Conversion surgery for initially unresectable locally advanced pancreatic ductal adenocarcinoma after chemotherapy followed by carbon-ion radiotherapy: a case report.

BACKGROUND: Recent advances in chemotherapy and chemoradiotherapy have enabled conversion surgery (CS) to be performed for selected patients with initially unresectable locally advanced (LA) pancreatic ductal adenocarcinoma (PDAC). Many studies indicate CS might extend the survival of patients with initially unresectable LA PDAC. However, several clinical questions concerning CS remain, such as the optimal preoperative treatment. Carbon-ion radiotherapy (CIRT) is a unique radiotherapy that offers higher biological effectiveness than conventional radiotherapy. Here, we report a long-term survival case with initially unresectable LA PDAC who underwent CS after chemotherapy followed by CIRT. CASE PRESENTATION: The patient was a 72-year-old Japanese woman with unresectable LA pancreatic head cancer with tumor contact to the superior mesenteric artery (SMA). She underwent four courses of chemotherapy (gemcitabine plus nanoparticle albumin-bound paclitaxel). However, the lesion did not shrink and tumor contact with the SMA did not improve after chemotherapy. Because the probability of achieving curative resection was judged to be low, she underwent radical dose CIRT, and chemotherapy was continued. She complained of vomiting 2 months after CIRT. Although imaging studies showed no tumor growth or metastasis, a duodenal obstruction which was speculated to be an adverse effect of CIRT was observed. She could not eat solid food and a trans-nasal feeding tube was inserted. Therapeutic intervention was required to enable enteral nutrition. We proposed several treatment options. She chose resection with the expectation of an anti-tumor effect of chemotherapy and CIRT rather than course observation with tube feeding or bypass surgery. Therefore, subtotal-stomach-preserving pancreatoduodenectomy with portal vein resection was performed as CS. Pathological examination of the resected specimen revealed an R0 resection with a histological response of Evans grade IIA. Postoperatively, she recovered uneventfully. Adjuvant chemotherapy with tegafur/gimeracil/oteracil (S1) was administrated. At the time of this report, 5 years have passed since the initial consultation and she has experienced no tumor recurrence. CONCLUSIONS: The present case suggests that multidisciplinary treatment consisting of a combination of recent chemotherapy and CIRT may be beneficial for unresectable LA PDAC. However, further studies are required to assess the true efficacy of this treatment strategy.

Preoperative frailty as a predictive factor for postoperative complications in patients with pancreatic cancer.

Frailty is considered one of the most important indicators of a patient's general condition. However, only a few studies have investigated the association between preoperative frailty and postoperative complications in pancreatic cancer. Therefore, this study aimed to examine this association in patients with pancreatic cancer. We retrospectively reviewed 52 consecutive patients who underwent pancreatectomy for pancreatic cancer between July 2019 and March 2021. Patients were classified into two groups according to the presence of postoperative complications. Their characteristics and clinical parameters, including physical function, were analyzed. Patients with postoperative complications had a higher prevalence of frailty (58.8% vs 14.3%, p = 0.003) and a shorter 6-min walk distance (380 m vs 436 m, p = 0.020) than those without postoperative complications. Logistic regression analysis identified preoperative frailty as the only independent risk factor for complications after pancreatectomy (p = 0.002). Preoperative frailty is associated with postoperative complications of pancreatectomy. Since preoperative frailty can be easily evaluated, it is a useful predictor of postoperative complications after pancreatectomy.

Mixed neuroendocrine-non-neuroendocrine neoplasm of the ampulla of Vater: a case report.

BACKGROUND: Mixed neuroendocrine-non-neuroendocrine neoplasms of the ampulla of Vater are rare and heterogenous, making it difficult to achieve a definitive preoperative diagnosis. Herein, we describe a patient in whom a provisional diagnosis of mixed neuroendocrine-non-neuroendocrine neoplasm of the ampulla of Vater was made preoperatively. CASE PRESENTATION: Computed tomography revealed an enhancing periampullary tumor in a 69-year-old man with obstructive jaundice. Subsequent duodenoscopy revealed an ulcerated lesion in the swollen ampulla of Vater, from which six biopsies were collected. Pathological examination revealed adenocarcinoma in five of them. The remaining one was a neuroendocrine neoplasm according to immunohistochemical analysis. With a provisional diagnosis of mixed neuroendocrine-non-neuroendocrine neoplasm of the ampulla of Vater, the patient underwent subtotal stomach-preserving pancreaticoduodenectomy with modified Child's reconstruction and was discharged without complications. Pathological examination revealed both adenocarcinoma and neuroendocrine carcinomas, each accounting for ≥ 30% of the tumor, resulting in a definitive diagnosis of mixed neuroendocrine-non-neuroendocrine neoplasm of the ampulla of Vater. Lymph node metastases with neuroendocrine components were also observed. Adjuvant chemotherapy was not administered because of the patient's renal dysfunction. Liver and lymph node metastases were detected 2 months after surgery, the neuroendocrine component being considered responsible for that relapse. The patient underwent platinum-based chemotherapy at 50% dosage, which initially resulted in significant tumor shrinkage; however, he died 6 months after surgery. CONCLUSIONS: While these tumors' heterogeneity make definitive preoperative diagnosis of mixed neuroendocrine-non-neuroendocrine neoplasm of the ampulla of Vater difficult, the possibility of this disease can be considered by careful examination. Further study is needed to establish the optimal diagnostic criteria and treatment strategy.

Gallbladder Ciliated Foregut Cyst Suspected of Malignancy Preoperatively.

BACKGROUND: Gallbladder ciliated foregut cysts (CFCs) of the lower diaphragm are extremely rare. Furthermore, they are rarely suspected of malignancy preoperatively. Case Presentation. A 50-year-old woman was referred to our hospital for further examination and treatment of a gallbladder tumor that was detected using abdominal ultrasonography (US). After a close inspection, she was diagnosed with a gallbladder tumor that was possibly malignant. Accordingly, open whole layer cholecystectomy was performed because intraoperative US revealed a tumor located on the intraperitoneal side of the gallbladder, and a rapid intraoperative pathological diagnosis identified no malignancy. A postoperative pathological examination revealed a cystic lesion with thin walls covered with ciliated epithelium, which laid on a connective tissue with smooth muscle fibers. Based on the above results, the final pathological diagnosis was CFC of the gallbladder without malignancy. CONCLUSIONS: Cases of gallbladder CFC can be considered as cysts requiring treatment owing to CFCs' potential for malignant transformation and high-frequency symptoms.

Double mapping with subareolar blue dye and peritumoral green dye injections decreases the false-negative rate of dye-only sentinel node biopsy for early breast cancer: 2-site injection is more accurate than 1-site injection.

BACKGROUND: The optimum sentinel node biopsy (SNB) mapping method for breast cancer remains to be determined. No matter which mapping agents are used, 2-site injection may be superior to 1-site injection in limiting the false-negative rate. METHODS: We examined whether a double-mapping method with subareolar injection of blue dye and peritumoral injection of green dye would decrease the false-negative rate of dye-only SNB in 145 patients with early breast cancer. RESULTS: The identification rate for blue-dyed and/or green-dyed (including mixed color-dyed) lymph nodes was 96.6% (140/145). Sensitivity and specificity were 95.1% (39/41) and 100% (99 of 99), respectively. Accuracy was 98.6% (138/140) with a false-negative rate of 4.9% (2/41). There were 4 patients in whom nodes of each color were found, but nodes of only 1 color were shown to be positive. The primary tumors of these 4 patients and of the 2 patients with false-negative results were located in the upper-outer quadrant of the breast. When only blue-dyed or green-dyed nodes (including mixed color-dyed nodes) were counted, the false-negative rates were 10.3% (4/39) for the subareolar mapping technique and 10.0% (4/40) for the peritumoral mapping technique. CONCLUSIONS: The double-mapping method based on subareolar and peritumoral injections decreases the false-negative rate of dye-only SNB for early breast cancer. Variations in lymphatic channels may exist in the lateral half of the breast and thus may influence identification of positive sentinel nodes. This finding should be taken into account in cases of multicentric breast cancer.

Evaluation of a dysfunctional and short-lived subset of monocyte-derived dendritic cells from cancer patients.

Monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood monocytes of 12 healthy volunteers (hMo-DCs) and 11 patients (pMo-DCs) with malignancies by culture for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4. In this study, we focused on the cytogram pattern by FACS analysis. A gate (R1) was set up by which more than 95% of hMo-DCs were contained. Mo-DCs having lower side scatter than the R1 (R2) comprised 4.5% of hMo-DCs and 24.2% of pMo-DCs. Expressions of antigen presentation-related molecules and phagocytic ability in the R2 of pMo-DCs were lower than those in the R1 population. The R2, but not R1, in pMo-DCs decreased in number between days 7 and 14, and expression levels of antigen presentation-related molecules in the living pMo-DCs on day 14 increased. The 11 patients received dendritic cell vaccine therapy with autologous, tumor-pulsed mature Mo-DCs (day 7). The low R2 group (R2 < or = 10%, 3 patients) had a significantly higher positive delayed-type hypersensitivity reaction against autologous tumor-pulsed Mo-DCs than that of the high R2 group (R2 > 10%, 8 patients) (p<0.001). These results indicate that the R2 of pMo-DCs may be a dysfunctional and short-lived subset.

Inactivation of protease-activated receptor-1 by proteolytic removal of the ligand region in vascular endothelial cells.

Proteolysis plays an important role in inactivating protease-activated receptor-1 (PAR1). We aimed to determine the cleavage site(s) responsive for the proteolytic inactivation of PAR1 in human umbilical vein endothelial cells. Fura-2 fluorometry revealed that the preceding stimulation with trypsin abolished the subsequent [Ca(2+)](i) response to thrombin, while the responses to PAR1-activating peptides remained intact. On the other hand, thrombin had no effect on the subsequent response to trypsin. The immunostaining with antibodies against the residues 35-46 (SPAN12) and 51-64 (WEDE15) revealed the broad boundaries of cleavage. Trypsin removed both epitopes from the cell surface within 3 min, while thrombin removed the epitope of SPAN12. The longer incubation with thrombin removed the epitope of WEDE15. However, PAR1-activating peptides thereafter induced an attenuated but significant elevation of [Ca(2+)](i). Not only the receptor internalization as observed with a confocal microscope, but also an additional cleavage was thus suggested to contribute to the thrombin-induced removal of the epitope of WEDE15. The analyses of the PAR1 mutants identified three cleavage sites for trypsin; residues 41-42, 70-71 and 82-83. The cleavage at the latter two sites was suggested to dominate that at the former, and thus remove the ligand region (residues 42-47). The inactivation of PAR1 due to proteolytic removal of the ligand region may contribute not only to the inactivation of PAR1 by proteases such as trypsin, but also to the termination of the intracellular signaling initiated by thrombin in the vascular endothelial cells.

Three-dimensional two-layer collagen matrix gel culture model for evaluating complex biological functions of monocyte-derived dendritic cells.

Dendritic cell-like cells (Mo-DCs) generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used as tools to treat cancer patients (DC-vaccines). Because Mo-DCs have multiple antigen presentation-related functions, including phagocytosis, migration, cytokine production, and T cell stimulation, establishment of a method for simultaneously evaluating the various functions of Mo-DCs is important. We developed a new in vitro three-dimensional two-layer collagen matrix culture model that consists of a collagen gel containing Mo-DCs as the lower layer and a collagen gel containing necrotic GCTM-1 tumor cells and/or T cells as the upper layer. We used this system to observe simultaneously multiple functions of Mo-DCs by phase-contrast or fluorescence microscopy and to assess IL-12 secretion during more than 2 weeks of culture. We also observed interactions between Mo-DCs and necrotic GCTM-1 or T cells on an individual cell basis by time-lapse videomicroscopy. In addition, we collected Mo-DCs from the collagen gels by collagenase treatment and analyzed the expression of antigen presentation-related molecules such as HLA-DR, CD80, CD83, and CD86 on Mo-DCs. This model may be a useful tool for evaluation of the various functions of Mo-DCs used as DC vaccines and for studies of the complex behaviors of Mo-DCs in vivo.

Interleukin 1beta enhances invasive ability of gastric carcinoma through nuclear factor-kappaB activation.

PURPOSE: We examined the role of interleukin (IL)-1beta in activation of nuclear factor kappaB (NF-kappaB) and the biological function of activated NF-kappaB in gastric carcinoma cells. EXPERIMENTAL DESIGN: Human gastric carcinoma cell line GCTM-1 was used to examine NF-kappaB activation by immunostaining and electrophoretic mobility shift assay. Matrix metalloproteinase (MMP)-9 expression, which plays an important role in tumor invasion, was assessed by semiquantitative reverse transcription-PCR, Western blotting, and immunostaining. The invasive ability of GCTM-1 cells was measured by Matrigel invasion assay. In vivo expression of IL-1beta and MMP-9 and activation of NF-kappaB in 10 surgically resected gastric carcinoma specimens were examined immunohistochemically. RESULTS: IL-1beta enhanced NF-kappaB activation, MMP-9 expression, and the invasive ability of GCTM-1. A NF-kappaB inhibitor, pyrrolidine dithiocarbamate, suppressed both MMP-9 expression and invasiveness of IL-1beta-treated GCTM-1 cells. IL-1beta did not increase the invasive ability of GCTM-1 cells transfected with MMP-9 antisense oligonucleotide. Concomitant expression of IL-1beta and nuclear NF-kappaB was observed in 3 of 10 gastric carcinoma specimens. Cells producing IL-1beta were tumor-infiltrating macrophages in two specimens and gastric carcinoma cells in one specimen. CONCLUSIONS: One of the molecules that may play a role in NF-kappaB activation in some gastric carcinomas is IL-1beta. The present results suggest that IL-1beta increases the invasive ability of carcinoma cells through activation of NF-kappaB and the resulting MMP-9 expression.

Exosomes secreted from monocyte-derived dendritic cells support in vitro naive CD4+ T cell survival through NF-(kappa)B activation.

We investigated the effect of exosomes secreted from human monocyte-derived dendritic cells (Mo-DCs), which are generated from PBMCs in response to treatment with GM-CSF and IL-4, on naive CD4+ T cell survival in vitro. Exosomes isolated from culture supernatants of Mo-DCs (>90% purity) were purified with anti-HLA-DP, -DQ, -DR-coated paramagnetic beads. Purified exosomes prolonged the survival of naive CD4+ T cells (>98% purity) in vitro. Treatment with neutralizing mAb against HLA-DR significantly decreased the supportive effect of purified exosomes on CD4+ T cell survival. Exosomes increased nuclear translocation of NF-(kappa)B in naive CD4+ T cells, and NF-(kappa)B activation was significantly suppressed by anti-HLA-DR mAb or NF-(kappa)B inhibitor pyrrolidine dithiocarbamate (PDTC). In addition, PDTC inhibited the effect of exosomes on naive CD4+ T cell survival. Thus, exosomes secreted by Mo-DCs appear to support naive CD4+ T cell survival via NF-(kappa)B activation induced by interaction of HLA-DR and TCRs.

Monocyte-derived dendritic cells that capture dead tumor cells secrete IL-12 and TNF-alpha through IL-12/TNF-alpha/NF-kappaB autocrine loop.

This study focused on the question of how monocyte-derived dendritic cells (Mo-DCs) that capture dead tumor cells (Mo-DCs-Tum) secrete interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-alpha). Mo-DCs-Tum showed higher secretions of IL-12 and TNF-alpha than were shown by Mo-DCs. Enhanced nuclear factor-kappa B (NF-kappaB) activation was also induced in Mo-DCs-Tum within 6 h. The NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed both IL-12 and TNF-alpha secretions from Mo-DCs-Tum. Administration of recombinant TNF-alpha or IL-12 enhanced IL-12 or TNF-alpha secretion respectively in Mo-DCs-Tum. Addition of anti-TNF-alpha or anti-IL-12 neutralizing antibody decreased NF-kappaB activation and IL-12 or TNF-alpha secretion in Mo-DCs-Tum. These results suggest that TNF-alpha or IL-12 secretion induces NF-kappaB activation, and it stimulates further TNF-alpha and IL-12 secretions, i.e., an IL-12/TNF-alpha/NF-kappaB autocrine loop, in Mo-DCs-Tum. Thus, Mo-DCs-Tum secrete a large amount of IL-12 and TNF-alpha through accelerated NF-kappaB activation induced by the IL-12/TNF-alpha/NF-kappaB autocrine loop.

Interferon-gamma suppresses transforming growth factor-beta-induced invasion of gastric carcinoma cells through cross-talk of Smad pathway in a three-dimensional culture model.

We reconstituted a three-dimensional gastric carcinoma model similar to invasive gastric carcinoma tissue. This model consists of a human gastric carcinoma cell line, GCTM-1, a human fibroblast cell line, TIG-1-20, and transforming growth factor-beta (TGF-beta)-containing type I collagen gel. Using this model, we were able to observe the growth of the two cell types, especially carcinoma cell invasive growth, in real time for more than 30 days. TGF-beta and TIG-1-20 were essential for GCTM-1 invasive growth and proliferation, respectively. TGF-beta induced the enhanced expression of matrix metalloproteinase 9 (MMP9) and urokinase-type plasminogen activator (uPA) in GCTM-1 at both the protein and enzymatic activity levels. The TGF-beta-induced invasion of GCTM-1 was inhibited by MMP9- or uPA-antisense (AS) oligonucleotide transfection to GCTM-1. When exogenous interferon-gamma (IFN-gamma) was added to this model, TGF-beta-dependent GCTM-1 invasion was significantly inhibited, concomitant with the decreased expression of MMP9 and uPA. The intracellular signal transduction of Smad was examined to analyse the mechanism of the inhibitory effect of IFN-gamma. TGF-beta accelerated the phosphorylation of Smad2/3 and nuclear translocation of the Smad2/3-Smad4 complex in GCTM-1, but these TGF-beta-induced effects were significantly inhibited by IFN-gamma-induced Smad7 expression. When GCTM-1 was cotransfected with AS oligonucleotide of Smad2 and Smad3, the TGF-beta-induced invasion of GCTM-1 disappeared. In addition, the inhibitory effect of IFN-gamma on TGF-beta-dependent GCTM-1 invasion vanished by the AS oligonucleotide of Smad7 transfection. These results indicate that IFN-gamma inhibits TGF-beta-dependent GCTM-1 invasion through cross-talk in the Smad pathway. IFN-gamma may be a new therapeutic tool for TGF-beta-expressed invasive carcinomas.

Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells.

1 Using fura-2 fluorometry of [Ca(2+)](i) in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca(2+)](i) elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP specifically attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher affinity PAR1-AP (haPAR1-AP) (Ala-pF-Arg-Cha-HArg-Tyr-NH(2)) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.

Dysfunctional and short-lived subsets in monocyte-derived dendritic cells from patients with advanced cancer.

Dendritic cells (DCs) are antigen-presenting cells specialized for the induction of the primary T-cell response. Tumor immunotherapy using DCs loaded with tumor antigens is under way for patients with several types of advanced malignancies. In this study, DC-like cells (Mo-DCs) were generated from peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. The antigen-presenting abilities, including capture of apoptotic tumor cells, IL-12 secretion, expression of antigen-presentation-related molecules (HLA-ABC, HLA-DR, and CD80), and mixed leukocyte reaction, of Mo-DCs from 37 patients with advanced cancer (pMo-DCs) were compared to those of 20 healthy volunteers (hMo-DCs). Seven days after the initial culture, no significant difference was found in either the number or the size of Mo-DC-forming colonies between the two groups. However, most of the antigen-presenting abilities of pMo-DCs were weaker than those of hMo-DCs on day 7. On day 14, both number and size of colonies were significantly decreased in pMo-DCs but not in hMo-DCs. Interestingly, the antigen-presenting abilities of the remaining pMo-DCs gradually strengthened with time and by day 14 no significant difference was observed between pMo-DCs and hMo-DCs. These results indicate that pMo-DCs contain dysfunctional and short-lived Mo-DC subsets.

A large quantity of CD3-/CD19-/CD16- lymphocytes in malignant pleural effusion from a patient with recurrent cholangio cell carcinoma.

Tumor infiltrating lymphocytes (TILs) are candidates for adoptive cellular immunotherapy. Here we report on a patient whose TILs presented unusual lymphocyte antigens. Pleural effusions were collected from a 47-year-old man with recurrent cholangio cell carcinoma and malignant effusion. Effusion-associated lymphocytes (EALs) were separated by Ficoll-Hypaque gradient, and the EAL phenotype was determined by flow cytometry. The percentage of positive cells was determined for each lymphocyte-related differentiation antigen. The percentages of CD3+, CD19+, and CD16+ lymphocyte subpopulations among EALs were 20%, 7%, and 3%, respectively. Nearly 70% of EALs were CD3-/CD19-/CD56-/CD16- cells. The phenotypes of peripheral blood lymphocytes (PBLs) collected simultaneously from the patient's peripheral blood were CD3+ (52%), CD19+ (20%), and CD16+ (20%). When EALs were cultured in medium without pleural effusion, T cell-related antigens, but not B cell- or natural killer (NK) cell-related antigens, were newly expressed on EALs, and this expression reached a plateau after 48 h in culture. The proportions of CD3+, CD19+, and CD16+ cells were 69%, 7%, and 3%, respectively. However, when EALs were cultured in medium with pleural effusion, increased expression of T cell-related antigens was not observed; the proportions of CD3+, CD19+, and CD16+ cells were 16%, 6%, and 1%, respectively. Neither total cell numbers nor cellular viability of EALs changed significantly after in-vitro culture, suggesting that significant proliferation or death of EALs did not occur during the culture period. Co-culture of the patient's PBLs with autologous pleural effusion for 96 h did not alter the expression of lymphocyte-related antigens on the PBLs. These results indicate that expression of T cell-related antigens, but not B cell- or NK cell-related antigens, on EALs was blocked temporarily by the malignant pleural effusion. This is the first report concerning the existence of a large quantity of unclassified lymphocytes in which the T cell-related antigens were reversibly masked in the malignant pleural effusion.