Interactions between CD47 and signal regulatory protein alpha (SIRPα) transmit the “don’t eat me signal” and inhibits phagocytosis. These expressions are associated with the clinicopathological features in several malignancies; however, it has not been investigated in soft tissue sarcoma (STS).

This study aimed to elucidate the relationships between the expression of CD47 and SIRPα in STS and its clinicopathological features.

Immunohistochemistry (IHC) staining was used in 128 cases of STS to study CD47 and SIRPα expression, and their relationships with the clinicopathological features of STS were assessed statistically.

CD47 was primarily expressed by tumor cells, and SIRPα in interstitial cells. In undifferentiated pleomorphic sarcoma (UPS), high levels of CD47 were observed somewhat more frequently in patients aged 60 years and older (p=0.0743), tumor diameters ≧5cm, (p=0.0660), and FNCLCC grade 3(p=0.0660). In leiomyosarcoma (LMS), higher proportions of women were observed in cases with high CD47 (p=0.0805), and higher proportions of FNCLCC grade 2 were observed in malignant peripheral nerve sheath tumor (MPNST) (p=0.0517). In terms of prognosis, overall survival (OS) was significantly worse in patients with high SIRPα, LMS (p=0.0128), MPNST (p=0.0179) and alveolar soft part sarcoma (ASPS) (p=0.0253). OS was significantly poorer in MPNST with high levels of both CD47 and SIRPα (p=0.0005).

Our results suggest that SIRPα expression plays an important role in the tumor microenvironment, and that it may be an indication for effective treatment with molecular targeted drugs.

BACKGROUND: Factor V (FV) deficiency is a monogenic inherited coagulation disorder considered to be an ideal indication for gene therapy. To investigate the possibility of therapeutic application of genome editing, we generated induced pluripotent stem cells (iPSCs) from a FV-deficient patient and repaired the mutation of factor V gene (F5) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) approach. METHODS: The patient’s peripheral blood mononuclear cells were reprogrammed for iPSCs. The targeting vector was designed with homology arms against F5 containing the corrected sequence. Cas9 ribonucleoprotein (RNP) complex and targeting vector were electroporated into iPSCs. Gene-edited iPSCs were differentiated into hepatocyte-like cells (HLCs). RESULTS: The mutation of F5 in patient-derived iPSCs was repaired by CRISPR/Cas9. In concentrated culture supernatants of patient-derived iPS-HLCs, neither FV antigen nor activity was detected, while in those of gene-corrected iPS-HLCs, FV antigen and specific activity were 67.0 +/- 13.1 ng/mL and 173.2 +/- 41.1 U/mg, respectively. CONCLUSIONS: We successfully repaired the F5 mutation using the CRISPR/Cas9 system and confirmed the recovery of FV activity with gene-corrected iPS-HLCs. In addition to being a promising modality of gene therapy, gene-edited iPSCs are also a promising means of elucidating pathophysiology.

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-cell leukemia virus type 1 (HTLV-1). HTLV-1-associated mRNA, including HBZ and tax, is deeply involved in the pathogenesis of ATLL. Using 88 ATLL tissue samples, we performed in situ mRNA analysis of HBZ and tax, and investigated their association with clinicopathological characteristics of ATLL. The median value of HBZ signals (/ 1000 ATLL cells) was 795.2 (range: 0.4-4013.1) and of tax signals (/ 1000 ATLL cells) was 5.1 (range: 0.1-891.2). The low expression HBZ group displayed a significant increase in the number of skin lesions (P=.0283). The high expression tax group displayed a significant increase in the number of PD-1-positive tumor-infiltrating lymphocytes (P<.0001). In addition, we identified patients with very high expression of tax signals (400 or more signals/ 1000 ATLL cells). These patients displayed significant reductions in the expression of HLA class I (P=.0385) and β2M (P=.0124). Moreover, these patients displayed significantly poor overall survival (median survival time [MST] 7.7 months, 95% confidence interval [CI] [4.7-NA]), compared with patients with less than 400 tax signals (MST 22.6 months, 95% CI [13.7-41.7]) (P=.0499). These results suggest that Tax-mediated treatment of ATLL should be performed carefully in the high expression tax group. More detailed studies could elucidate the clinicopathological significance of HBZ and tax mRNA expressions in ATLL.

　With advances in minimally invasive diagnostic procedures, there is increasing demand for predictive biomarker testing on cytology samples. Similar to histology specimens, cell blocks (CBs) are embedded in paraffin, retain some tissue architectural detail, and yield multiple slides. CBs play a significant role in providing ancillary testing, including immunocytochemistry and molecular analysis. To the best of our knowledge, there is currently no standardized CB processing method worldwide. In our present study, we investigated nucleic acid quality in sodium alginate CBs.
　First, we investigated the influence of nucleic acid quality and protein expression in sodium alginate FFPE-CB produced under different formalin fixing conditions. Cell lines were fixed in two types of formalin solution, namely 10% neutral buffered formalin (NBF) and 10% non-neutral buffered formalin (NNBF), for various times, and FFPE-CBs were produced using sodium alginate. DNA integrity number (DIN) was investigated to assess DNA quality. Immunohistochemical staining differences were also evaluated. DNA quality decreased in samples fixed in both formalin solutions in a time-dependent manner. DNA quality obtained from cell lines fixed in 10% NBF for 3 and 6 hours was higher than that in samples fixed for 24 hours or more. On immunostaining for cytokeratin, cell lines fixed with both types of formalin solutions for 3 and 6 hours showed good staining. The Ki-67 labeling index in samples fixed with formalin solution for 24 hours or more were of lower quality than those fixed for 3 and 6 hours. Our data showed that relatively high-quality DNA and stable protein expression can be obtained from cytological samples fixed with 10% NBF for 3 to 6 hours using the sodium alginate FFPE-CB method.
　Second, we investigated the nucleic acid quality in sodium alginate CBs prepared from liquid-based cytology (LBC) specimens, and evaluated the impact of long-term storage of the LBC-CBs. We analyzed the nucleic acid quality in LBC-CBs prepared from effusion fluid in 19 patients with lung or ovarian cancer from January to March in 2020. The significance of long-term storage of the LBC-CBs on the nucleic acid quality was also analyzed. The cytological findings of the LBC-CBs were satisfactory and the CB area was related to the DNA quantity (r=0.808). In regards to the impact of long-term storage, the DIN could be calculated in 16 of the 19 cases (84.2%) and the DNA quality was relatively good. The length of time that the samples had been stored (days) prior to preparation of the LBC-CBs had no significant effect on the DIN (r=-0.176), whereas long-term sample storage (years) adversely affected the DNA quality (P<0.001). The nucleic acid quality in LBC-CBs was excellent, and if an adequate amount of DNA is obtained, the samples prepared by this method will be available for a variety of genetic analyses.

　In recent years, the treatment strategy for advanced or recurrent non-small lung cancer (NSCLC) has changed drastically with the development of immune checkpoint inhibitors (ICIs). These ICIs target the programmed death (PD)-1/ PD-1 ligand 1 (PD-L1) pathway, and restore the immune system’s capacity to recognize and eliminate tumors, thus improving treatment outcome in NSCLC patients. Nevertheless, since it has been reported that only a limited number of NSCLC patients show marked and durable responses to anti-PD-1/PD-L1 therapy, there has been a need for biomarkers that can predict and monitor the clinical benefits of anti-PD-1/PD-L1 therapy.
　Since anti-PD-1 therapy targets the immune system, biomarkers associated with immune responses must be developed. For example, the level of PD-L1 expression on tumor cells, as assessed by immunohistochemistry (IHC), has already been highlighted as a potential biomarker predictive of the response to PD-1 inhibitor. However, the reliability of PD-L1 expression as such a biomarker appears to be limited because it can be quite heterogeneous, even within the same tumor, and may change dynamically and drastically according to circumstances. In addition, biopsy of tumors to assess IHC-based PD-L1 expression requires invasive procedures such as bronchoscopy or video-assisted thoracoscopy, and can sometimes be problematic depending on the size and location of the tumor being investigated. By contrast, markers present in blood can be assessed with a minimal degree of invasiveness, and unlike biopsies, blood samples can be repeated and sequentially studied, thus providing dynamic information during treatment. These advantages of blood sampling would be well suited to patients receiving ICIs, in view of the dynamic nature of the antitumor immune response. Therefore, we are using peripheral blood samples to investigate biomarkers that can predict and monitor the efficacy of anti-PD-1/PD-L1 therapy.

Background and purpose) Adenocarcinoma is the most common histological type of non-small cell lung cancer (NSCLC), and various biomarkers for predicting its prognosis after surgical resection have been suggested, particularly in early stage lung adenocarcinoma. Periostin [also termed POSTN, PN, or osteoblast specific factor (OSF 2)] is an extracellular matrix protein, the expression of which is associated with tumor invasiveness in patients with NSCLC. In the present study, thin-section computed tomography (CT) findings prior to surgical resection, as well as the histological features, including periostin expression, were assessed to determine their value in terms of predicting outcomes following resection of T1 invasive lung adenocarcinoma.
Patients and methods) A total of 73 patients who underwent surgical resection between January 2000 and December 2009 were enrolled. A total of seven parameters were assessed in the thin-section CT scans: i) contour; ii) part solid ground glass nodule or solid nodule; iii) percentage of solid component (the CT solid score); iv) presence of air bronchogram and/or bubble like lucencies; v) number of involved vessels and the vi) shape and vii) number of linear strands between the nodule and the visceral pleura. Two chest radiologists independently assessed the parameters. Periostin expression was evaluated on the basis of the strength and extent of staining. Univariate and multivariate analyses were subsequently performed using the Cox proportional hazards model.
Results) There was a substantial to almost perfect agreement between the two observers with regard to classification of the seven thin-section CT parameters (κ=0.64～0.85). In the univariate analysis, a CT solid score >80%, pathological lymphatic invasion, tumor (T) and lymph node (N) status and high periostin expression were significantly associated with recurrence (all P<0.05). Multivariate analysis showed that a CT solid score >80% and high periostin expression were risk factors for recurrence (P=0.002 and 0.011, respectively). The cumulative recurrence rates among the three groups (both negative, CT solid score >80% or high periostin expression, or both positive) were significantly different (log rank test, P<0.001).
Conclusion) The CT solid score on preoperative thin-section CT scans and histological periostin expression on resected tumor specimens may assist in predicting postoperative recurrence of T1 invasive lung adenocarcinoma.

Malignant gliomas are the most representative and frequent malignant tumor of the central nervous system. In recent years the diagnosis of malignant brain tumors has become more objective compared with conventional pathological diagnosis because of advancements in molecular and genetic analysis. We can now use the results of several representative genetic alterations to obtain a final diagnosis, however, there are still some cases in which the classification is very difficult. Particularly, some rare pediatric brain tumors are frequently difficult to classify into an existing category even by molecular-based genetic analysis. Therefore, we have experienced difficulty in planning the clinical treatment and estimating the prognosis in patients with pediatric brain tumors. To help clarify these issues we are now actively working on genetic analysis by means of Onco-panel, Foundation One in Kurume University Hospital, and we also plan to collect and analyze the results of genetic analysis, and clinical data on sensitivity to the drug treatment and patient survival. As another approach, we will review conventional samples and clinical data in patients with gliomas, taking advantage of the large number of glioma samples that have been saved for study.

　Elucidation of novel mechanisms and relevant biomarkers involving malignant progression and drug resistance in cancer can contribute to further improvement of anticancer therapeutics. Our present study uses three independent approaches to target N-Myc Downstream Regulated Gene 1 (NDRG1) and Y-box binding protein-1 (YB-1). First, enhanced expression of NDRG1 suppresses cell proliferation of glioblastoma (GBM) cells through inactivation of the GSK3β/β-catenin signaling pathway. Treatment with Differentiation Inducing Factor-1 (DIF-1) enhances NDRG1 expression, followed by suppression of cell proliferation and tumor growth by GBM in vitro as well as in vivo. Secondly VEGF-induced angiogenesis is specifically blocked in mouse cornea and aorta sprouting models for angiogenesis when NDRG1 is silenced. NDRG1 plays its critical role in VEGF-induced angiogenesis through activation of PLCγ1/ERK signaling pathway after VEGF binding to VEGFR in endothelial cells. Third, enhanced expression of YB-1 induces suppression of ERα expression in breast cancer cells, followed by the appearance of tumors resistant to antiestrogen therapeutic drugs. Treatment with inhibitors of PI3K/AKT and RAS/MEK/ERK pathways blocks YB-1 activation and overcomes resistance to antiestrogens in breast cancer.