Engineering CD3/CD137 Dual Specificity into a DLL3-Targeted T-Cell Engager Enhances T-Cell Infiltration and Efficacy against Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

CH7233163 Overcomes Osimertinib-Resistant EGFR-Del19/T790M/C797S Mutation.

Osimertinib is the only EGFR-tyrosine kinase inhibitor (TKI) capable of overcoming EGFR-T790M-mutated NSCLC, but osimertinib-resistant EGFR triple mutations (Del19/T790M/C797S or L858R/T790M/C797S) have been reported. Although allosteric EGFR TKIs (e.g., EAI-045) that potentially overcome L858R/T790M/C797S have been identified, there are no effective inhibitors against Del19/T790M/C797S. In this study, we identified CH7233163 as having the potential to overcome EGFR-Del19/T790M/C797S. CH7233163 showed potent antitumor activities against tumor with EGFR-Del19/T790M/C797S in vitro and in vivo In addition to EGFR-Del19/T790M/C797S, the characterization assays showed that CH7233163 more selectively inhibits various types of EGFR mutants (e.g., L858R/T790M/C797S, L858R/T790M, Del19/T790M, Del19, and L858R) over wild type. Furthermore, crystal structure analysis suggested that CH7233163 is a noncovalent ATP-competitive inhibitor for EGFR-Del19/T790M/C797S that utilizes multiple interactions with the EGFR's αC-helix-in conformation to achieve potent inhibitory activity and mutant selectivity. Therefore, we conclude that CH7233163 is a potentially effective therapy for osimertinib-resistant patients, especially in cases of EGFR-Del19/T790M/C797S.

Computed tomography findings in Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly: comparison with EBV-negative DLBCL.

OBJECTIVE: This study aimed to compare CT findings in patients with Epstein-Barr virus positive (EBV+) diffuse large B-cell lymphoma (DLBCL) of the elderly with CT findings in patients with Epstein-Barr virus negative (EBV-) DLBCL. METHODS: We retrospectively identified 9 consecutive patients with EBV+ DLBCL (6 males and 3 females; aged 72-83 years, mean: 76.2 years) and 39 consecutive patients with EBV- DLBCL (19 males and 20 females; aged 53-91 years, mean: 71.3 years) who had undergone CT examinations between September 2007 and August 2016. In each type of disease, clinical and CT findings were evaluated. RESULTS: No significant differences in B symptom incidence or serum lactate dehydrogenase levels were found between the two diseases. However, the prognosis of patients with EBV+ DLBCL was significantly poorer than that of those with EBV- DLBCL (p < 0.05). Nodal and extranodal necrosis was found in 6 (66.7%) of 9 patients with EBV+ DLBCL and in 6 (15.4%) of 39 patients with EBV- DLBCL (p < 0.005). CONCLUSION: The CT finding of nodal and extranodal necrosis was significantly more frequent in patients with EBV+ DLBCL than in patients with EBV- DLBCL. Advances in knowledge: This is the first report on the CT findings in patients with EBV+ DLBCL of the elderly. Different CT findings are present in EBV+ DLBCL and EBV- DLBCL.

Dedifferentiated chordoid meningioma with rhabdomyosarcomatous differentiation on the middle cranial fossa.

A 46-year-old woman presented with headache and right hemiparesis. MRI demonstrated a mass in the left middle fossa. Total resection was performed. A histological examination of the tumor specimen showed several characteristic morphological features. A chordoid meningioma showing an epithelial-like palisade arrangement was observed. An anaplastic short spindle cell tumor exhibiting a fascicular pattern was considered to be a rhabdomyosarcoma. After conventional radiotherapy, the tumor was well controlled without any neurological deficit for 20 months. When subsequent recurrences were observed, the patient was treated by surgery, stereotactic radiosurgery and chemotherapy. Thirty-two months after the initial treatment, the patient died due to intracranial dissemination and an autopsy was performed. The histological examination of the recurrent and autopsy specimens showed a prominent sarcoma component. This case appears to be the first reported intracranial tumor diagnosed as a dedifferentiated chordoid meningioma with rhabdomyosarcomatous differentiation.

Fucosylation is associated with the malignant transformation of intraductal papillary mucinous neoplasms: a lectin microarray-based study.

PURPOSE: Intraductal papillary mucinous neoplasm (IPMN) is an intraductal mucin-producing pancreatic neoplasm with the potential for malignant transformation. Changes in glycans expressed on the cell surface and glycotransferases play important roles in malignant transformation. We conducted this study to analyze glycan alterations in IPMNs by using a lectin microarray and to identify the factors associated with altered glycans and their relationships with malignant transformation. METHODS: Using a lectin microarray, we evaluated glycan expression in 22 samples of IPMN with carcinoma, obtained from curative resections performed in our department. We also used immunohistochemistry to investigate fucosyltransferase 8 (Fut 8) protein expression, which is associated with glycan alterations in IPMNs. RESULTS: The lectin microarray demonstrated that only two lectins, Aleuria aurantia lectin (AAL) and Aspergillus oryzae L-fucose-specific lectin (AOL), which bind to fucose, exhibited significant sequential increases from normal pancreatic duct to adenoma and carcinoma. Similarly, Fut 8 protein expression, which is associated with AAL and AOL, sequentially and significantly increased from the normal pancreatic duct to adenoma and carcinoma. CONCLUSIONS: Lectin microarray analysis suggested that fucosylation is associated with the malignant transformation of IPMNs.

KIT (CD117) Expression in Benign and Malignant Sweat Gland Tumors.

KIT (CD117, c-kit) is a receptor tyrosine kinase involved in the tumorigenesis of several neoplasms. KIT is expressed by the secretory cells of normal sweat glands. We studied the KIT expression and KIT mutational status in various benign and malignant tumors of eccrine and apocrine glands. We included a total of 108 cases comprising 10 benign and 6 malignant sweat gland tumors, and KIT expression was immunohistochemically detected (positive rate): 10 syringomas (0%), 8 poromas (25%), 20 mixed tumors (40%), 21 spiradenomas (43%), 1 cylindroma (0%), 5 hidradenomas (40%), 7 syringocystadenoma papilliferum cases (0%), 1 papillary hidradenoma (100%), 2 tubulopapillary hidradenomas (50%), 8 hidrocystomas (29%), 2 adenoid cystic carcinomas (100%), 5 porocarcinomas (20%), 6 apocrine carcinomas (33%), 10 extramammary Paget diseases (30%), 1 spiradenocarcinoma (100%), and 1 syringocystadenocarcinoma papilliferum (0%). Most KIT-positive cells were luminal cells, arising from glandular structures. We performed polymerase chain reaction-single-strand conformation polymorphism for detecting KIT mutational status. All cases showed no mutations at hot spots for KIT (exons 9, 11, 13, and 17). KIT mutation does not seem to be mechanism for KIT expression, but the expression may be from native sweat glands.

Establishment of new predictive markers for distant recurrence of colorectal cancer using lectin microarray analysis.

We evaluated the clinical benefits of novel predictive markers for distant recurrence with colorectal cancer using lectin microarray analysis of cell surface glycan modifications. Glycoproteins were extracted from formalin-fixed, paraffin-embedded tumor specimens and normal epithelium from 53 consecutive curatively resected stage I-III colorectal cancer cases and then subjected to lectin microarray to obtain lectin-glycan interaction (LGI) values. In addition, clinicopathological factors associated with distant recurrence were identified. LGI values that were associated with distant recurrence were validated with an additional 55 curatively resected stage II colorectal cancer cases. LGI values for Agaricus bisporus (ABA) lectin, prominent in cancer tissues, were statistically associated with distant recurrence. ABA lectin staining exhibited strikingly intense signals in the cytoplasm and apical surfaces of cancer cells, while weak staining was observed in the supranuclear regions of normal epithelium. This ABA tumor/normal LGI ratio may be a new predictive biomarker for distant recurrence of curatively resected colorectal cancer.

Inhibition of metastasis of rhabdomyosarcoma by a novel neutralizing antibody to CXC chemokine receptor-4.

Rhabdomyosarcoma is the most common soft tissue sarcoma affecting children, and the overall cure rate of children with metastatic disease remains below 30%. The CXC chemokine receptor-4 (CXCR4)/stromal cell-derived factor-1 (SDF1) axis has been implicated in the promotion of metastatic potential in several tumors. In this study, we developed a novel anti-CXCR4 mAb, CF172, and investigated its antimetastatic activity against rhabdomyosarcoma cells in vitro and in vivo, to evaluate its potential as a therapeutic antibody to treat rhabdomyosarcoma. The CF172 molecule showed a specific binding reactivity against human CXCR4, as well as a specific neutralizing activity against CXCR4/SDF1 signal transduction. Using CF172, we determined that SJCRH30 rhabdomyosarcoma cells expressed high levels of CXCR4. In addition, CF172 was found to inhibit the SDF1-induced migration activity of SJCRH30 cells in vitro. Using xenograft models of SJCRH30 cells, we carried out in vivo efficacy studies for peritoneal and lymph node metastasis, which were clinically observed in rhabdomyosarcoma. These studies indicated that CF172 significantly decreased both types of metastasis of SJCRH30. In conclusion, we found that a novel anti-CXCR4 mAb, CF172, with specific reactivity against human CXCR4, prevented peritoneal metastasis and lymph node metastasis of rhabdomyosarcoma in animal models. These results suggest that CF172 is a potential antimetastasis therapeutic antibody for rhabdomyosarcoma treatment.

Mammalian target of rapamycin signaling activation patterns in pancreatic neuroendocrine tumors.

BACKGROUND: Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway dysregulation has been implicated in the development of various human cancers. However, expression of mTOR cascade components in pancreatic neuroendocrine tumors (PNETs) has not been fully explored. The aim of this study was to assess the expression of mTOR pathway in PNETs using immunohistochemistry. METHODS: From December 1984 to April 2012, we surgically treated 42 patients with PNETs. We used immunohistochemistry to evaluate expression of mTOR, phosphorylated mTOR (p-mTOR), p70S6 kinase (S6K), phosphorylated S6 ribosomal protein (p-S6rp), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and phosphorylated 4E-BP1 (p-4E-BP1) in the resected specimens. The relation between the expression of these molecules and clinicopathological characteristics was investigated. RESULTS: We identified the expression of mTOR (28.6%), p-mTOR (52.4%), S6K (52.4%), p-S6rp (40.5%), 4E-BP1 (81.0%), and p-4E-BP1 (26.2%) in PNETs. The expression of mTOR, p-mTOR, S6K, and p-S6rp was significantly associated with tumor invasion, proliferation, and an advanced-stage. Particularly, the expression of p-mTOR was related to clinically relevant factors such as tumor size, vascular invasion, extrapancreatic invasion, lymph node and/or distant metastasis, mitotic count, and European Neuroendocrine Tumor Society TNM staging as well as the 2004 and 2010 World Health Organization (WHO) classification. In addition, p-S6rp expression was related to vascular invasion, extrapancreatic invasion, lymph node and distant metastasis, mitotic count, and the 2010 WHO classification. In contrast, no significant relation between 4E-BP1 activation and clinicopathological factors was observed. The expression of p-mTOR was strongly correlated with that of p-S6rp (r = 0.474, P = 0.002). CONCLUSIONS: Our results suggest that activation of the mTOR/S6K signaling pathway plays a significant role in tumorigenesis and progression of PNET.

Significant expression of thyroid transcription factor-1 in pulmonary squamous cell carcinoma detected by SPT24 monoclonal antibody and CSA-II system.

In contrast to the usefulness of thyroid transcription factor-1 (TTF-1) in distinguishing primary adenocarcinoma of the lung from metastatic lesions, TTF-1 expression in pulmonary squamous cell carcinoma is reported to be at low level and not a suitable immunohistochemical marker. We hypothesized that the highly sensitive detection system, CSA-II, can visualize even faint expression of TTF-1 in pulmonary squamous cell carcinoma. In this study, 2 commercially available clones of TTF-1 monoclonal antibody, 8G7G3/1 and SPT24, were used for staining 38 cases of pulmonary squamous cell carcinoma, in combination with the CSA-II and the conventional detection system, EnVision. The combined use of the 8G7G3/1 clone with EnVision and CSA-II showed a positive reaction in only 1 and 4 cases, respectively. The use of SPT24 clone showed positive staining in 5 cases with EnVision and in 20 of 38 cases (52.6%) with the CSA-II. Interestingly, positive staining by the SPT24-CSA-II technique of samples from tissue blocks preserved for <2 years was 73.6% compared with only 31.5% in those preserved for >2 years. In addition, a 6-month preservation of the cut sections resulted in stain fading and decreased positivity (50%), compared with freshly cut sections. We conclude that the use of the SPT24 monoclonal antibody with the CSA-II system can detect even weak expression of TTF-1 in pulmonary squamous cell carcinoma. This staining technique can potentially allow the discrimination of primary squamous cell carcinoma of the lung from metastatic lesions, especially in freshly prepared paraffin sections.

Solid variant of keratocystic odontogenic tumor of the mandible: report of a case with a clear cell component and review of the literature.

This is a case report on a solid variant of keratocystic odontogenic tumor arising in the mandible, which aggressively infiltrated into the cancelous spaces and involved the periosteal connective tissue of the mandible. The patient was a 57-year-old woman with an ill-defined radiolucent lesion having a moth-eaten pattern in the left molar region of the mandible. Computed tomography scans revealed that the tumor penetrated the buccal cortical plate of the mandible. Histologically, the lesion was characterized by multicystic spaces lined with a thin layer of keratinizing squamous epithelium, which contained basal cells with palisaded hyperchromatic nuclei. Lumina were filled with concentric layers of parakeratin. An additional feature was the appearance of a conspicuous clear cell component showing intraluminal papillary proliferation or forming small cord-like nests in the fibrous stroma. The patient underwent segmental mandibulectomy followed by reconstruction using a titanium plate. A 20-year follow-up revealed no recurrence of the tumor.

PLAG1 and CYLD do not play a role in the tumorigenesis of adenoid cystic carcinoma.

The pleiomorphic adenoma gene 1 (PLAG1) gene is activated in a subset of pleomorphic adenomas of the salivary gland by gene fusion. Germ­line mutation in cylindromatosis (CYLD), a tumor suppressor gene, causes familial cylindromatosis and Brook­Spiegler syndrome. In the present study, aberrations in PLAG1 and CYLD were investigated in adenoid cystic carcinoma (ACC) of the salivary gland. Reverse­transcription PCR and PCR direct sequencing were performed to detect gene fusion of PLAG1 and mutation of CYLD in 34 ACC tissues. No PLAG1 fusion was detected in ACC. However, silent mutation of CYLD was detected in 2 cases of ACC, but no missense mutation was detected in ACC. These results suggest that PLAG1 and CYLD do not play a role in ACC tumorigenesis.

Cutaneous horn malignant melanoma.

A 73-year-old Japanese woman presented with cutaneous horn on the right cheek. The resected tumor was 9 mm in diameter, with 14 mm protrusion, and showed exophytic growth with marked papillomatosis. Histopathology showed proliferation of atypical melanocytes with melanin pigments in the epidermis and dermis under the cutaneous horn. These cells were confined to the base of the cutaneous horn, and did not spread to the surrounding epidermis. The final diagnosis was cutaneous horn malignant melanoma. This pathological entity is considered a specific form of verrucous melanoma, and might be added to the list of cutaneous horn-forming lesions.

Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D.

Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti-VEGF-D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF-D, as well as a specific inhibitory activity against the binding of human VEGF-D to VEGFR-3. In addition, cVE199 was found to inhibit the biological activity of VEGF-D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK-N-DZ, abundantly expressed VEGF-D, an in vivo efficacy study was performed using a xenograft model of SK-N-DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK-N-DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF-D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti-VEGF-D monoclonal antibody, cVE199, with specific reactivity against human VEGF-D, prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF-D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti-metastasis therapeutic antibody in neuroblastoma treatment.

Bronchial stump aspergillosis after stapled lobectomy for lung cancer.

Aspergillus causes several pulmonary complications, but bronchial stump aspergillosis (BSA) is very rare. To date, 31 cases of bronchial stump aspergillosis have been reported in the English, German, and Japanese literature. The bronchial stump was closed by hand-sewn suturing in most cases, but we report herein two cases of BSA that developed after stapled closure of the bronchial stump.

Ovarian small cell carcinoma complicated by carcinomatous meningitis.

Meningeal metastasis is rare in the clinical course of ovarian carcinoma and its prognosis is extremely poor. We experienced a case of carcinomatous meningitis from metastatic ovarian small cell carcinoma. A 33-year-old woman with atypical genital bleeding, was diagnosed with a right ovarian tumor and referred to our department. She underwent a total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and lymphadenectomy. It was an optimal debulking surgery. She was diagnosed with ovarian carcinoma classified as Stage IIIc according to the Féderation Internationale de Gynécologie et d'Obstétrique classification system. Histological findings showed small cell carcinoma of the pulmonary type. The tumor was bilateral with paraaortic lymph node involvement. The patient was treated with irinotecan and cisplatin (CPT-P therapy). After 4 courses of CPT-P therapy, multiple liver metastases and Virchow's lymph node metastases were found. She was treated with amrubicin as a second-line chemotherapy, but the treatment was ineffective. Five months after surgery, the patient complained of severe headache and nausea. Lumbar puncture was performed and cytology was positive. Magnetic resonance brain imaging indicated meningeal thickening. The patient was diagnosed with meningeal metastasis and received 19-Gy whole cranial irradiation. In spite of these treatments, her disease progressed rapidly and she was often drowsy. She died of aspiration pneumonia 6 months after surgery.

α-amylase crystalloid granuloma of the parotid gland: case report and review of the literature.

We report a case of α-amylase crystalloid granuloma of the parotid gland in a 65-year-old Japanese woman. Histopathologically, the lesion comprised cystlike dilatation of the ducts and foreign body granulomas, with deposits of numerous crystalloid structures. The crystalloids were eosinophilic and varied in size and shape. Immunohistochemically, the crystalloids were positive for α-amylase. Immunoelectron microscopy showed the crystalloids to be cuboidal or rectangular in shape with irregularly shaped central spaces. We discuss this rare condition and review the literature on α-amylase crystalloids.

Expression and mutational status of RON in neoplastic lesions of the breast: analysis of MSP/RON signaling in ductal carcinoma in situ and invasive ductal carcinoma.

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to MET and involved in tumorigenesis. We investigated the roles of aberrations in RON and its ligand, macrophage-stimulating protein (MSP), in invasive ductal carcinoma (IDC, n = 81), ductal carcinoma in situ (DCIS, n = 26), and in benign lesions (n = 20) of mammary gland. Expression of RON and MSP was evaluated by immunohistochemistry and the mutational status of a region containing the proteolytic cleavage site in exon 1 and each exon of the kinase domain (exon 14-20) of RON was screened by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. The proportion of cases positive for RON expression was significantly different between malignant [86% (92/107)] and benign [40% (8/20)] lesions. RON expression was positive in both IDC and DCIS [90% (73/81) and 73% (19/26), respectively], whereas MSP expression was present in 54% (44/81) of IDC and absent in DCIS. RON expression correlated significantly with the histological grade of DCIS. No mutations were detected in the examined regions of RON in breast cancer samples as confirmed by PCR-SSCP. The findings suggest the involvement of RON expression in the development of breast cancer, and that an autocrine/paracrine loop of RON seems to affect tumor invasiveness.