet aggregation, has been reported to be one of the possible prognostic factors of oral squamous cell carcinoma (OSCC). However, the biological significance of podoplanin is largely unclear. The aim of this study was to develop a practical model for the prediction of prognosis using the grade of podoplanin expression, and also to evaluate the biological function of podoplanin. Eighty-two specimens of patients with previously untreated OSCC, who underwent either biopsy or surgery, were histopathologically and immunohistochemically analyzed. These 82 cases were composed of 66 well-differentiated, 10 moderately differentiated and 6 poorly differentiated OSCC. Podoplanin was successfully immunostained in 78 specimens, and was detected in most cases, but the frequency of positive cells varied. The prognosis of patients with more than 50 % podoplanin-positive tumor cells was significantly poorer than that of the other patients. Multivariate hazards regression analysis suggested that a linear combination of covariates, OSCC patients with more or less than 50 % podoplanin expression, age of more or less than 70 years old, mode of invasion and T3, T4 or T2 versus T1 of the UICC T-stage classification was the most effective model for evaluating the prognosis of OSCC patients. Additionally, podoplanin expression had a significant relationship to UICC clinical stage and the expression of Ki-67. An effective regression model using podoplanin expression was developed for evaluating the prognosis of OSCC and the biological significance of podoplanin was suggested to be associated with the growth and/or progression of OSCC.

Prediction of outcome of patients with oral squamous cell carcinoma using vascular invasion and the strongly positive expression of vascular endothelial growth factors.

Vascular invasion and lymph node metastasis have been used as histopathological prognosticators of cancers including oral squamous cell carcinoma (OSCC). In addition to metastatic potential via blood vessels, tumor-induced angiogenesis might also be associated with prognosis. However, the efficacy of combined evaluation of vascular invasion and angiogenesis-associated molecules for the prognosis of OSCC remains obscure. This is also the case in lymph node metastasis and lymphovasculogenesis-associated molecules. The aim of this study was to examine factors related to prognosis to improve the accuracy of prognostic prediction of OSCC using vasculogenesis-associated markers. Ninety specimens of patients from 1991 to 2002 with previously untreated OSCC, who underwent either biopsy or surgery, were histopathologically and immunohistochemically analyzed using antibodies for vascular endothelial growth factor (VEGF)-A, VEGF-C, cyclooxygenase (COX)-2 and Midkine. The ninety cases were composed of 72 well-differentiated, 12 moderately differentiated and 6 poorly differentiated OSCC. Efficient models of prognostic prediction were evaluated by extensive statistical analyses. The presence of vascular invasion or lymph node metastasis was confirmed to be significantly associated with poor prognosis in the univariate analysis. Multivariate logic regression analysis suggested that patients with the strongly positive expression of either VEGF-A or VEGF-C had a significant association with poor prognosis even in patients without vascular invasion and in early-stage patients. Neither COX-2 nor Midkine contributed to predict the prognosis of the patients. The strongly positive expression of VEGF-A or VEGF-C was suggested to reinforce the histopathological diagnosis of vascular invasion and improve the accuracy and efficacy of prognostic prediction of OSCC.

Autophagic compartments gain access to the MHC class II compartments in thymic epithelium.

The presentation of self-peptides in the context of MHC molecules by thymic epithelial cells (TECs) is essential for T cell repertoire selection in the thymus. However, the underlying mechanisms of this process have not been fully elucidated. To address whether autophagy, a catabolic process involving the degradation of a cell's components through the lysosomal machinery, intersects the MHC class II-restricted Ag presentation pathway in TECs, we investigated the colocalization of LC3, a peculiar autophagy marker molecule, with MHC class II compartments in in vitro-established TEC lines by immunofluorescence microscopy and Western blotting analyses. We found that in both cortical and medullary TEC lines, LC3 was colocalized with the H2-DM-positive lysosomal compartments, in which MHC class II plus class II-associated invariant chain peptides complexes are formed. Furthermore, our analysis of thymic cryosections from 1-day-old mice revealed that LC3 colocalizes with the H2-DM-positive compartments in TECs. These results strongly suggest that the cytoplasmic self-Ags gain access to the H2-DM-positive compartments via the autophagic process in the thymus.

Intraosseous carcinoma of the mandible composed of cells expressing myoepithelial cell-associated antigens.

Carcinomas are rarely observed centrally in mandibular and maxillary bone. Etiology of intraosseous squamous cell carcinoma is thought to involve derivation from remnants of the odontogenic epithelium. Mucoepidermoid carcinoma is also rarely observed centrally in the mandibular and maxillary bone. Previous analyses raised the possibility that the tumor arose from entrapped salivary elements or from metaplastic odontogenic epithelia. Herein is described an intramandibular carcinoma with characteristic various morphologies. The tumor was composed of epidermoid cells, clear cells, mucous cells and intermediate cells, which were surrounded by cellular-rich collagenous stroma. The epidermoid cells, clear cells and mucous cells formed solid sheets, and the clear cells and intermediate cells formed trabecular structures. Sparse cell regions were partly accompanied by clear cells and mucous cells. Immunohistochemistry was positive for myoepithelial markers in the trabecular portion of the tumor. These findings suggest that this is a unique case of intramandibular mucoepidermoid carcinoma that includes tumor cells expressing myoepithelial cell-associated antigens.

Midkine expression correlating with growth activity and tooth morphogenesis in odontogenic tumors.

Midkine (MK; a low molecular weight heparin-binding growth factor) is a multifunctional cytokine. MK plays a role in morphogenesis of many organs including teeth through epithelial-mesenchymal interactions. We immunohistochemically examined MK expression in various human odontogenic tumors. There was no difference in positive rate and intensity of MK between benign odontogenic tumors and their malignant counterparts. Ameloblastoma showed MK localization in the peripheral columnar cells in budding processes from the parenchyma, which frequently expressed proliferating cell nuclear antigen. MK was also preferentially expressed in keratinized cells in acanthomatous ameloblastoma and keratocystic odontogenic tumor. In odontogenic mixed tumors except for odontoma, intense immunoreactivity to MK was found in epithelial follicles, the surrounding odontogenic ectomesenchymal tissue, and the basement membrane between them. Intensity in the odontogenic ectomesenchyme decreased in relation to distance from the epithelial follicles. No expression was found in tumor cells associated with production of dental hard tissues in odontogenic mixed tumors including odontoma. These findings suggested that MK is involved in the reciprocal interaction between odontogenic epithelium and odontogenic ectomesenchymal tissue in areas without dental hard tissue formation in odontogenic mixed tumors. Coexpression of MK and proliferating cell nuclear antigen was also observed in epithelial follicles and highly cellular nodules in the ectomesenchyme of odontogenic mixed tumors. MK is considered to mediate growth activity of odontogenic tumors and cell differentiation of odontogenic mixed tumors through molecular mechanisms similar to those involved in morphogenesis of the tooth.

Mechanism of action of dipropofol and synergistic action with other antibacterial agents in vitro.

The mode of action of dipropofol and its antibacterial activity in combination with other antibiotics against Gram-positive and -negative bacteria were investigated. Dipropofol showed a bactericidal effect against Staphylococcus aureus 209P, and inhibited the incorporations of 3H-glutamate and 3H-leucine into S. aureus 209P and Bacillus subtilis PCI219 cells. These results indicated that the mechanism of action of dipropofol was mediated by the inhibition of protein synthesis or amino acid incorporation. A synergistic effect was performed by checkerboard titration with Muller-Hinton agar plates containing dipropofol (0.39 microg/ml, 1/4 x MIC) and various concentrations of nine other antibiotics in vitro. The synergism against vancomycin resistant Enterococcus faecium was confirmed in the combination of dipropofol with rifampicin. The MIC of rifampicin was decreased from 0.39 microg/ml to <0.005 microg/ml by the addition of dipropofol. This combinational effect in reversing vancomycin resistance of enterococci highlights novel drug targets and has importance in the design of new therapeutic regimens against resistant pathogens.

Trapping effect of eugenol on hydroxyl radicals induced by L-DOPA in vitro.

Many researchers have stated that eugenol might inhibit lipid peroxidation at the stage of initiation, propagation, or both, and many attempts have been made to elucidate the mechanism of its antioxidant activity. Nevertheless, details of its mechanism are still obscure. This study was carried out to investigate the trapping effect of eugenol on hydroxyl radical generated from L-3,4-dihydroxyphenylalanine (DOPA) in MiliQ water and the generation mechanism of the hydroxyl radical by this system which uses no metallic factor. This was studied by adding L-DOPA and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to phosphate buffered saline (PBS) or MiliQ water, and the generation of hydroxyl radical was detected on an ESR spectrum. By this method, the effect of antioxidants was detected as a modification of ESR spectra. We found that the eugenol trapped hydroxyl radicals directly, because it had no iron chelating action, did not trap L-DOPA semiquinone radical and inhibited hydroxyl radicals with or without iron ion.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.

Regulation of osteoclastogenesis by three human RANKL isoforms expressed in NIH3T3 cells.

Receptor activator of nuclear factor-kappaB ligand (RANKL) induces osteoclastogenesis by binding with the receptor, receptor activator of nuclear factor-kappaB in the presence of macrophage colony-stimulating factor. Three human RANKL isoforms, hRANKL1, hRANKL2, and hRANKL3, were identified. hRANKL1 was identical to previously reported RANKL and possessed intracellular, transmembrane, and extracellular domains, hRANKL2 did not have the intracellular domain, and hRANKL3 did not have the intracellular and transmembrane domains. When bone marrow macrophages were cultured with NIH3T3 cells expressing hRANKL1, osteoclasts were formed, but when cultured with NIH3T3 cells expressing hRANKL2 or hRANKL3, no tartrate resistant acid phosphatase-positive cell was observed. In the coculture system, coexpression of hRANKL3 with hRANKL1 significantly inhibited the formation of osteoclasts by hRANKL1, but coexpression of hRANKL2 with hRANKL1 did not affect the osteoclastogenesis by hRANKL1 significantly. These results suggest that the activity of osteoclastogenesis by hRANKL1 is regulated by the attenuator, hRANKL3.

Multimerization of the receptor activator of nuclear factor-kappaB ligand (RANKL) isoforms and regulation of osteoclastogenesis.

The receptor activator of nuclear factor-kappaB ligand (RANKL), a member of the tumor necrosis factor family, is a transmembrane protein, which is known as an essential initiation factor of osteoclastogenesis. Previously, we identified three RANKL isoforms. RANKL1 was identical to the originally reported RANKL. RANKL2 had a shorter intracellular domain. RANKL3 did not have the intracellular or transmembrane domains and was suggested to act as a soluble form protein. Here, we show that RANKL forms homo- or heteromultimers. NIH3T3 cells transfected with RANKL1 or RANKL2 form mononuclear tartrate-resistant acid phosphatase-positive preosteoclasts in an in vitro osteoclastogenesis assay system. Coexpression of RANKL1 and RANKL2 induces multinucleated osteoclasts. RANKL3 has no effect on the formation of preosteoclasts or osteoclasts but significantly inhibits fusion of preosteoclasts when coexpressed with RANKL1 and RANKL2. These findings imply the presence of multiple multimeric structures of RANKL, which may regulate bone metabolism.

Poor prognosis of colorectal cancer in patients over 80 years old is associated with down-regulation of tumor suppressor genes.

UNLABELLED: GOALS, BACKGROUND: The elderly population has been increasing during the last half a century and it would be important to know how aging influences the occurrence and biologic behavior of cancers. STUDY: We investigated clinicopathologic characteristics of colorectal cancer in 1354 patients who underwent colorectal cancer resection and compared the results between extremely elderly patients (over 80 years old) and middle-aged/elderly patients (40 to less than 80 years old). Furthermore, we also examined expression of tumor suppressor genes and Cox-2 using frozen samples of colorectal cancer obtained from 62 patients ranging in age from 45 to 87 years. RESULTS: The results obtained in the extremely aged patients were: (1) higher ratio of women, (2) higher incidence at the proximal site, (3) higher incidence of cases with deeper invasion, (4) higher incidence of cases with lymph node metastasis (5) poorer survival rate as compared with middle-aged/elderly patients, and (6) lower mRNA expression levels of p27 and p53. CONCLUSIONS: These findings taken together suggest that poor prognosis of colorectal cancer in patients over 80 years is associated with down-regulation of mRNA expression of some tumor suppressor genes.

Hepatocellular carcinoma with osteoclast-like giant cells: possibility of osteoclastogenesis by hepatocyte-derived cells.

Giant cell tumor (GCT) of bone is a primary osteolytic tumor that is characterized by the formation of osteoclast-like giant cells. In addition to GCT of bone, extraskeletal GCT are known to be formed in several soft tissues. Giant cells in GCT of bone were suggested to be identical to osteoclasts, but the characterization of giant cells in extraskeletal GCT remains incomplete. In this study, a case of sarcomatoid hepatocellular carcinoma with osteoclast-like giant cells was analyzed. Immunohistochemistry revealed the expression of almost all markers of osteoclasts: tartrate-resistant acid phosphatase, CD68, CD51, CD54 and matrix metalloprotease-9, in osteoclast-like giant cells in the tumor. In situ hybridization revealed the expression of receptor activator of nuclear factor-kappa B (RANK) in the giant cells and receptor activator of nuclear factor-kappa B ligand (RANKL) in the tumor cells. The hepatic origin of the sarcomatoid hepatocellular carcinoma cells was confirmed by the expression of albumin. This is the first report suggesting that hepatocyte-derived cells possess the potential for osteoclastogenesis. In addition, these findings suggest that osteoclast-like cells in the hepatocellular carcinoma were formed by the same mechanism as osteoclastogenesis in bone.