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BACKGROUND: We previously showed that total tumor resection enhances metastatic growth in a syngeneic metastatic mouse model of neuroblastoma. In this study, we further investigated which surgical factors contributed most to metastatic growth. METHODS: Tumor cells derived from MYCN transgenic mice were subcutaneously injected into wild-type mice. Mice were randomly assigned to receive partial resection (PR group), subcutaneous implantation of a sponge (Sp group), or observation (Obs group). The lymph node metastasis volume and the frequency of lung metastasis were compared 14 days after assignment by measuring C-reactive protein (CRP) and interleukin-6 (IL-6) levels. RESULTS: The lymph node metastasis volume in the Sp group was larger than in the Obs group (148.4 [standard deviation {SD}: 209.5] vs. 10.2 [SD 12.8] mm3). The frequency of lung metastasis was greater in the Sp group than in the PR group (11.9 [SD 12.2] vs. 6.6 [SD 4.0] counts/slide). The CRP level in the Sp group was higher than in the PR group (2.3 [SD 0.5] vs. 1.5 [SD 0.4] µg/mL), and the IL-6 level in the Sp group was higher than in the PR or Obs groups (28.4 [SD 34.5] vs. 12.4 [SD 19.0] vs. 5.4 [SD 8.1] pg/mL). CONCLUSION: Metastatic growth may be enhanced by systemic inflammation.
Subject(s)
C-Reactive Protein , Disease Models, Animal , Inflammation , Lung Neoplasms , Neuroblastoma , Animals , Neuroblastoma/pathology , Mice , Lung Neoplasms/pathology , Lung Neoplasms/secondary , C-Reactive Protein/metabolism , Inflammation/pathology , Interleukin-6 , Lymphatic Metastasis , Mice, TransgenicABSTRACT
BACKGROUND: Long-term urinary outcomes after anorectal malformation (ARM) repair are affected by surgical approach and sacral anomalies. This study aimed to compare laparoscopic-assisted anorectoplasty (LAARP) and posterior sagittal anorectoplasty (PSARP) in terms of urinary complications. METHODS: Between 2001 and 2022, 45 patients were treated with LAARP or PSARP. The rectourethral fistula and inflow angle between the fistula and rectum was confirmed by preoperative colonography. The incidence of urinary complications and treatment were compared between the two groups. RESULTS: Four patients (14%) had remnant fistula and five patients (17%) had neurogenic bladder dysfunction in LAARP group, while three patients (18%) had urethral injury in PSARP group. All patients with remnant fistula were asymptomatic and followed without treatment. The incidence of remnant fistula improved between earlier decade and later decade. In all cases with urethral injury, suture repair was performed and no postoperative leakage was noted. All five patients with neurogenic bladder dysfunction had spine abnormalities that required clean intermittent catheterization (CIC) and two were free from CIC finally. CONCLUSIONS: It is important to check inflow angle preoperatively to prevent remnant fistula. For PSARP, meticulous dissection is required when separating fistula from urethra because they create common wall. The most contributing factor to neurogenic bladder is sacral anomalies. Preoperative evaluation and postoperative urinary drainage are important.
Subject(s)
Anorectal Malformations , Laparoscopy , Rectal Fistula , Urethral Diseases , Urinary Bladder, Neurogenic , Urinary Fistula , Humans , Infant , Rectum/surgery , Rectum/abnormalities , Anorectal Malformations/complications , Anorectal Malformations/surgery , Anorectal Malformations/epidemiology , Urinary Bladder, Neurogenic/etiology , Laparoscopy/adverse effects , Treatment Outcome , Rectal Fistula/surgery , Rectal Fistula/complications , Urinary Fistula/etiology , Urinary Fistula/surgery , Urethral Diseases/etiology , Urethral Diseases/surgery , Postoperative Complications/etiology , Urethra/surgery , Retrospective Studies , Anal Canal/abnormalitiesABSTRACT
The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.
Subject(s)
Biomarkers, Tumor , Homovanillic Acid , Neuroblastoma , Vanilmandelic Acid , Humans , Neuroblastoma/urine , Neuroblastoma/diagnosis , Male , Female , Risk Assessment , Child, Preschool , Biomarkers, Tumor/urine , Infant , Homovanillic Acid/urine , Vanilmandelic Acid/urine , Child , Catecholamines/urine , Case-Control Studies , Dopamine/urine , Dopamine/analogs & derivatives , Chromatography, LiquidABSTRACT
Introduction: Intractable lymphatic anomalies (LAs) include cystic lymphatic malformation (LM; macrocystic, microcystic, or mixed), generalized lymphatic anomaly, and Gorham-Stout disease. LAs can present with severe symptoms and poor prognosis. Thus, prospective studies for treatments are warranted. We conducted a prospective clinical trial of sirolimus for intractable LAs. Methods: This was an open-label, single-arm, multicenter, prospective trial involving five institutions in Japan. All patients with LAs received oral sirolimus once daily, and the dose was adjusted to ensure that the trough concentration remained within 5-15 ng/mL. We prospectively assessed the drug response (response rate for radiological volumetric change in target lesion), performance state, change in respiratory function, visceral impairment (pleural effusion, ascites, bleeding, pain), laboratory examination data, quality of life (QOL), and safety at 12, 24, and 52 weeks of administration. Results: Eleven patients with LAs (9 generalized lymphatic anomaly, 1 cystic LM, 1 Gorham-Stout disease) were treated with sirolimus, of whom 6 (54.5%; 95% confidence interval: 23.4-83.3%) demonstrated a partial response on radiological examination at 52 weeks of administration. No patients achieved a complete response. At 12 and 24 weeks of administration, 8 patients (72.7%) already showed a partial response. However, patients with stable disease showed minor or no reduction after 12 weeks. Adverse events, such as stomatitis, acneiform dermatitis, diarrhea, and fever, were common with sirolimus. Sirolimus was safe and tolerable. Conclusion: Sirolimus can reduce the lymphatic tissue volume in LAs and may lead to improvements in clinical symptoms and QOL.
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Introduction: Biliary atresia (BA) is a cholestatic hepatopathy caused by fibrosing destruction of intrahepatic and extrahepatic bile ducts, and its etiology has not been clearly revealed. In BA, liver fibrosis progression is often observed even after Kasai portoenterostomy (KPE), and more than half of cases require liver transplantation in their lifetime in Japan. Macrophages play an important role in liver fibrosis progression and are classically divided into proinflammatory (M1) and fibrotic macrophages (M2), whose phenotypic transformation is called "macrophage polarity." The polarity has been reported to reflect the tissue microenvironment. In this study, we examined the relationship between macrophage polarity and the post-KPE clinical course. Materials and methods: Thirty BA patients who underwent KPE in our institution from 2000 to 2020 were recruited. Multiple immunostainings for CD68, CD163, CK19, and α-SMA were carried out on liver biopsy specimens obtained at KPE. ROC curves were calculated based on each clinical event, and the correlation with the clinical data was analyzed. Results and discussion: The M2 ratio, defined as the proportion of M2 macrophages (CD163-positive cells), was correlated inversely with the occurrence of postoperative cholangitis (AUC: 0.7602). The patients were classified into M2 high (n = 19) and non-high (n = 11) groups based on an M2 ratio value obtained from the Youden index ( = 0.918). As a result, pathological evaluations (Metavir score, αSMA area fraction, and CK19 area fraction) were not significantly different between these groups. In mild liver fibrosis cases (Metavir score = 0-2), the M2 non-high group had a significantly lower native liver survival rate than the high group (p = 0.02). Moreover, 4 out of 8 cases in the M2 non-high group underwent early liver transplantation within 2 years after KPE. Conclusions: Non-M2 macrophages, including M1 macrophages, may be correlated with postoperative cholangitis, and the M2 non-high group in mild liver fibrosis cases had a significantly lower native liver survival rate than the high group, requiring early liver transplantation in this study. Preventing advanced liver fibrosis is a key factor in improving native liver survival for BA patients, and liver macrophages may play important roles in liver homeostasis and the promotion of inflammation and fibrosis.
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BACKGROUND: Tumor recurrence, anorectal and urinary dysfunction, and lower limb dysfunction after surgery are observed in infantile sacrococcygeal teratoma (SCT). In this paper, a multi-institutional retrospective observational study was conducted to clarify the long-term functional prognosis in Japan. METHODS: This study was conducted using a paper-based questionnaire distributed to 192 facilities accredited by the Japanese Society of Pediatric Surgeons, covering patients who underwent radical surgery at less than 1 year old and who survived for at least 180 days after birth from 2000 to 2019. RESULTS: A total of 355 patients were included in this analysis. Altman type was I-II in 248 and type III-IV in 107, and the median maximum tumor diameter was 6.1 (range: 0.6-36.0) cm. There were 269 mature teratomas, 69 immature teratomas, and 10 malignant tumors. Total resection was performed in 325, subtotal or partial resection in 27, and surgical complications were noted in 54. The median postoperative follow-up was 6.6 (0.5-21.7) years. Eighty-three patients (23.4 %) had functional sequelae, including 62 (17.5 %) with anorectal dysfunction, 56 (13.0 %) with urinary dysfunction, and 15 (4.2 %) with lower limb motor dysfunction. Recurrence occurred in 42 (11.8 %) at a median age of 16.8 (1.7-145.1) months old. Risk factors for dysfunction included preterm delivery, a large tumor diameter, Altman type III-IV, incomplete resection, and surgical complications. Risk factors for recurrence included immature teratoma or malignancy, incomplete resection, and surgical complications. CONCLUSIONS: Postoperative dysfunction was not low at 23.4 %, and 11.8 % of the patients experienced recurrence occurring more than 10 years after surgery, suggesting the need for periodic imaging and tumor markers evaluations in patients with risk factors. It is necessary to establish treatment guidelines for best practice monitoring of the long-term quality of life. LEVEL OF EVIDENCE: Level II Retrospective Study.
Subject(s)
Pelvic Neoplasms , Spinal Neoplasms , Teratoma , Child , Humans , Infant , Japan/epidemiology , Neoplasm Recurrence, Local/epidemiology , Neoplasm Recurrence, Local/pathology , Pelvic Neoplasms/epidemiology , Pelvic Neoplasms/surgery , Quality of Life , Retrospective Studies , Sacrococcygeal Region/pathology , Spinal Neoplasms/pathology , Teratoma/epidemiology , Teratoma/surgery , Teratoma/complications , Child, Preschool , Adolescent , Young Adult , AdultABSTRACT
BACKGROUND: It has been hypothesized that the origin of early-onset endometriosis could be from endometrial mesenchymal stem cells (eMSCs) in neonatal uterine blood (NUB). There is no information on the possible mechanistic basis linking an association between NUB/neonatal endometrium and development of early-onset endometriosis. In this study we performed a series of experiments to clarify the mechanistic link between NUB and/or neonatal endometrium and development of early-onset endometriosis. METHODS: We retrospectively collected postmortem neonatal endometria (n = 15) and prospectively collected NUB (n = 18) of female babies for the analysis of different biological markers including eMSCs. Immunohistochemical analysis of neonatal endometria was performed to examine the expression patterns of ovarian steroid receptors (ER/PGR), decidualization (prolactin, IGFBP1), pre-decidualization (Glycodelin A, α-SMA), proliferation (Ki-67 index), vascularity (CD31 + cells), immunocompetent CD68+, CD45+, CD56 + cells and some putative markers of eMSCs. Cell transfer method and immunocytochemistry were used to investigate the eMSCs and/or endometrial cells in NUB. RESULTS: Immunohistochemical analysis of postmortem neonatal endometria revealed variable staining response to ER/PGR, decidual markers, and substantial proliferative and angiogenic activity. A moderate to strong immunoexpression of Glycodelin-A was found in both neonatal and adult endometria. The tissue infiltration of CD56+, CD45 + and CD68 + immunocompetent cells was significantly low in neonatal endometria than that in adult endometria (p = 0.0003, p < 0.0001, p = 0.034, respectively). No eMSCs or even endometrial cells were detected in NUB. However, a variable expression of some phenotypes of eMSCs (CD90/CD105) was found in neonatal endometria. CONCLUSIONS: Based on our serial experiments we did not find any supporting evidence for the role of NUB in early-onset endometriosis. Neonatal endometria showed variable expression of ovarian steroid receptors, decidualization, and a substantial amount of proliferative and angiogenic activity. As an alternative mechanism, a significantly less tissue accumulation of immunocompetent cells in neonatal endometria may explain the survival of ER + and PGR + cells should they make entry into the pelvis and consequent development of early endometriosis with the onset of ovarian function. Future study with large sample size and application of modified technological tools is warranted to test the NUB hypothesis and to clarify their biological or clinical significance. TRIAL REGISTRATION: not applicable.
Subject(s)
Endometriosis , Humans , Female , Endometriosis/metabolism , Retrospective Studies , Glycodelin/metabolism , Endometrium/metabolism , Uterine Hemorrhage/metabolismABSTRACT
BACKGROUND/AIM: Using the tyrosine hydroxylase (TH)-MYCN mouse neuroblastoma (NB) model, we have previously reported the accumulation of mouse mesenchymal stem cells (mMSCs) on tumors in vivo and the antitumor effect of mMSCs transfected with a small molecule (IFN-ß) expression gene. In this study, we have developed novel MSCs secreting anti-disialoganglioside GD2 antibody (anti-GD2-MSCs) and evaluated their antitumor effects in vitro. MATERIALS AND METHODS: We generated an anti-GD2 antibody construct (14.G2a-Fcx2-GFP) incorporating FLAG-tagged single-chain fragment variable against GD2 fused to a linker sequence, a fragment of the constant portion of human IgG1, and GFP protein. The construct was lentivirally transduced into mMSCs and the transduction efficiency was assessed by GFP expression. The secretion of FLAG-tagged anti-GD2 antibody was detected by Western blotting using anti-FLAG antibody. Antibody binding capacity was confirmed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using human NB cells and human natural killer (NK) cells to assess whether the antitumor activity was enhanced in the presence of the produced antibodies. RESULTS: The transduction efficiency of anti-GD2-MSCs was more than 90%. anti-GD2-MSCs secreted antibodies extracellularly and these antibodies had high affinity to GD2-expressing human NB cells. ADCC assays showed that the addition of antibodies secreted from anti-GD2-MSCs significantly increased the cytotoxic activity of NK cells against NB cells. CONCLUSION: Newly developed anti-GD2-MSCs produced functional antibodies that have affinity to the GD2 antigen on NB cells and can induce ADCC-mediated cytotoxicity. Anti-GD2-MSCs based cellular immunotherapy has the potential to be a novel therapeutic option for intractable NB.
Subject(s)
Antibodies, Monoclonal , Mesenchymal Stem Cells , Mice , Humans , Animals , Antibodies, Monoclonal/pharmacology , Killer Cells, Natural , Antibody-Dependent Cell Cytotoxicity , Immunotherapy , Gangliosides/genetics , Gangliosides/metabolismABSTRACT
BACKGROUND: Intrahepatic bile duct (IHBD) stones are one of the most common late complications of Roux-en-Y hepaticojejunostomy for congenital biliary dilatation (CBD). We report the current treatment strategies for IHBD stones and their outcomes in our institute. METHODS: Between 1983 and 2021, 117 patients with CBD were surgically treated in our institute. Our treatment strategies included oral ursodeoxycholic acid (UDCA), double-balloon endoscopic retrograde cholangiography (DB-ERC), percutaneous cholangio-drainage (PTCD), and open surgery. A retrospective study was conducted using medical charts. RESULTS: Postoperative IHBD stones were identified in 12 of 117 patients with CBD (10.2%). Five patients received UDCA, and small stones were successfully resolved in two cases. DB-ERC was performed eight times in five patients, but the endoscope could not reach the porta hepatis due to a long jejunal loop in two of five patients. One patient presented with severe acute pancreatitis induced by prolonged DB-ERC. PTCD was performed in three patients, two of whom finally underwent open surgery due to unsuccessful lithotomy. Open surgery was eventually performed in three patients. Lithotomy was performed in one patient; lithotomy with strictureplasty was performed in another patient. The other patient was diagnosed with intrahepatic cholelithiasis with adenocarcinoma. He underwent left lobectomy and died of carcinomatous peritonitis. CONCLUSIONS: Oral UDCA may be effective for small stones. Although DB-ERC should be considered as a first-line interventional therapy for lithotomy, it may not be feasible due to a long jejunal loop, and pancreatitis may occur. Long-term follow-up and early detection and treatment for IHBD stones may yield a good prognosis.
Subject(s)
Choledochal Cyst , Pancreatitis , Male , Humans , Retrospective Studies , Acute Disease , Bile Ducts, Intrahepatic/surgery , Ursodeoxycholic AcidABSTRACT
PURPOSE: Mouse IgG anti-disialoganglioside GD2 antibody-secreting mouse mesenchymal stem cells (anti-GD2-MSCs) were developed, and their anti-tumor effects were validated in an in vivo neuroblastoma mouse model. METHODS: Anti-GD2 antibody constructs were generated, incorporating FLAG-tagged single-chain fragment variables against GD2 fused to a linker sequence, and a fragment of a stationary portion was changed from human IgG to mouse IgG and GFP protein. The construct was lentivirally introduced into mouse MSCs. A syngeneic mouse model was established through the subcutaneous transplantation of a tumor tissue fragment from a TH-MYCN transgenic mouse, and the homing effects of anti-GD2-MSCs were validated by In vivo imaging system imaging. The syngeneic model was divided into three groups according to topical injection materials: anti-GD2-MSCs with IL-2, IL-2, and PBS. The tumors were removed, and natural killer (NK) cells were counted. RESULTS: Anti-GD2-MSCs showed homing effects in syngeneic models. The growth rate of subcutaneous tumors was significantly suppressed by anti-GD2-MSCs with IL-2 (p < 0.05). Subcutaneous tumor immunostaining showed an increased NK cell infiltration in the same group (p < 0.01). CONCLUSION: Anti-GD2-MSCs using mouse IgG showed a homing effect and significant tumor growth suppression in syngeneic models. Anti-GD2-MSC-based cellular immunotherapy could be a novel therapeutic strategy for intractable neuroblastoma.
Subject(s)
Mesenchymal Stem Cells , Neuroblastoma , Humans , Mice , Animals , Gangliosides/therapeutic use , Interleukin-2/therapeutic use , Neuroblastoma/metabolism , Disease Models, Animal , Mice, Transgenic , Immunoglobulin G/therapeutic useABSTRACT
BACKGROUND: The leading pathology of biliary atresia (BA) is inflammatory and fibrous obstruction of extrahepatic bile duct, but the pathogenesis remains unclear. IL13 is a cytokine associated with allergies and inflammatory fibrosis, and periostin induces fibrogenesis by stimulation with IL13. We analyzed the involvement of IL13 and periostin in inflammatory fibrosis in the extrahepatic bile duct of BA patients. MATERIALS AND METHODS: Surgically resected tissues from the hepatic hilar area of BA patients were immunostained with CD45, α-SMA, IL13 and periostin and statistically analyzed. Fibroblasts from the resected tissue were cultured with recombinant IL13, and periostin production was analyzed by quantitative polymerase chain reaction and Western blotting. RESULTS: IL13 was stained in 93% of large and micro bile ducts, and 92.1% matched with the CD45 location (p = 0.006) around the large bile ducts. Periostin staining correlated with the localization of IL13 and αSMA (p < 0.001) around the large bile ducts. Periostin mRNA and protein were upregulated by IL13 stimulation in cultured fibroblasts. CONCLUSION: IL13 was associated with induced periostin expression by fibroblasts, playing a vital role in the pathogenesis of fibrogenesis around the extrahepatic bile duct in BA.
Subject(s)
Bile Ducts, Extrahepatic , Biliary Atresia , Cell Adhesion Molecules , Interleukin-13 , Humans , Bile Ducts/surgery , Bile Ducts/pathology , Biliary Atresia/surgery , Biliary Atresia/metabolism , Fibrosis , Interleukin-13/metabolism , Liver/metabolism , Cell Adhesion Molecules/metabolismABSTRACT
BACKGROUND: One of the most frequent complications after repair of esophageal atresia (EA) is gastroesophageal reflux disease (GERD). Although GERD-associated EA is known to often require anti-reflux surgery, the predicting factors remain unclear. We retrospectively analyzed EA in our institution. METHODS: Of 65 children with EA treated in our hospital from 1995 to 2018, 45 with Gross C type EA, followed for over 1 year, were enrolled in this study. The patients were divided into fundoplication and non-fundoplication groups and compared in terms of their clinical features. RESULTS: The fundoplication and non-fundoplication groups included 13 and 32 cases, respectively. On univariate analysis, gestational age, body weight, prenatal diagnosis, polyhydramnios, re-do surgery, and gap length of the esophagus differed significantly between the groups (P < 0.05). CONCLUSION: Early delivery, low body weight, and a long gap length are, are considered to be risk factors for fundoplication. However, the present study further showed that prenatal diagnosis and polyhydramnios were also significant contributing factors. The presence of a prenatal diagnosis and polyhydramnios may induce preterm delivery, therefore, cases of polyhydramnios due to suspected EA should be managed to prevent early delivery. Better understanding of the postnatal course after surgery is required, especially for prenatal diagnosis cases.
Subject(s)
Esophageal Atresia , Gastroesophageal Reflux , Child , Esophageal Atresia/complications , Esophageal Atresia/surgery , Fundoplication/adverse effects , Gastroesophageal Reflux/diagnosis , Gastroesophageal Reflux/etiology , Gastroesophageal Reflux/surgery , Humans , Infant, Newborn , Retrospective Studies , Risk Factors , Treatment OutcomeABSTRACT
BACKGROUND: We previously reported the in vitro and in vivo antitumor effects of trametinib, a MEK inhibitor, on neuroblastoma with MAPK pathway mutations. As we observed eventual resistance to trametinib in our previous study, we evaluated the combination therapy of CA3, a YAP inhibitor, with trametinib, based on a recent report suggesting the potential involvement of YAP in the mechanism underlying the resistance to trametinib in neuroblastoma. METHODS: SK-N-AS cells (a neuroblastoma cell line harboring RAS mutation) were treated with CA3 in vitro and subjected to a viability assay, immunocytochemistry and flow cytometry. Next, we analyzed the in vitro combination effect of CA3 and trametinib using the CompuSyn software program. Finally, we administered CA3, trametinib or both to SK-N-AS xenograft mice for 10 weeks to analyze the combination effect. RESULTS: CA3 inhibited cell proliferation by both cell cycle arrest and apoptosis in vitro. Combination of CA3 and trametinib induced a significant synergistic effect in vitro (Combination Index <1). Regarding the in vivo experiment, combination therapy suppressed tumor growth, and 100% of mice in the combination therapy group survived, whereas the survival rates were 0% in the CA3 group and 33% in the trametinib group. However, despite this promising survival rate in the combination group, the tumors gradually grew after seven weeks with MAPK reactivation. CONCLUSION: Our results indicated that CA3 and trametinib exerted synergistic antitumor effects on neuroblastoma in vitro and in vivo, and CA3 may be a viable option for concomitant drug therapy with trametinib, since it suppressed the resistance to trametinib. However, this combination effect was not sufficient to achieve complete remission. Therefore, we need to adjust the protocol to obtain a better outcome by determining the mechanism underlying regrowth in the future.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Extracellular Signal-Regulated MAP Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neuroblastoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Female , Mice, Nude , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Pyridones/pharmacology , Pyridones/therapeutic use , Pyrimidinones/pharmacology , Pyrimidinones/therapeutic use , S Phase/drug effects , Survival Analysis , Transcription Factors/metabolism , Xenograft Model Antitumor Assays , YAP-Signaling ProteinsABSTRACT
PURPOSE: We reported the in vitro and in vivo anti-tumor effects of trametinib, an MEK inhibitor, on neuroblastoma. However, long-term trametinib administration for bulky tumors failed to prevent local relapse. In this study, we established a local minimal residual disease (L-MRD) model to develop an optimal clinical protocol. METHODS: We prepared an l-MRD model by implanting neuroblastoma cells (SK-N-AS) into the renal capsule of nude mice with total tumorectomy or sham operation 14 days later. These mice received post-operative administration of trametinib or vehicle for eight weeks. Relapse was measured once weekly. Flow cytometry was performed with SK-N-AS cells treated by trametinib. RESULTS: Tumorectomy+trametinib dramatically suppressed relapse, and all mice survived during trametinib administration, while other treatments failed to suppress relapse. The survival rates for other groups were 20% in sham+trametinib, 17% in tumorectomy+vehicle, and 0% in sham+vehicle. Relapse occurred in the tumorectomy+trametinib group after withdrawal of trametinib administration. Flow cytometry revealed G1 arrest in SK-N-AS cells treated with trametinib. CONCLUSION: These findings suggested that trametinib was able to suppress relapse from minimal residual tumor cells. Therefore, we propose that trametinib be administered as an option for maintenance therapy after surgical and chemotherapeutic treatments for neuroblastoma in future clinical protocols.
Subject(s)
Neoplasm Recurrence, Local , Neuroblastoma , Animals , Cell Line, Tumor , Mice , Mice, Nude , Neoplasm Recurrence, Local/drug therapy , Neoplasm, Residual , Neuroblastoma/drug therapy , Protein Kinase Inhibitors , Pyridones/pharmacology , Pyridones/therapeutic use , Pyrimidinones/therapeutic useABSTRACT
PURPOSE: The majority of relapsed neuroblastomas have mitogen-activated protein kinase (MAPK) pathway activating mutations. We previously showed the in vitro and in vivo anti-tumor effects of MAPK/ERK kinase (MEK) inhibitors in MAPK-activated neuroblastoma. We herein assessed the correlation between MAPK activation and the prognosis in neuroblastoma patients using phosphorylated extra-cellular signal-regulated kinase (pERK) immunohistochemistry to establish the protocol for the clinical administration of MEK inhibitors. METHODS: Neuroblastoma samples from patients treated in our hospital were immunostained with pERK. The clinical outcomes were retrospectively collected from medical records. The correlation between pERK positivity and the prognosis was analyzed. RESULTS: Regarding pre-chemotherapeutic specimens, there were no differences in the pERK status between tumors with a good and bad prognosis in both the nuclei and cytoplasm. Regarding post-chemotherapeutic specimens, one of eight tumors with a good prognosis and four of six tumors with a poor prognosis showed pERK-positive nuclear staining (p = 0.0909) and five of eight tumors with a good prognosis and four of six tumors with a poor prognosis showed pERK-positive cytoplasmic staining (p > 0.9999). CONCLUSION: These findings suggested post-chemotherapeutic-not pre-chemotherapeutic-nuclear pERK-positive neuroblastoma tends to be associated with a poor prognosis and may be a potential therapeutic target for MEK inhibitor treatment.
Subject(s)
Antineoplastic Agents/therapeutic use , Extracellular Signal-Regulated MAP Kinases/metabolism , Immunohistochemistry/methods , Neuroblastoma/metabolism , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Phosphorylation , Prognosis , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Retrospective StudiesABSTRACT
BACKGROUND/AIM: Malignant rhabdoid tumor (MRT) is a rare, aggressive neoplasm found in young children, caused by inactivation of a single gene, SNF5 (INI1, SMARCB1). MRT cases with multifocal tumors at diagnosis are categorized as synchronous MRT, often with a germline mutation of SNF5. The aim of this study was to establish new models useful in clarifying the biological basis of synchronous MRT. MATERIALS AND METHODS: We established two novel MRT cell lines, designated as KP-MRT-KS and KP-MRT-KSa, derived from different lesions and at a different time from a synchronous multifocal 7-month-old female MRT patient. RESULTS: Both cells showed typical morphology of MRT, with a compound genomic mutation in exons 2 and 5 of the SNF5 gene. The exon 2 mutation was found in the germline. CONCLUSION: These cell lines could serve as powerful tools for unveiling the molecular mechanism of refractory synchronous MRT.
Subject(s)
Neoplasms, Multiple Primary/pathology , Rhabdoid Tumor/pathology , Base Sequence , Carcinogenesis/genetics , Carcinogenesis/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Cluster Analysis , DNA Methylation/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Infant , Neoplasms, Multiple Primary/genetics , Rhabdoid Tumor/diagnostic imaging , Rhabdoid Tumor/genetics , SMARCB1 Protein/genetics , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Pulmonary hypoplasia is an important cause of morbidity and mortality in infants with congenital diaphragmatic hernia (CDH). This study aimed to verify our hypothesis that the abnormal development of bronchial cartilage as well as alveolar immaturity, might play a central role in hypoplasia of the lung in human CDH. METHOD: We retrospectively analyzed autopsied lungs from 10 CDH cases and compared with nine age-matched controls to assess the bronchial cartilage and alveolar maturity using morphological techniques. RESULT: Ki-67 and thyroid transcription factor-1 (TTF-1) expression in the alveoli significantly increased in bilateral lungs with CDH. The shortest distance from the bronchial cartilage to the pleura was significantly shorter in ipsilateral (left) lungs with CDH, showing a positive correlation with the radial alveolar count (RAC). Regarding the small bronchial cartilages less than 20 000 µm2 , the average cartilage area significantly decreased in left lungs with CDH, and tended to decrease in right lungs with CDH. In addition, cartilage around the bronchi less than 200 µm in diameter tended to be smaller in left lungs with CDH. In contrast, regarding the cartilage around the bronchi 200 to 400 µm in diameter, the ratio of the total cartilage area relative to the bronchial diameter tended to be higher in left lungs with CDH, although there was a large variation. CONCLUSIONS: These opposite directional cartilage abnormalities around the distal and more proximal bronchi support our hypothesis that abnormal development of bronchial cartilage might play an important role in the hypoplastic lung in CDH.
Subject(s)
Bronchi/abnormalities , Cartilage/abnormalities , Hernias, Diaphragmatic, Congenital , Female , Hernias, Diaphragmatic, Congenital/metabolism , Humans , Infant, Newborn , Ki-67 Antigen/metabolism , Male , Pulmonary Alveoli/metabolism , Retrospective Studies , Thyroid Nuclear Factor 1/metabolismABSTRACT
Emerging data have suggested that sirolimus may be a treatment option for complicated vascular anomalies (VAs). The present study aimed to investigate the immunologic effects of sirolimus treatment for 6 months in patients with VAs. Blood samples obtained from the patients enrolled in 2 multicenter studies to investigate the efficacy of sirolimus for VAs before and after sirolimus treatment for 6 months were used. Data for total white blood cell count, absolute lymphocyte count, serum immunoglobulins (Igs) levels (IgG, IgA, IgM), lymphocyte proliferation assays with mitogens including phytohemagglutinin and concanavalin A, and flow cytometric analysis of lymphocyte subsets were evaluated. A total of 18 patients with VAs receiving sirolimus treatment were included in the study. Comparisons of white blood cell, absolute lymphocyte count, IgG, IgA, IgM, and reaction rates of phytohemagglutinin and concanavalin A revealed no significant differences before and after treatment. No significant differences were observed in the absolute counts of lymphocyte subtypes before and after treatment, except for regulatory T-cell counts, which were significantly decreased after treatment. Severe infections were not observed during sirolimus treatment. The immunologic parameters assessed in the present study were hardly affected by sirolimus treatment for 6 months in patients with VAs.
Subject(s)
Immunosuppressive Agents/therapeutic use , Lymphocytes/immunology , Sirolimus/therapeutic use , T-Lymphocytes, Regulatory/immunology , Vascular Malformations/drug therapy , Adolescent , Adult , Child , Child, Preschool , Female , Follow-Up Studies , Humans , Infant , Lymphocytes/drug effects , Male , Prognosis , T-Lymphocytes, Regulatory/drug effects , Vascular Malformations/immunology , Vascular Malformations/pathology , Young AdultABSTRACT
Human teratoma is a germ cell tumor that contains normal tissues (e.g., hair, skin or cartilage) differentiated from embryonal germ layers. Because of the feature of this tumor, we hypothesized that human teratomas contain multipotent stem cells that can develop into various non-cancerous normal tissues. In this study, we cultured neurospheres originally derived from a human infantile teratoma tissue, and the sphere cells were found to possess the characteristics of neural stem cells. Tumor tissues were obtained from an infantile immature teratoma at the time of surgical resection. In the primary cell culture, colonies were formed in two weeks and were individually cultured in serum-free conditioned neural stem cell medium (NSC medium). Colonies changed into spheres and grew in smooth round forms, or attached to the bottom of the dishes and extended processes and filaments around. Sphere cells were dissociated into single cells, and new spheres (secondary spheres) were formed in NSC medium. Cell differentiation was induced by culturing cells in serum-containing medium (differentiation medium), as cells spread and attached to the bottom of dishes and changed form. The expression of Nestin, Sox2, CXCR4, and (stem cell markers), ß3-tubulin (a neural marker) GFAP (a glial marker) CNPase, SOX10 (oligodendrocyte markers) and NF-L in cells was analyzed by immunofluorescence and a Q-PCR. Nestin, SOX2, CXCR4 were abundant in both primary and secondary spheres. Neural and glial markers (ß3-tubulin and GFAP, respectively) were increased in cells cultured in differentiation medium while stem cell markers were diminished. The oligodendrocyte markers SOX10 and CNPase were also found in both spheres and differentiated cells. In conclusion, spheres with the characteristics of neural stem cells were obtained from the primary culture of a human infantile teratoma. These spheres are considered to have the potential to undergo a natural course of neural development in humans.
Subject(s)
Antigens, Differentiation/metabolism , Multipotent Stem Cells/metabolism , Neural Stem Cells/metabolism , Teratoma/metabolism , Humans , Multipotent Stem Cells/pathology , Neural Stem Cells/pathology , Teratoma/pathologyABSTRACT
PURPOSE: Mesenchymal stem cells (MSCs) are reported to migrate toward damaged tissues or tumors. We previously reported the in vivo short-term (1 day) tumor-homing effect of xenogeneic human MSCs (hMSCs) using the TH-MYCN mouse neuroblastoma model (MYCN-TgM). In this study, we analyzed the long-term tumor-homing effect of allogeneic mouse MSCs (mMSCs) and explored the antitumor effect and drug delivery function of mMSCs. METHODS: mMSCs were administered intraperitoneally (i.p.) to MYCN-TgM and traced by an in vivo imaging system (IVIS). We administered green fluorescent protein (GFP)-transduced mMSCs into MYCN-TgM i.p. and examined the cell survival by immunohistochemistry. We also administered interferon beta-transduced mMSCs (mMSCs-IFN-ß) to MYCN-TgM i.p. and measured the concentration of IFN-ß in the tumor and organs by an enzyme-linked immunosorbent assay (ELISA). The survival curves of MYCN-TgM administered every week was analyzed. RESULTS: The IVIS revealed the accumulation of fluorescence was observed in the tumor both in vivo and after excision. Immunohistochemistry using anti-GFP antibody revealed that the mMSCs existed within the tumor until 14 days but not in the organs. The ELISA showed increased concentrations of IFN-ß only in the tumors, with the values gradually diminishing over 14 days. The mMSCs-IFN-ß group survived significantly longer than the control group (p < 0.03), while the mMSCs-alone group did not show a survival advantage. CONCLUSIONS: Allogeneic mMSCs showed a homing ability for mouse neuroblastoma and existed within the tumor for as long as two weeks. This may be a candidate drug delivery vehicle for antitumor agents against neuroblastoma.