Prognostic Impact of Tumor-Infiltrating Lymphocytes, Tertiary Lymphoid Structures, and Neutrophil-to-Lymphocyte Ratio in Pulmonary Metastases from Uterine Leiomyosarcoma.

BACKGROUND: The presence of tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) in tumor tissue has been related to the prognosis in various malignancies. Meanwhile, neutrophil-to-lymphocyte ratio (NLR) as a systemic inflammation marker also has been associated with the prognosis in them. However, few reports have investigated the relationship between pulmonary metastases from sarcoma and these biomarkers. METHODS: We retrospectively recruited 102 patients undergoing metastasectomy for pulmonary metastases from uterine leiomyosarcoma at Okayama University Hospital from January 2006 to December 2019. TILs and TLSs were evaluated by immunohistochemical staining of surgically resected specimens of pulmonary metastases using anti-CD3/CD8/CD103/Foxp3/CD20 antibodies. NLR was calculated from the blood examination immediately before the most recent pulmonary metastasectomy. We elucidated the relationship between the prognosis and these factors. Because we considered that the status of tumor tissue and systemic inflammation were equally valuable, we also assessed the impact of the combination of TILs or TLSs and NLR on the prognosis. RESULTS: As for TILs, CD3-positive cells and CD8-positive cells were correlated with the prognosis. The prognosis was significantly better in patients with CD3-high group, CD8-high group, TLSs-high group, and NLR-low group, respectively. The prognosis of CD8-high/NLR-low group and TLSs-high/NLR-low group was significantly better than that of CD8-low/NLR-high group and TLSs-low/NLR-high group, respectively. CONCLUSIONS: CD3-positive TILs, CD8-positive TILs, TLSs, and NLR are correlated with the prognosis, respectively. The combination of CD8-positive TILs or TLSs and NLR may be the indicators to predict the prognosis of patients with pulmonary metastases from uterine leiomyosarcoma.

Survival and prognostic factors in patients undergoing pulmonary metastasectomy for lung metastases from retroperitoneal sarcoma.

BACKGROUND: Soft-tissue sarcomas are rare malignancies that consist of many different histologic subtypes and arise in various locations in the body. In patients with lung metastases from retroperitoneal sarcomas, the long-term outcomes and prognostic factors are unknown. This study is a retrospective review of patients undergoing pulmonary metastasectomy for retroperitoneal sarcoma metastases at one institution, with the purpose of determining prognostic factors and clinical outcomes. METHODS: This is a single-center, retrospective cohort study of patients undergoing pulmonary metastasectomy for lung metastases from various sarcomas at Okayama University Hospital from January 2006 to December 2018. The Kaplan-Meier method and log-rank test were used for the analyses, and cut-off values of continuous variables were determined by a receiver operating characteristic curve analysis. RESULTS: Twenty-four patients underwent the first pulmonary metastasectomy for lung metastases from retroperitoneal sarcoma in our hospital. Leiomyosarcoma was the most common histologic subtype of retroperitoneal sarcoma (79.2%, n = 19). Median overall survival was 49.9 months, and the 3-year and 5-year survival rates after the first pulmonary metastasectomy were 62.5% and 26.4% respectively. In univariate analysis, age ≥56 years, disease-free interval < 15 months, and size of metastasis (≥ 27 mm) were associated with poor survival. CONCLUSION: Pulmonary metastasectomy can be considered as an effective management strategy in retroperitoneal sarcoma patients with lung metastases in appropriately selected cases, just as it is for other sarcomas.

A Simple Prognostic Benefit Scoring System for Sarcoma Patients with Pulmonary Metastases: Sarcoma Lung Metastasis Score.

BACKGROUND: Pulmonary metastasectomy could be considered one of the treatment options for disease control in sarcoma patients with pulmonary metastases; however, there is little consensus regarding the suitable criteria for predicting the likely outcomes in these patients. The aim of this study was to establish a prognostic benefit scoring system based on preoperatively examined prognostic factors for sarcoma patients with pulmonary metastases. METHODS: This was a single-center, retrospective cohort study conducted in a cohort of 135 sarcoma patients who underwent a first pulmonary metastasectomy at Okayama University Hospital between January 2006 and December 2015. Based on the results of a multivariable logistic regression analysis performed to determine the factors influencing 3-year mortality, a Sarcoma Lung Metastasis Score was created and its correlation with 3-year survival was analyzed. RESULTS: The results of the multivariate analysis revealed significant differences in the disease-free interval (< 2 years vs. ≥ 2 years; odds ratio (OR) 4.22, 95% confidence interval (CI) 1.67-10.70), maximum tumor diameter (≥ 15 mm vs. < 15 mm; OR 3.86, 95% CI 1.75-8.52), and number of pulmonary metastases (≥ 6 vs. < 6; OR 2.65, 95% CI 1.06-6.620). The Sarcoma Lung Metastasis Score, which was defined as the total score of these three factors, reliably predicted 3-year survival (score: 0, 89.5%; 1, 63.2%; 2, 39.0%; 3, 10.5%). CONCLUSIONS: Our newly proposed simple Sarcoma Lung Metastasis Score appears to be a useful prognostic predictor for sarcoma patients with pulmonary metastases, in that it could be helpful for the selection of appropriate treatments for these patients.

The neutrophil-to-lymphocyte ratio as a novel independent prognostic factor for multiple metastatic lung tumors from various sarcomas.

PURPOSE: Sarcomas are among the most refractory malignant tumors and often recur as pulmonary metastasis. Although the presence of a high neutrophil-to-lymphocyte ratio (NLR) has been associated with the prognosis of several malignancies, the relationship between the NLR and sarcoma with pulmonary metastasis is unclear. We investigated the impact of the NLR in patients who underwent surgical resection for metastatic lung tumors from various sarcomas. METHODS: The subjects of this retrospective study were 158 patients with metastatic lung tumors from various sarcomas, who underwent initial pulmonary metastasectomy between 2006 and 2015. We examined the clinicopathological variables, including the NLR and the characteristics of surgical procedures. Survival was estimated by the Kaplan-Meier method and prognostic factors were evaluated by multivariate analysis. RESULTS: Multivariate analysis revealed significantly better survival of the group with an NLR < 2.26 immediately before the most recent pulmonary metastasectomy, in addition to such factors as the largest resected lesion being < 22 mm, a disease-free interval of > 2 years, and 3 or more pulmonary metastasectomies. CONCLUSION: The NLR immediately before the most recent pulmonary metastasectomy is a novel independent prognostic factor, which may be helpful when considering repeated pulmonary metastasectomy.

Double deletion of calponin 1 and calponin 2 in mice decreases systemic blood pressure with blunted length-tension response of aortic smooth muscle.

Calponin is a family of actin filament-associated regulatory proteins. Among its three isoforms, calponin 1 is smooth muscle specific and calponin 2 is expressed in smooth muscle and certain non-muscle cells. Previous studies showed that calponin 1 knockout mice had detectable changes in the contractility of urogenital smooth muscle whereas other smooth muscles were less affected. To investigate the possibility that calponins 1 and 2 have overlapping functions in smooth muscle, we examined the effect of double knockout of calponin 1 and calponin 2 genes (Cnn1 and Cnn2) on smooth muscle functions. The results showed for the first time that calponin 1 and calponin 2 double knockout in mice does not cause lethality. The double knockout mice showed decreased systemic blood pressure, decreased force development and blunted length tension response in endothelial-removed aortic rings. A compensatory increase of calponin 1 was found in smooth muscle of Cnn2-/- mice but not vice versa. Cnn1-/- and Cnn2-/- double knockout aortic smooth muscle exhibits faster relaxation than that of wild type control. Double deletion or co-suppression of calponin 1 and calponin 2 in vascular smooth muscle to blunt myogenic response may present a novel approach to develop new treatment for hypertension.

[The Cutting Edge of Sarcoma Genomics].

Sarcoma is well-known rare cancer with few therapeutic options. Recent comprehensive genomic analyses of adult soft tissue sarcoma revealed few somatic mutations and massive copy number variations(CNVs)by the specific chromosomes. Those features are quite different from the genomics of carcinoma such as lung and colon cancers in which driver and passenger mutations play a central role in the pathogenesis. Furthermore, it has been demonstrated that substantial population of sarcoma patients has pathological germline variants of cancer predisposition genes. These findings imply that, in addition to somatic mutations, inherited germline variants may play a role in the disease state of sarcoma via dosage effects. On this basis, we also discuss on the prospect and limitation of the precision medicine of sarcoma.

[A case of sarcomatoid malignant peritoneal mesothelioma responding to combination chemotherapy of cyclophosphamide, vincristine, adriamycin and dacarbazine(CYVADIC)].

A 66-year-old woman was seen at our hospital because of abdominal fullness. A computed tomography(CT)revealed massive tumors in abdominal cavity. The patient underwent surgery consisting of tumorectomy, segmental gastrectomy, partial resection of small intestin, transverse colectomy, left oophorectomy and gastrostomy. By using immunohistochemical staining, the patient was diagnosed as sarcomatoid malignant peritoneal mesothelioma. Rapidly abdominal fullness occurred as of 22 days after the operation, and an abdominal CT revealed the massive recurrent tumors. We started a combination chemotherapy of cyclophosphamide, vincristine, adriamycin and dacarbazine (CYVADIC). The recurrent tumors showed remarkable reduction after the two courses of CYVADIC chemotherapy. Although we next started carboplatin and paclitaxel combination chemotherapy, she died due to rapidly progression of the disease with disseminated intravascular coagulation after 132 days of the operation. Malignant mesothelioma, especially sarcomatoid mesothelioma, is known to have a poor prognosis. However, our case suggests that we could improve the prognosis of sarcomatoid malignant mesothelioma by aggressive chemotherapy.

Role of H1-calponin in pancreatic AR42J cell differentiation into insulin-producing cells.

Basic or h1-calponin is a smooth muscle-specific, actin-binding protein that is involved in the regulation of smooth muscle contractile activity. We found in this study the expression of mRNA and protein for h1-calponin in AR42J-B13 cells, which is a useful model for investigating islet beta-cell differentiation from pancreatic common precursor cells. Following treatment of AR42J cells with activin A and hepatocyte growth factor, the protein levels of h1-calponin decreased in a time-dependent manner during the course of the cell differentiation. When h1-calponin was continuously overexpressed by utilizing recombinant adenovirus-mediated gene transfer, the percentage of cell differentiation in h1-calponin overexpressing cells was markedly suppressed as compared with that in the cells without overexpression (6.7 +/- 2.5 vs. 28.6 +/- 3.2%, P < 0.001, Student's t test). Finally, overexpression of h1-calponin (65.6 +/- 3.4), or that lacking actin-binding domain (55.9 +/- 3.4%), significantly (P < 0.001) suppressed the activin A-stimulated transcriptional activity of activin responsive element (ARE), whereas calponin homology-domain disruption mutant did not (100.6 +/- 1.9%). These results suggest that regulation of h1-calponin is involved in the regulation of differentiation of AR42J cells into insulin-producing cells at least partly through modulating ARE transcriptional activity.

Mutations affecting somite formation in the Medaka (Oryzias latipes).

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.

Mutations affecting liver development and function in Medaka, Oryzias latipes, screened by multiple criteria.

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Malignant phyllodes tumor metastasized to the right ventricle: a case report.

Cardiac metastasis of malignant phyllodes tumor is very rare. We herein report a rare case that developed cardiac metastasis from malignant phyllodes tumor. A 38-year-old woman underwent lumpectomy, and the final pathological findings showed the 5-cm malignant phyllodes tumor partially containing 1 cm of squamous cell carcinoma. Four months after the first surgery, a local recurrence of malignant phyllodes tumor and distant metastases to the bone, lung, pulmonary main trunk, and right ventricle were detected. Mass reduction surgery of cardiac metastasis of the malignant phyllodes tumor was performed to avoid sudden death. In immunohistochemical findings, the tumor was suspected to be originated in myoepithelial cells because of the expression of smooth muscle lineage including α-smooth muscle actin and Calponin1 and highly malignant characteristics showing MIB-1 and p53 highly positive with angiogenesis. Further studies are needed to clarify the effective treatment to these tumors.

Aberrant methylation and silencing of the calponin gene in human sarcoma cells.

BACKGROUND: We have recently developed a new strategy for the treatment of sarcomas by using human calponin promoter to drive replication and oncolysis of herpes simplex virus (HSV-1). In order to apply this therapy to a broad spectrum of sarcoma cells, it is essential to elucidate silencing mechanisms of the calponin promoter in a subset of sarcoma cells: MATERIALS AND METHODS: In this study, we investigated the mechanisms of calponin inactivation in human sarcoma tissues and cell lines through the analysis of CpG methylation of promoter region by bisulfite modification, because the 5'-flanking region of the human calponin gene was originally identified as a DNA fragment bound to the conserved methyl-CpG binding domain (MBD) of MeCP2. Furthermore, we examined the correlation between DNA methylation and expression of the calponin gene by treatment with the demethylating agent 5-Aza-CdR, and in vitro 5'CpG methylation in a luciferase reporter gene construct. RESULTS: Here, we have provided evidence that DNA hypermethylation of the specific 5'CpG sites in exon 1 is a mechanism accompanying decreased calponin expression in both human sarcoma tissues and cell lines. CONCLUSION: These results suggest that DNA methylation may be an important mechanism regulating calponin transcription in human sarcoma cells, and thus could potentially affect the efficacies of treatment of this malignancy utilizing the human calponin promoter.

Altered mechanical properties in smooth muscle of mice with a mutated calponin locus.

The mechanical properties of smooth muscles in aorta and vas deferens were studied in mice with a mutated basic calponin locus to learn the physiological function of calponin. The intact smooth muscles were stimulated with high KCl and the force development was compared between calponin deficient (knockout, KO) mice and wild type (WT) ones. The isometric force induced by various concentrations of high KCl was lower in KO than in WT both in aorta and in vas deferens. The length-force relations were compared between KO and WT. The active isometric force in KO was significantly lower at most muscle lengths examined than in WT without the change in resting force both in aorta and in vas deferens. In vas deferens, the rate of force development after quick release in length at the peak force was significantly faster in KO than in WT. The above results show that the force development is lower and the rate of cross-bridge cycle is faster in KO mice than in WT ones, suggesting that calponin plays basic roles in the control of the contraction of smooth muscle.

Increased locomotor activity, increased food and water intake and decreased PVN neurons in H1 calponin gene-deficient mice.

Calponin (h1 or basic) is an actin-binding protein that is expressed abundantly in smooth muscle. Our previous study using h1 calponin-null mutant mice demonstrated that h1 calponin inhibits the shortening velocity of smooth muscle contraction without significantly affecting the amplitude of force production. Furthermore, early onset of osteogenesis and increased bone formation have been reported in mutated mice. In the present study, we examined the effect of h1 calponin depletion on the metabolism and behavior of mice and found that the mutated mice showed increased locomotor activity, as well as increased intake of food and water, associated with the decreased number of neurons in the paraventricular nucleus of the hypothalamus (PVN).

A Single-Center, Open-Label, Randomized, Parallel-Group Study Assessing the Differences Between an Angiotensin II Receptor Antagonist and an Angiotensin-Converting Enzyme Inhibitor in Hypertensive Patients with Congestive Heart Failure: The Research for Efficacy of Angiotensin II Receptor Antagonist in Hypertensive Patients with Congestive Heart Failure Study.

BACKGROUND: Angiotensin II receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) have been used to treat congestive heart failure (CHF). According to a MEDLINE search, however, few studies are available on the clinical differences between ARBs and ACEIs in CHF. OBJECTIVE: To examine the clinical differences between an ARB (candesartan cilexetil) and an ACEI (lisinopril) in the treatment of CHF, we investigated exercise capacity, ventricular function, and neurohormonal levels in hypertensive patients with CHF before and after treatment with these agents. METHODS: Patients with symptoms of CHF (New York Heart Association functional class II-III and left ventricular ejection fraction [LVEF] ≤45%) complicated by hypertension (systolic blood pressure [BP] ≥140 mm Hg or diastolic BP ≥90 mm Hg) were eligible for this single-center, open-label, randomized, parallel-group study. They were given either the ARB or the ACEI for 24 weeks. A cardiopulmonary exercise test and echocardiography were performed. Clinical findings and cardiac events in addition to the CHF symptoms were investigated. Neurohormonal levels were measured before and after 24 weeks of treatment with the study drug. The primary end point of this study was exercise capacity, which was measured using peak oxygen consumption (VO2). RESULTS: Forty-two patients with CHF were enrolled and 38 (28 men, 10 women; mean [SD] age, 69.0 [8.2] years) completed the study. None of these patients had definite progression of the CHF symptoms. In the ARB-treated patients, mean (SD) peak VO2 (mL/min/kg) and LVEF (%) increased from 14.1 (2.9) to 15.3 (3.4) and from 34.4 (9.5) to 41.8 (9.5), respectively. In the ACEI group, the peak VO2 did not change, but the LVEF (%) increased from 34.2 (10.2) to 40.4 (13.0). However, the differences between ARB and ACEI were not clarified because of the possibility of a small sample size. CONCLUSIONS: Although this study was not powered to show differences in efficacy between the ARB and ACEI in this study, our findings suggest that both ARB and ACEI had beneficial effects in hypertensive patients with CHF. Some unidentified differences in hemodynamic characteristics were found between the ARB and the ACEI groups.

[Development of incurable cancer cell targeted agents using viral technology].

The safety and anti-tumor effect of oncolytic virus have been reported in a clinical study conducted in Japan. We have engineered a novel multimutated tumor-specific oncolytic herpes virus, harboring a smooth muscle-specific calponin promoter. Since tumor cells present in a hypoxic environment are known to be resistant to chemotherapy and radiotherapy, we also engineered a novel oncolytic herpes virus targeting a specific tumor microenvironment, which harbors a gene encoding a fusion protein of oxygen-dependent degradation (ODD) domain of HIF1α and ICP4, a master viral transcription factor required for replication. The recombinant virus selectively replicates in and disrupts the target tumor cells, including human sarcoma and malignant mesothelioma cells which are unresponsive to chemotherapy and molecular targeted therapy. We confirmed significant anti-tumor effects of the novel viruses in vivo in an allogeneic experimental model of an undifferentiated pleomorphic sarcoma (malignant fibrous histiocytoma; MFH) spontaneously generated in immunocompetent Fischer rats. Our viruses, manufactured in the Master Virus Seed Stock in the Good Manufacturing Practice facility will become novel agents that enable tumor cells unresponsive to conventional treatment to be disrupted.